Immunodiagnostics and Patient Safety

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Krystyna Sztefko Immunodiagnostics and Patient Safety

Patient Safety Edited by

Oswald Sonntag and Mario Plebani

Volume 3

Krystyna Sztefko

Immunodiagnostics and Patient Safety

DE GRUYTER

Author Prof. Krystyna Sztefko Jagiellonian University Clinical Biochemistry Department P-A Pediatric Institute UJ CM ul. Wielicka 265 30-663 Kraków Poland )3". sE )3". Library of Congress Cataloging-in-Publication Data Sztefko, Krystyna. Immunodiagnostics and patient safety / by Krystyna Sztefko. p. ; cm. — (Patient safety) Includes bibliographical references and index. ISBN 978-3-11-024947-7 (alk. paper) 1. Immunoassay. 2. Patients—Safety measures. I. Title. II. Series: Patient safety. [DNLM: 1. Immunoassay—methods. 2. Diagnostic Techniques and Procedures. 3. Immunochemistry. 4. Patient Care—standards. 5. Safety Management—methods. QW 525.5.I3] QP519.9.I42S98 2011 616.07'56—dc22 2011002655 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de. © 2011 Walter de Gruyter GmbH & Co. KG, Berlin/New York. The publisher, together with the authors and editors, has taken great pains to ensure that all information presented in this work (programs, applications, amounts, dosages, etc.) reflects the standard of knowledge at the time of publication. Despite careful manuscript preparation and proof correction, errors can nevertheless occur. Authors, editors and publisher disclaim all responsibility and for any errors or omissions or liability for the results obtained from use of the information, or parts thereof, contained in this work. The citation of registered names, trade names, trade marks, etc. in this work does not imply, even in the absence of a specific statement, that such names are exempt from laws and regulations protecting trade marks etc. and therefore free for general use. Project management: Dr. Petra Kowalski. Production editor: Heike Jahnke. Production manager: Ulrike Swientek. Typesetting: Apex CoVantage, LLC Cover image: Comstock/Getty Images. Printing and binding: Hubert & Co GmbH & Co KG, Göttingen. Printed in Germany U Printed on acid-free paper. www.degruyter.com

Contents

Preface ................................................................................................................ Abbreviations ......................................................................................................

ix xi

Part I – Immunoassay: Theory, practice, and patient safety .................................

1

1

Immunochemical methods – Basic principles and definitions ....................... 1.1 Antibody as immunoassay reagent ......................................................... 1.2 Antigen as immunoassay reagent ........................................................... 1.3 The course of the immunochemical reaction ......................................... 1.4 Immunoassay formats ............................................................................ 1.5 Labels in immunochemical reactions .................................................... 1.6 Separation of bound and unbound fractions .......................................... 1.7 Other technologies based on the antigen-antibody reaction .................. 1.8 Basic theory of immunoassay and patient safety ....................................

3 3 7 7 9 14 14 15 16

2

Immunoassay standardization ....................................................................... 2.1 Reference materials for small molecules ................................................ 2.2 Reference materials for proteins ............................................................ 2.3 Reference methods ................................................................................ 2.4 Reference methods for heterogeneous proteins...................................... 2.5 Traceability ............................................................................................ 2.6 The uncertainty of measurement ............................................................ 2.7 Epitope as a solution for better standardization...................................... 2.8 Harmonization of immunoassays .......................................................... 2.9 Immunoassay standardization and the patient’s safety ...........................

17 17 18 19 20 20 21 23 24 24

3

Immunoassay calibration and calibration curve fitting .................................. 3.1 Calibration process................................................................................ 3.2 Commutability problem ........................................................................ 3.3 Matrix effect .......................................................................................... 3.4 Calibration and patient safety ................................................................

27 27 28 28 29

4

Reference intervals and immunoassay ........................................................... 4.1 General problems with reference intervals for analytes measured by immunoassay.................................................................... 4.2 Limitation of different approaches to reference intervals ........................ 4.3 Reference intervals and patient safety ....................................................

31 31 32 33

Laboratory preanalytical and analytical phase of immunoassay .................... 5.1 Laboratory preanalytical factors and immunoassay ................................ 5.2 Blood collection devices ....................................................................... 5.3 Anticoagulants ......................................................................................

35 35 36 37

5

vi

Contents

5.4 5.5 5.6 6

Hemolysis, lipemia, hyperbilirubinemia, paraproteinemia, and immunoassay................................................................................ Analyte stability in fresh and frozen serum samples ............................. Analytical phase in immunoassay measurement ..................................

Human natural antibodies and immunoassay ................................................ 6.1 The human immune system ................................................................. 6.2 Chemical structure of immunoglobulins .............................................. 6.3 Antigen-combining site and complementarity-determining regions ...... 6.4 Genes for immunoglobulin variable regions ........................................ 6.5 Diversification of immunoglobulins in vivo ......................................... 6.6 Natural antibody against exogenous (nonself ) and endogenous (self ) antigens .................................................................. 6.7 Natural antibodies and patient safety ...................................................

7 Immunoassay interference – How to recognize, eliminate, or reduce it.................................................................................................... 7.1 Definition and prevalence of interference in immunoassay .................. 7.2 Cross-reactivity versus interference in immunoassay ............................ 7.3 Analyte specific and nonspecific binding proteins as a source of interference in immunoassay ......................................... 7.4 Autoantibodies as a source of interference ........................................... 7.5 Nonimmune IgG complexes as a source of interference ...................... 7.6 Interference from heterophilic antibodies ............................................ 7.7 Detection of heterophilic antibodies in the patient’s sample ................ 7.8 Methods used for removal or inactivation of interfering heterophilic antibodies ........................................................................ 7.9 High-dose effect (hook effect, prozone effect) ...................................... 7.10 Low-dose hook effect .......................................................................... 7.11 Interference from heterophilic antibodies and patient safety ................ 7.12 Interpretation of immunoassay results is an art .................................... 8

37 38 40 43 43 44 45 46 47 48 51

53 53 54 59 63 66 69 78 89 92 95 96 97

Immunoassay and patient safety .................................................................... 8.1 Fallibility of immunoassays .................................................................. 8.2 Know your immunoassay..................................................................... 8.3 Basic knowledge on critical points in immunoassay for physicians ...... 8.4 Immunoassay in research..................................................................... 8.5 Who is responsible for the patient’s safety? ..........................................

103 103 105 110 113 113

Part II – Immunochemistry measurements in practice: Examples of problems in some current immunoassays...........................

115

Example 1 Example 2

Parathyroid hormone (PTH) – Heterogeneity as a major problem in PTH measurement by immunochemistry........................ Human chorionic gonadotropin (hCG) – Problems of heterogeneity and lack of standardization ....................................

117 123

Contents

Example 3 Troponin measurement by immunoassay – Problem of low assay sensitivity and interference from heterophilic antibodies ............................................................ Example 4 Aldosterone and proteolytic renin activity (PRA) – Are they useful together? .................................................................. Example 5 Thyroglobulin measurement – Autoantibody problem ...................... Example 6 Prolactin measurement by immunoassay – Heterogeneity and macroform problems................................................................. Example 7 Thyroid function tests – Most frequently measured and most difficult to interpret: Reference interval for thyroid-stimulating hormone (TSH) and problems of free thyroid hormone fraction measurement ................................. Index ...................................................................................................................

vii

129 137 141 145

149 161

Preface

Among the measurement systems used by medical laboratories, immunochemistry is one of the most important as a great variety of substances of both low and very high molecular weights, such as proteins, peptides, nonprotein hormones (amine derivatives, steroids), therapeutic drugs, vitamins, and oligonucleotides, can be measured by this technique. Due to the principle of immunochemistry, the immune reaction between antigen and antibody, immunochemical methods are very sensitive and highly specific toward the determined molecule (antigen or antibody). As a result of these merits, immunoassay is now a very widely used technique serving both physicians responsible for the patients’ care and medical researchers doing research in medical and pharmaceutical sciences. The popularity and wide acceptance of immunochemistry is to some extent dimmed by a serious drawback: susceptibility to interference from sample constituents, both of endogenous and exogenous origin. All interference appearing in immunoassay causing inaccuracy in the analyte measurement may jeopardize the patient’s safety. Heterophilic interference is the most dangerous because it is directly related to the patient’s sample. Fallibility of the immunochemistry measurement system because of unpredicted patient-related interference does not disqualify this technique. The merits of immunoassay still overcome its flaws. Because no alternative technique exists for the fast measurement of very small concentrations of many clinically important analytes present in serum or plasma samples with good sensitivity and specificity, as well as with reasonable accuracy and precision, the method will remain for many more years one of the most popular analytical tools. As for now, all efforts should be done to understand, recognize, and eliminate the interference in order to improve accuracy of immunochemistry measurement. The consciousness of the possibility of erroneous immunoassay results due to interference and good knowledge of basic immunochemistry among physicians and laboratory professionals is the only way to protect the patient from misdiagnosis and mistreatment caused by erroneous laboratory results. The book contains two parts. The first part, “Immunoassay: Theory, practice, and patient safety,” chapters 1 to 4, covers the basic principles of immunoassay technique along with the assay standardization and calibration, as well as the main problems connected with reference intervals for the analytes most frequently measured by immunochemistry. Chapter 5 deals with the laboratory preanalytical phase of immunochemistry measurement including information on factors influencing the test results, like sampling the blood, use of anticoagulants and other tube additives, as well as on typical interference like lipemia, hyperbilirubinemia, and paraproteinemia, common with other clinical chemistry techniques. Most important from the point of view of the patient’s safety, issues covered by the first part of this book are the immunoassay interference from heterophilic antibodies, autoantibodies, nonimmune complexes, and binding proteins. For better understanding the background of interference from different heterophilic antibodies, the information concerning the natural antibodies present in serum or plasma samples is discussed in chapter 6. Information on immunoassay interference in relation to assay format and the methods used for detection, elimination, and/or reduction of heterophilic interference are covered by chapter 7. The last chapter

x

Preface

of part 1 (chapter 8) deals with some recommendations for laboratory professionals, physicians, and scientists concerning the means leading to improvement of the patient’s safety both from the laboratory and clinical side. Theoretical problems discussed in part 1, connected with difficulties of interpretation of results due to lack of assay standardization, diversity of reference intervals, low assay sensitivity, heterogeneity of peptides/proteins, presence of interference from heterophilic antibodies or autoantibodies and abnormality in binding proteins are demonstrated as they appear in the most characteristic examples of immunoassay applications in part 2. As examples, the problems with immunochemistry measurement of the following analytes have been chosen: parathyroid hormone, human chorionic gonadotropin, troponin, aldosterone and proteolytic renin activity, thyroglobulin, prolactin, and thyroid function tests. For each of the chosen analytes, different analytical problems are seen, and the most recent information on the measurement of these analytes is included. Hopefully, this book will be useful for both laboratory professionals using the immunochemistry techniques and physicians taking advantage of the immunochemistry results in everyday practice. Laboratory professionals will find the information on current analytical problems connected with immunoassay and advice on how to detect, reduce and/or eliminate interference originating from the presence in the sample of heterophilic antibodies and from other sources. Physicians will find information helping them to understand the source of fallibility of immunochemistry measurements and explain difficulties encountered sometimes in the interpretation of the laboratory results obtained by this method. The book will also be useful for scientists doing research in medical sciences who will be warned that the results obtained by immunochemistry methods are not always reliable and the research conclusions may not always be valid. I greatly appreciate the opportunity given to me by the editors of the book series on patient safety to take advantage of my experience in immunochemistry measurements and interpretation of immunoassay results. In writing the book, my main intention was to draw the attention of all users of immunochemical methods to the possibility of unexpected errors inherently connected with the principle of these methods—the reaction between antigen and antibody. Although a different scope of knowledge about immunochemistry methods is needed for laboratory professionals, physicians, and scientists, all users should be aware that erroneous results can be obtained even in healthy individuals, and for the time being, there is nothing that can be done to overcome the fallibility of these methods. The only remedy accessible at present is continuous improvement of knowledge, sharing the information on unexpected erroneous patient results, pushing the immunoassay manufacturers’ toward improvements of assay design in order to make them more resistant to any kind of interference, and close cooperation between all medical professionals responsible for patient care. All this is necessary to protect patient safety.

Abbreviations

ACS AFP AutoAb BP CBG CDR CK-BB CK-MB CRP FSH FT4 HAAAs HAMAs HARAs HBR hCG HGH hsCRP IFCC LC/MS LH LOD MGUS MS NSB POC PRA PTH RF SDS SHBG T3 T4 TBG Tg Tn TPO TRAAb TSH

Antigen-Combining Site A-Fetoprotein Autoantibodies Binding Protein Cortisol-Binding Globulin Complementarity-Determining Region Isoenzyme BB of Creatine Kinase Isoenzyme MB of Creatine Kinase C-Reactive Protein Follicle Stimulating Hormone Free Thyroxin Human Antianimal Antibodies Human Antimouse Antibodies Antirabbit Antibodies Heterophilic Blocking Reagent Human Chorionic Gonadotropin Human Growth Hormone Highly Sensitive C-Reactive Protein International Federation of Clinical Chemistry and Laboratory Medicine Liquid Chromatography/Mass Spectrometry Luteinizing Hormone Limit of Detection Monoclonal Gammapathy of Unknown Significance Mass Spectrometry Nonspecific Binding Point of Care Proteolytic Renin Activity Parathyroid Hormone Rheumatoid Factor Sodium Dodecyl Sulfate Sex-Hormone-Binding Globulin Triiodothyronine Thyroxin Thyroxin-Binding Globulin Thyroglobulin Troponin Thyroid Peroxidase Antibodies Against Thyroid-Stimulating Hormone Receptor Thyroid-Stimulating Hormone

Part I Immunoassay: Theory, practice, and patient safety

1 Immunochemical methods – Basic principles and definitions

During recent years, immunochemical methods became the basic analytical technique used for the determination of molecules that are important from the clinical point of view, such as proteins, peptides, nonprotein hormones (amine derivatives, steroids), therapeutic drugs, vitamins, and oligonucleotides. Since the beginning of the use of immunochemical methods more than 50 years ago, their specificity and analytical sensitivity have been much improved, and automatization has made possible performing a huge number of determinations in a very short time. These methods have found application not only in large central laboratories, but also in point-of-care-testing systems located in clinical wards, outpatient clinics, pharmacies, and doctors’ offices. Immunochemistry like no other analytical technique has had a tremendous impact on the development of the theoretical backgrounds of many medical disciplines, such as endocrinology, immunology, immunohematology, oncology, and cardiology. Due to the introduction of immunochemical methods, the measurement of clinically important biochemical markers has become possible, without which the diagnosis, monitoring, and prevention of many diseases would not be conceivable. All immunochemical methods are based on a highly specific reaction between antigen and antibody – the binding of one or several unique conformations of an antigen molecule (epitope) by a corresponding antigen-combining site on an antibody molecule. Epitopes and antigen-combining sites may be defined only by their mutual complementarity and not by their inner properties. Complementarity between epitope and antigen-combining site is characterized by specificity of interaction, and this forms the basis of immunochemical methods. The great analytical potential of immunochemical methods is closely related to the unique properties of antibodies in recognizing the proper structure on the analyte molecule in a mixture of many similar chemical structures. However, binding between the antigen and antibody indicates only that at least one of the substances present in the reaction mixture contains the epitope structure; it does not mean that the molecule recognized by the antibody possesses biological activity. It is the chemical structure rather than bioactivity that is measured by immunochemistry methods, and this is a very well-known limitation of these methods. Both the antigen and the appropriate antibody must be present in the reagent mixture for an immunochemical reaction to take place, but for the quantitative evaluation of this reaction, the addition of labeled antigen or labeled antibody is required, making it possible to follow the course of the reaction.

1.1 Antibody as immunoassay reagent Animal antibodies (immunoglobulins) used in immunoassays are composed of two light and two heavy polypeptide chains held together by disulfide bonds. Such a structure contains two antigen-binding sites made up of fragments of both the heavy and the light chains (antigen-binding domains). Each individual antibody molecule possesses unique

4


binding specificities and can distinguish very small differences in chemical structure between antigens. This is because the amino acid sequence in the amino-terminal domains of light and heavy chains (variable regions) varies according to the binding specificity of the antibody. Detailed information about the structure of natural antibodies, antibody classes, valence, and diversification is presented in chapter 6. Similar classes of immunoglobulins are found in other mammals and are probably homologous to those occurring in human beings. Regardless of the immunoassay format, the most important reagent in immunochemical methods is the antibody directed toward the determined antigen, which is usually called the capture antibody, or first antibody. It originates from species other than humans (mouse, rabbit, sheep, or goat). Assay reagent antibody can be directed toward the whole antigen molecule or against individual domains of the antigen molecule. The following types of antibodies are currently used in the immunochemical methods: 1. Polyclonal antibodies. Polyclonal antibodies are the heterogeneous mixture of immunoglobulins of similar antigenic specificity but different affinity. Some of this heterogeneity results from the production of antibodies that bind to different epitopes on the immunizing antigen. However, even antibodies directed at a single antigenic determinant can be markedly heterogeneous, because polyclonal antibodies recognize not only epitopes present on immunogen, but also on other molecules, such as substances administered to the immunized animal together with immunogen (proteins and contaminants). In addition, polyclonal antibodies contain some sort of background from antibodies present in the animal’s blood before the immunization. Each consecutive immunization of the animal is connected with the changes in affinity of antibodies to the antigen and with the changes in the quantity and the kind of isotypes of antibodies. Although purification procedures for polyclonal antibodies are frequently necessary, their use in immunochemistry has some disadvantages that relate to their heterogeneity. First, each antiserum is different from all other antisera taken from the immunized animals, even if raised in a genetically identical animal by using identical preparation of antigen and the same immunization protocol. Second, antisera can be produced only in limited volumes, and thus it is impossible to use an identical serological reagent in a long-term or complex series of experiments or clinical tests. Besides, even antibody purified by affinity chromatography may contain a minor population of antibodies expressing cross-reactivity. 2. Monoclonal antibodies. Monoclonal antibodies represent the product of a single clone or plasma cell line. They are characterized by the same specificity (they recognize a single epitope) and by the same affinity. Monoclonal antibodies are generally of much lower affinity than polyclonal antibodies. Low-affinity antibodies are particularly susceptible to matrix effect and sample interference. The majority of monoclonal antibodies used in the immunochemical methods are murine antibodies. Efforts to use the same approach, as in the case of mouse monoclonal antibody, to produce human monoclonal antibodies have met with very limited success. The purification of monoclonal antibodies before use in immunochemistry methods is not required. Frequently, a mixture of monoclonal antibodies is used as assay capture antibody. Monoclonal antibodies are not useful in traditional precipitation methods. 3. Antibody fragments. Antibody fragments are frequently used in order to reduce nonspecific binding (NSB) and/or interference in the immunochemical reaction between

1.1 Antibody as immunoassay reagent

5

antigen and antibody. It is known that immunoglobulins can be degraded hydrolytically by a variety of proteases. For clinical and diagnostic procedures, the digestion of IgG with papain or pepsin is used (fFig. 1.1). The digestion of IgG by papain results in three fragments: two identical Fab fragments (antigen-binding fragments) endowed with antigen-binding activity, and the third, Fc fragment (fraction crystallizable), which activates complement by the classical pathway. The Fab fragment consists of an intact light chain and part of a heavy chain from the amino-terminal part of the antibody. Fragment Fc consists of two fragments, each being half of a heavy chain from the carboxy terminus. If pepsin is used for IgG digestion, two Fab fragments remain linked, F(ab’)2, but the Fc fragment is digested into smaller fragments. The Fab and F(ab’)2 fragments have exactly the same antigen-binding characteristics as the original antibody because they retain much of the three-dimensional structure of the parent molecule. Fab and F(ab’)2 fragments are used as capture antibody in many immunochemical methods. 4. Chimeric antibodies. Chimeric human antibodies are produced by manipulation of the DNA exons coding for the IgG variable (V) and constant (C) region domains. The molecules are made up of domains from different species. This means that V regions of heavy and light chains are usually of mouse origin, whereas C regions are human (about 70% of the entire molecule is human). In a humanized version, only Fab fragments are coded by animal sequences, while the rest of the molecule (about 90%) is of human origin. By this approach, 100% human antihuman monoclonal antibodies can be obtained. However, it needs to be mentioned that

Fig. 1.1: Immunoglobulin G (IgG) hydrolysis by papain and pepsin yielding different antibody fragments.

6


the more human is monoclonal antibody, the greater is the chance of a decreased immunogenicity and loss of antigen specificity. 5. Recombinant antibodies. Recombinant single-chain antibodies (scFv fragments) are truncated Fab comprising the variable domain of a heavy chain linked by a stretch of synthetic peptide to a V domain of a light chain. The unique characteristics of antibodies in determining the quality and possibilities of analytical application in the immunochemical methods are as follows: (a) High specificity of antibody toward the substance bound, which makes possible the determination of antigens in the presence of many closely related substances in samples of biological materials at very low concentrations of the order of 1012 mol/L. (b) High energy of noncovalent bonds between antibody and antigen, which makes possible the reaction between these two reactants (1). Originally, only polyclonal antibodies were used in immunochemical methods. Later, monoclonal antibodies were applied, and now, monoclonal antibodies, recombined antibodies, or antibody fragments are frequently used. Most important is that antibodies used in the immunochemical methods should have appropriate specificity and affinity in order to enable the proper reaction kinetics between antigen and antibody. Many polyclonal antibodies can be obtained quickly and easily by animal immunization, but they have low specificity and contain contaminants that interfere with the binding reaction of antibody with antigen. Even highly purified polyclonal antibodies contain antibodies directed against different epitopes, which may be advantageous in case of heterogeneity of some peptides or proteins determined by immunoassay. In contrast to polyclonal antibodies, monoclonal antibodies could be used in immunoassay without purification. However, the purity of monoclonal antibodies does not always guarantee their specificity. These antibodies have a unique specificity for a defined epitope, and it may happen that the reaction will take place only between antibody and peptide/protein fragments containing this epitope. The whole peptide molecule may not be recognized because, for instance, of peptide chain folding. However, monoclonal antibodies may not recognize the same antigen in different models of immunochemical methods due to the occurrence of conformational changes of the antigen molecule. Therefore, after the introduction of monoclonal antibodies to routine use in immunoassays, the results of the determination of some antigens were sometimes much lower than those obtained with the use of polyclonal antibodies. The same antibody used in immunoassay may have one, two, or more antigencombining sites for totally different (nonrelated) epitopes. Nonetheless, the situations when different binding sites in the antibody molecule are situated very closely to each other or the fragment of the amino acid sequence of one binding site is identical to that of another binding site are quite common. This may be described as partial overlapping of binding sites. In such situations, the binding of one epitope may block the binding of another epitope for which the antigen-combining site is placed nearby. This means that the assay antibody may bind not only the antigen used for animal immunization, but also other molecules with structure similar to the epitope. Whereas the binding of antibody with antigen occurs usually with a high affinity, there are infrequent situations when the antibody has higher affinity to heterogeneous antigens than to the antigen used for immunization. This phenomenon, observed for instance when antibodies directed against closely related immunogen analogs are tested, is known as heterospecificity.

1.2 Antigen as immunoassay reagent

7

1.2 Antigen as immunoassay reagent The term immunogen describes any substance (a protein or a molecule coupled to a carrier) that when introduced to the organism of another species elicits the production of antibodies in the host. Haptens are the substances that alone are not able to elicit antibody production, but when they are conjugated with immunogenic protein carrier and injected into an animal cause the production of three types of antibodies: carrierspecific, hapten-specific, and conjugate-specific antibody. An antigen is defined as any substance that can bind to a specific antibody. The distinction between an immunogen and an antigen should thus be made. All antigens have the potential to elicit production of specific antibodies, but some need to be attached to the immunogen to do so. Thus, all immunogens are antigens, but not all antigens are immunogenic (2). The antigens commonly found in blood serum may or may not contain posttranslational modifications. The antigenicity of protein is localized in a discrete region of the molecule (antigenic determinant, epitope) recognized by the corresponding antideterminant (antigencombining site) on the antibody. The best-known classification of epitopes distinguishes between sequential epitopes (continuous), containing about 3–6 amino acids, and spatial epitopes (discontinuous), composed of 12–15 amino acid residues. It is known on the basis of experimental data that only 3–5 amino acid residues of the epitope, defined from the structural point of view, contribute significantly to the energy of binding. Exchange of only 1 amino acid residue within the epitope results in a decrease of the affinity constant by two to three orders of magnitude, whereas substitution of 1 amino acid in any other place within the area of the contact of antigen with antibody brings about a much smaller change of affinity. Distinguishing between continuous and discontinuous epitopes depends on whether all amino acid residues belonging to the epitope are in the polypeptide chains neighboring each other. Continuous epitope corresponds to short polypeptide fragments composed of few amino acids. On the contrary, discontinuous epitope is composed of amino acid residues that are not neighboring in the polypeptide chain but are spatially close due to its folding. Although this classification of epitopes has been commonly accepted, nevertheless the borders between the two kinds of epitopes are not very sharp. The epitope recognized by the antigen-combining site may be described as continuous or discontinuous structure, but in fact, the binding process takes place on the level of individual atoms. It has been accepted that 90% of all protein epitopes have discontinuous structure, because it is not only the primary structure of the protein, but also its conformation that is important. This is the reason for considerable differences in the course of the immunochemical reaction for native antigen and synthetic protein antigen, because these two antigens possess common primary amino acid structure but may not have the same secondary structure. Complementarity between groups of atoms in the antigen and groups of atoms in the antigen-combining site of the antibody molecule is the main factor responsible for antibody specificity (2).

1.3 The course of the immunochemical reaction Protein molecules naturally repel each other in aqueous solution due to hydrophilic nature of their surface. This repulsive force acts over a distance of 20–30 A˘; thus the original forces acting between the antigen epitope and the antigen-combining site in the antibody must overcome this barrier. The number and strength of the noncovalent bonds

8


linking the antigen with the antibody are determined by the three-dimensional structure of both the epitope and its contact surface on the antibody. Binding of antibodies with protein antigens occurs over large sterically and electrostatically complementary areas. The antigen-antibody reaction involves several different forces. Electrostatic forces act between the oppositely charged amino acid side chains; Van der Waals dipole-dipole interaction is seen between electric dipoles formed when fluctuations in electron clouds around molecules oppositely polarize neighboring atoms; hydrophobic forces cause hydrophobic groups to exclude water molecules and pack themselves together; and hydrogen bonds are formed when hydrogen is shared between electronegative atoms, mainly nitrogen and oxygen, and polarization of the electron cloud on both acceptor and donor is observed. Covalent bonds, the most common bonds formed in many molecules in the course of chemical reactions, never occur in the antigen-antibody interaction. Water reinforces the complementarity and the interaction between antigen and antibody (3). Changes in pH and high salt concentration can disrupt antigen-antibody binding by weakening electrostatic forces and/or hydrogen bonds. The strength of a hydrophobic interaction is related to the surface area hidden from water. The contribution of each of these forces to the overall interaction between antibody and antigen depends on the particular antibody and antigen involved. In general, hydrophobic and Van der Waals forces operate over very short ranges and serve to pull together two complementary surfaces. Electrostatic forces and hydrogen bonds strengthen the overall interaction. Significant structural changes occur at the binding site of the antigen and antibody when the binding of these two reagents takes place (4). The mechanism for antigenantibody recognition is very complicated and cannot be simply compared to the formation of ionic or covalent bonding that exists in many chemical compounds. If the chemical structure on the antibody (a combination of atoms) comes close to the appropriate chemical structure on the antigen, then rearrangement of atoms takes place (the “induced fit” model for antigen-antibody recognition) for better fit of these two molecules (5,6,7). The magnitude of this inducibility ultimately affects the specificity of the antigen-antibody interaction. The better fit between the epitope on the antigen and the binding site on the antibody, the stronger are the noncovalent bonds formed. The interaction between the antibody and the antigen can be described by affinity and avidity. The strength of binding of the antibody to its antigen in terms of a single antigen-combining site on the antibody binding to a monovalent antigen is termed as its affinity, and it is the property of the antigen. The total binding strength of the antibody molecule with a variety of antigen-combining sites is called its avidity and is the property of the antibody (2). A high degree of steric complementarity exists between the epitope and the antigencombining site, but the binding of antibody with antigen in a reaction mixture is not a static phenomenon. It is a reversible equilibrium reaction subjected to the mass action law. The dissociation energy of a molecule is usually higher than the energy of association of molecules required for the formation of the associate, which means that higher energy is needed for disruption of the antigen-antibody complex than for its formation. The rate constants of association and dissociation have a direct influence on the course of the immunochemical reaction. Moving the reaction equilibrium in the direction of antigen-antibody complex formation or its dissociation depends, among other factors, on pH, ionic strength, and temperature. To gain the level of affinity of binding (107– 109 L/mol range) required for the immunochemical reaction, direct contact between only a few amino acids of antigen epitope and a few amino acids of antigen-combining site

1.4 Immunoassay formats

9

on the antibody molecule is sufficient. With great approximation, the immunochemical reaction can be described on the basis of the law of mass action, assuming that in a homogeneous solution only a pure antigen (one epitope) and antibody (one antigencombining site) are present. But it has to be accepted that the occurrence of allosteric changes does not affect the binding between antigen and antibody, the reaction is in equilibrium, and very low NSB is present. The ratio of the two constants (association constant and dissociation constant) equals the equilibrium constant, which represents the final ratio of analyte bound with antibody to unbound (free) analyte. Both association rate and dissociation rate constants are very important for following the course of immunochemical reactions of different antigen-antibody combinations.

1.4 Immunoassay formats Nomenclature and classification of immunochemical methods are sometimes misleading, inasmuch as there is approximately the same number of similarities as the number of differences between various groups of methods. All immunoassays can be divided according to which substrate is labeled (antigen or antibody), what is being measured (antigen or antibody), what label is used, and how the signal is measured. However, the concentration of the capture antibody is the key criterion for classifying immunochemistry methods. Based on this criterion, two main formats for immunoassay can be distinguished: competitive and noncompetitive (immunometric). 1. Competitive methods. These are the methods with limited amount of high-affinity capture antibody. Two separate method formats for antigen measurement can be applied: (a) competition between labeled and unlabeled antigen for the antigencombining site on the antibody when the immunochemical reaction is performed in liquid phase (fFig. 1.2a) and (b) competition between the antigen analog attached to solid phase and the sample antigen for antigen-combining site on the labeled antibody (fFig. 1.2b). (c) For antibody measurement, advantage is taken of the competition between the antigen attached to a solid phase and two antibodies, one from the patients’ sample and one labeled antibody that is added (fFig. 1.2c). In competitive methods performed in liquid phase, a fixed amount of labeled antigen and a variable amount of antigen from a standard solution or from an unknown sample compete for the fixed and limited amount of antibody. After the equilibrium of the reaction is reached, the separation of antigen bound to antibody from the unbound antigen must be performed by using specific or nonspecific precipitation agents. Then the bound or free analyte fraction (or both) is measured by the appropriate detection system. When the antigen-antibody complex is measured, the inverse relationship exists between the analyte concentration and the signal. In the analog competitive method, a fixed amount of analog attached to solid phase competes with the measured variable amount of analyte from the patient’s sample for a fixed amount of labeled antibody. After an appropriate incubation time and washout step, the signal is measured. In the analog format of competitive method, analogous to competitive methods performed in liquid phase, the measured signal is inversely proportional to the antigen concentration. If antibody is determined by competitive methods, the measured signal is also inversely proportional to the amount of antibody in the

10


Fig. 1.2a and b: Competitive immunoassay formats. (a) Liquid-phase competitive immunoassay: Competition between a fixed amount of the labeled antigen (tracer) and variable amounts of the measured (unlabeled) antigen for a limited number of antigen-combining sites on the assay antibody. After reaction equilibrium is reached, the bound antigen is separated from the free fraction, and the signal coming from the labeled-antigen bound fraction is measured. (b) Analog competitive immunoassay: Competition between a fixed amount of the solidphase antigen analog and variable amounts of the measured antigen for a limited number of antigen-combining sites on the labeled assay antibody. The complex of the antigen analog with the labeled assay antibody is measured.


11

Fig. 1.2c: Competitive immunoassay formats. (c) Competitive immunoassay for antibody measurement: Competition between a fixed amount of the labeled antibody and variable amounts of the measured antibody for a limited amount of antigen attached to the solid phase. The assay signal coming from the labeled antibody bound to the solid-phase antigen is measured. In all competitive methods, the assay signal is inversely related to the measured antigen (or antibody) concentration.

sample. The sensitivity of the competitive assays depends on the affinity of antibody, accuracy of signal measurement, and extent of NSB. In order to measure NSB, the NSB sample must contain all assay reagents except antibody. To improve the sensitivity of competitive methods, the sequential addition of assay reagents can be applied with the addition of antibody as the first reagent, followed by addition of labeled antigen. The precision of competitive methods depends on the error in pipetting of the biological specimen, labeled antigen, and antibody. According to “the antibodybinding site occupancy principle” in competitive assay, labeled antigen is bound to unoccupied antigen-combining sites on the antibody. 2. Noncompetitive methods (immunometric methods). These are the methods with an unlimited amount of capture antibody coupled to solid phase and an unlimited amount of labeled antibody. It should be clarified that although the amounts of both capture and labeled antibodies are unlimited and in excess in relation to antigen, their amount is constant and fixed. An antigen measured by such methods must contain at least two different antigenic determinants for at least two different antibodies used in the method. When the antigen is linked with two assay antibodies (capture and signal antibodies), a typical sandwich is formed. For this reason, for noncompetitive methods the name sandwich assay is frequently used. Separation of the capture antibody bound to analyte from other sample components is performed by a washing step, and then labeled antibody is added. The separation of the complex of solid-phase capture antibodyantigen-labeled antibody from unbound labeled antibody is performed by an

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additional washing step (heterogeneous assay – the separation of unbound labeled antibody is required). Noncompetitive methods can be also performed by simultaneous addition of capture antibody, antigen (serum sample), and labeled antibody, and the signal is directly modulated by the formation of a sandwich (homogenous assay – the separation of excess of unbound labeled antibody is not required). In all noncompetitive methods there is a linearly proportional relationship between the analyte concentration and resulting signal (positive dose-response curve). The sensitivity of the noncompetitive methods is determined by the error in the measurement of the signal and by the NSB of the labeled antibody to the capture (immobilized) antibody, as well as to the solid phase per se. Precision of immunometric method depends only on the exactness of the addition of the biological specimen, because both labeled and unlabeled antibodies are added in excess. The NSB should be as low as possible. In a noncompetitive assay, the labeled-antibody binding is confined to occupied sites on the capture antibody. There are three formats for noncompetitive (immunometric) assays: 1. The most common is the “forward two-step” method (fFig. 1.3a). In the first step, the antigen reacts with the solid-phase capture antibody. After proper incubation time, all other sample components are washed out, and the labeled antibody directed against the second epitope on the antigen is added. Then, one to three washing steps are usually applied to remove the unbound labeled antibody.

Fig. 1.3a: Noncompetitive immunoassay formats. (a) Forward two-step method, heterogeneous assay: The reaction between an unlimited amount of solid-phase capture antibody with variable amounts of antigen. After the washing-out step and addition of an unlimited amount of labeled antibody (signal antibody) followed by a second washout step, the typical sandwich (complex of capture antibody-antigen-labeled antibody) is formed and the signal is measured.


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2. In the “reverse two-step” method, the antigen reacts with the labeled antibody in liquid phase forming a soluble complex. Then after the proper incubation period of time, solid-phase capture antibody, which binds the complex of antigen bound with the labeled antibody, is added (fFig. 1.3b). Unbound labeled antibody is washed out.

Fig. 1.3b and c: Noncompetitive immunoassay formats. (b) Reverse two-step method, heterogeneous assay: Reaction between variable amounts of the antigen and an unlimited amount of the labeled antibody in liquid phase is performed as first; then after proper incubation time, an unlimited amount of capture antibody attached to solid phase is added. After washing out the unbound labeled antibody, the signal from the labeled antibody-antigen-solid-phase capture antibody complex is measured. (c) Homogenous noncompetitive immunoassay: Capture antibody, antigen, and signal antibody are mixed together in liquid phase, and the assay signal is modulated by the formation of the capture antibody-antigen-labeled antibody complex. In all noncompetitive methods, the assay signal is proportional to the measured antigen concentration.

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3. Finally, in the “simultaneous” (homogenous) assay (fFig. 1.3c), all the reagents – capture antibody, antigen, and labeled antibody – are mixed together simultaneously. The unique characteristics of the homogenous methods are that the signal is modified by the immunochemical reaction and no separation or washing steps are required.

1.5 Labels in immunochemical reactions In both competitive and noncompetitive assay formats, the most frequently used labels are the following: a. Radioactive isotopes (125I, 57Co, and 3H). The measurement of radioactivity is performed by using a gamma scintillation counter (125I, 57Co) or a liquid scintillation counter for beta emitters (3H). When using radioisotope assays (radioimmunoassay or immunoradiometric assay), it is important to keep in mind that due to radioactive decay, the assay kit has a short lifetime, on average 6–8 weeks for 125I . Also, a sufficiently long counting time is needed to keep the counting error below 1%. b. Enzymes (e.g., alkaline phosphatase, horseradish peroxidase, glucose-6-dehydrogenase, glucose oxidase, and B-lactamase). The quantification of enzyme immunoassays depends on the detection system used. c. Fluorescent labels (fluorescein isothiocyanate, lanthanide chelates, umbeliferone, and europium). Many applications using such labels can be mentioned (e.g., fluorescent polarization, laser-induced fluorescence, time-resolved fluorescence, and flow cytometry). d. Chemiluminescent labels (isoluminol, isoluminol derivatives, and acridinium esters). In chemiluminescence, the excitation event is caused by a chemical (e.g., oxidation or electrochemical reaction).

1.6 Separation of bound and unbound fractions All competitive and noncompetitive heterogeneous assays require separation of the antigen-antibody complex (bound fraction) from unbound, free analyte both unlabeled and labeled (competitive assay) or a washing step for removing all sample components other than analyte and free, unbound labeled antibody (noncompetitive assay). Usually, only the antibody-bound fraction is measured, although the measurement of the unbound fraction or both the bound and free fractions can be useful in practice (competitive methods). In early competitive liquid-phase assays, simple but nonspecific precipitation with dextran-coated charcoal adsorbing unbound analyte, ammonium sulfate, or PEG were used for separation; then more specific second antibody (doubleantibody methods) or PEG-assisted second antibody was applied. Most of the currently performed noncompetitive immunoassays rely on solid-phase separation with a washing step removing NSB molecules after the antigen was bound to the capture antibody. The washing step in a noncompetitive assay does not, however, eliminate interference, as it requires a compromise between removal of undesirable NSB agents and affecting the physical binding of the antigen to the capture antibody and the second reaction with the signaling antibody (8).

1.7 Other technologies based on the antigen-antibody reaction

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The efficiency of the separation step has a direct effect on assay sensitivity and overall quality. In a competitive assay, poor separation causes a high level of NSB due to the free labeled analyte associated with the bound fraction. Because the lowest signal generated in the assay (low binding of the labeled antigen to the antibody) is at the highest concentration of unlabeled analyte, the high level of NSB causes the inaccuracy of measurement at the low end of the calibration curve. In noncompetitive heterogeneous methods, the washing step removes all sample components except the analyte bound to the capture antibody. After the addition of labeled antigen and the formation of the sandwich, the washout step is required in order to remove the entire unbound labeled antibody. This step has a direct effect on assay sensitivity. Any residual labeled analyte adsorbed or attached to the solid phase increases the assay signal. Because in noncompetitive methods the lowest signal occurs at zero analyte concentration, even a very small amount of remaining labeled antibody decreases the assay analytical sensitivity.

1.7 Other technologies based on the antigen-antibody reaction Western blotting (immunoblotting) is the technique used to visualize antigens in a complex mixture. As the first step of the method, sample proteins are solubilized in a strongly denaturing charged detergent (sodium dodecyl sulfate, SDS). Then the solubilized proteins are separated according to molecular weight by SDS–polyacrylamide gel electrophoresis. Next, the separated proteins are transferred to a nitrocellulose membrane (blot), which is then incubated with the antibody. After antigen-antibody complex formation, all unbound antibody is washed out and anti-IgG coupled with enzyme label (e.g., horseradish peroxidase or alkaline phosphatase) is added. The position of the antigen bands that react with the test antibody is visualized on a nitrocellulose membrane by deposition of the colored material originating from the enzyme substrate. Flow cytometry, another technique based on the antigen-antibody reaction, is used to calculate the number of cells that express cell-surface antigens by coupling with the monoclonal antibodies labeled with fluorochromes. The flow cytometer is a device that sucks through a small orifice the cells ordered in the form of a linear flow (one by one) by hydrodynamic focusing. The flow of cells is then crossed by laser light eliciting fluorescence that reaches the dedicated detectors after being focused by a set of mirrors and lenses. This method makes it possible to detect the presence of cell elements to which the labeled antibody has been attached. During about 10 minutes, it is possible to evaluate tens of thousands of cells present in the examined mixture. The method is used mainly in hematology, in diagnostics of infectious diseases, and in diagnostics of immune deficiencies. In addition, methods aimed at the permeabilization of cell membranes (cellular permeabilization) have been designed. These methods make possible the detection of intracellular antigens, such as cytokines or growth factors. By using, for instance, nucleotide probes labeled with fluorochromes, it is possible to investigate the expression of some genes in the cells. Contemporary flow cytometers provide the possibility of not only detection of surface antigens, but also performing the required segregation and separation (sorting) of the cells containing a given antigen. During the past 50 years, great improvements in immunoassay technology have been witnessed. The requirement for more sensitive and faster analyte measurement pushed the research toward new applications of the antigen-antibody reaction. Based on the immunometric system, protein/peptide microarray or antibody microarray can

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be performed as forward-phase protein microarray or reversed-phase protein microarray formats (9). In the forward-phase protein microarray (protein/peptide microarray, antibody microarray), capture molecules (peptides, proteins, and antibodies) are immobilized on solid-surface microarray format rows and columns. After the addition of the biological sample, binding between the immobilized molecules and corresponding analyte occurs. In the protein/peptide microarray, binding can be visualized by adding labeled secondary antibody. In the antibody microarray, binding can be visualized either by direct labeling of the analyte or by the addition of labeled secondary antibody (sandwich assay). This technique can be performed on a planar microarray-based system or bead microarray-based system. In the reverse-phase protein microarray format, the entire proteome of cells immobilized in rows on a solid support can be visualized by using highly specific labeled antibodies. Both formats allow the measurement of many molecules of interest present in the sample at the same time.

1.8 Basic theory of immunoassay and patient safety Without knowledge of the basic principles of immunoassay, especially the assay format, any discussion of the serious problems that may occur during the analytical phase of the measurement, which may affect the patient’s result, is almost impossible. Assay performance and susceptibility of immunoassay measurement to interference depends strongly on reagent antibodies (capture and signal antibodies), assay format, and signal measurement. Thus, regardless of how the immunoassay measurement procedure is performed – manually or by automatic immunochemistry measurement system – laboratory professionals must know the basic principles of the assay used in the laboratory.

References 1. Davies C. Introduction to immunoassay principles. In: Wild D, ed. The Immunoassay Handbook. London: Nature Publishing Group; 2001: 3–40. 2. Murphy K, Travers P, Walport M. Janeway’s Immunobiology. 7th ed.: Garland Science, Taylor & Francis Group; New York and London 2008. 3. Braden BC, Poljak RJ. Structural features of the reactions between antibodies and protein antigens. FASEB J 1995;9: 9–16. 4. Stanfield RL, Takimoto-Kamimura M, Rini JM, Profy AT, Wilson IA. Major antigen-induced domain rearrangements in an antibody. Structure 1993;1: 83–93. 5. Wilson IA, Stanfield RL. Antibody-antigen interaction: New structures and new conformational changes. Curr Opin Struct Biol 1994;4: 857–67. 6. Wedemayer GJ, Patten PA, Wang LH, Schulz PG, Stevens RC. Structural insights into evolution of an antibody combining site. Science 1997;276: 1665–9. 7. Webster DM, Henry AH, Rees AR. Antibody-antigen interactions. Curr Opin Struct Biol 1994;4: 123–9. 8. Ismail AAA. A radical approach is needed to eliminate interference from endogenous antibodies in immunoassay. Clin Chem 2005;51: 25–6. 9. Yu X, Schneiderhan-Marra N, Joos TO. Protein microassays for personalized medicine. Clin Chem 2010;56: 376–87.

2 Immunoassay standardization

All analytical methods, including immunochemistry methods, require proper standardization to ensure that different assays used for the measurement of a particular analyte give the same results. To ensure the highest level of accuracy in routine measurement of any analyte, a reference measurement system must be established. Proper standardization is only possible if the molecules intended to be measured (measurand) present in reference material and in the biological sample are identical not only in relation to chemical conformation, properties, and heterogeneity but also concerning epitopes being recognized by immunoassay reagent antibodies (capture and label antibodies). In clinical chemistry, the primary standards used for method standardization should be homogenous, pure, and identical in structure and physical and chemical properties to the corresponding substance that is being measured in the biological sample. Because a vast variety of analytes can be measured by immunochemistry, many different analytical problems are encountered during standardization procedures both for well-defined, low-molecular-weight molecules and for many heterogeneous macromolecules. The hierarchical link between analytical methods (definitive, reference, and routine methods) and corresponding reference materials (primary reference material, secondary reference material, and calibrators) is well known for laboratories. However, the relationship between reference methods and reference materials works perfectly for small, chemically uncomplicated molecules, like electrolytes or glucose, but implementation of simple standardization procedures into immunoassay for complex molecules, as well as for very heterogeneous macromolecules, is challenging and not always possible to achieve.

2.1 Reference materials for small molecules Availability of well-defined standards (reference materials) and definitive or reference methods for assigning a certified value to a given reference material is the prerequisite for standardization (1). For simple molecules like steroids, thyroid hormones, biogenic amines, and certain drugs, the chemical composition, structure, and properties of which are well-known, highly purified reference materials (primary standards) are readily available. However, some of these molecules are present in the biological fluids not only in free form but also in the form bound to a variety of specific and nonspecific binding proteins. For this reason, standardization of procedures designed for free hormone measurement is quite difficult, and transferring the theoretical concepts into the practice presents some obstacles. For example, steroid hormones and thyroid hormones exist in blood as a free fraction and as a fraction bound to plasma proteins, so their concentration in serum water differs from that measured in serum. Because there is currently no method for direct measurement of analytes in serum water, the measurement of free fractions, for instance free thyroxin (FT4), requires equilibrium dialysis or ultrafiltration performed prior to the immunoassay measurement in order to obtain correct concentration results. It is almost impossible to meet the principles of metrology during the measurement of free fraction without disturbing the equilibrium that always exists

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between free and bound hormone fraction not only in vivo but also in vitro. Although in some methods, like the analog method for FT4 determination, measurements are based on calibrators with the known capacity of thyroxin-binding proteins, the problem of standardization of assay for the measurement of samples taken from patients with extremely high or very low concentrations or abnormal forms of specific and nonspecific binding proteins must still be solved. It has to be remembered that for molecules like thyroxin or cortisol, the concentration of the free hormone fraction present in blood is much lower as compared to the respective total hormone concentration, and more sensitive assays are required. Although International System (SI) units are recommended, traditional units (e.g., μg /dL for T4) are still in use for some analytes.

2.2 Reference materials for proteins For high-molecular-weight analytes such as proteins, primary standards can either be purified from natural sources (tissues, serum, or urine) or obtained by DNA recombination techniques. Their primary structure can be determined by amino acid analysis, and their molar concentration can be accurately assigned. For many complex proteins, the definition of the substance intended to be measured is very difficult because of potential intrinsic and acquired heterogeneity, both chemical and conformational. Also, during the process of protein purification, structural integrity of the analyte can be more or less altered, and in addition, purified proteins often contain different contaminants. In the case of proteins/peptides present in biological fluids in many different molecular forms, purified and recombinant proteins may not have the exact molecular composition and secondary or tertiary structure; thus purified reference material may differ from the recombinant reference material of protein in respect to antigenic activity. Therefore, recombinant protein, purified protein, and native protein may react differently not only in various assays using different antibodies but also in assays using the same reagent antibody (2). This is not the only obstacle in protein standardization. It is known that molecular heterogeneity is an inherent feature of all proteins. In any individual, heterogeneous composition of different forms of many proteins or peptides may be genetically established or may be disease related. Therefore, the molecular ratio of different forms of heterogeneous protein in healthy individuals may significantly differ from the composition of this protein seen when in the same individuals a particular disease is present. The differences can even be related to the disease stage. Such significant heterogeneity observed in patients’ samples can be due to different posttranslational biochemical processes like glycosylation, sialylation, polymerization, or aggregation. Besides many forms of hormones resulting from heterogeneity, patient samples may also contain prohormones, unusually hydrolyzed hormone fragments, free protein subunits, and complexes of protein with other macromolecules (e.g., complex of protein with immunoglobulin or with soluble receptor fragments) or inorganic components. As a result, for heterogeneous analytes it is almost impossible to prepare reference material that reflects all circulating forms of heterogeneous protein present in the biological sample, simply because frequently not only the molar ratio of different forms is unknown but also knowledge concerning all forms of protein is currently lacking. The question is which reference material for heterogeneous protein should be used to reflect the differences in composition of protein forms in different pathological states.

2.3 Reference methods

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Partial proteolytic degradation of the peptide chain, or nicking, is fairly common among hormones and other biological markers (e.g., human chorionic gonadotropin [hCG], troponin). Some analytes have different chemical and structural properties depending on the tissue origin (e.g., pancreatic and gut glucagon), or different forms in serum and urine are present (e.g., hCG). Some proteins present in biological samples can be immunoreactive without being biologically active, or the opposite may be true: some biologically active proteins are not immunoreactive, so they are not measured by immunoassay. Therefore, not only is information about the molecular forms needed for preparing the appropriate standard for accurate analyte measurement by immunoassay, but also it is very important to know what the reactivity of the assay reagent antibodies is in relation to the characteristics of the analyte in the sample and reference material. It is known that specificity and affinity of antibodies is not absolute and may be modified in the presence of other substances in biological samples. All that is needed to have the appropriate reference material for immunoassay standardization of clinically useful molecules is the exact definition of the substance intended to be measured, its biological and clinical function, and the influence of the matrix when the concentration values are assigned by reference method. The availability of matrix-based certified reference material (secondary standard) is very critical for the immunoassay standardization process. Matrix effect, by definition, is the effect of all other components of the analytical system, except for the analyte, on the value of the analyte being measured, with respect to a specified condition. Although matrix-supplemented reference material is usually commutable with normal serum, a problem arises when the determination of the analyte is performed in patient samples with altered proteins or other atypical interfering substances present. The matrix used for secondary reference materials usually has the following characteristics: optimal clarity, triglyceride concentration of less than 2 mg/dL (0.023 mmol/L), and no hemoglobin and bilirubin present. In addition, it should be tested for HIV-1 and 2, HTLV-1, HbsAg, and HCV, and it must be free from such components as rheumatoid factor and monoclonal antibodies, which are known causes of immunoassay interference (2). Although the matrix of serum-based secondary standards is similar to that of biological samples, this does not completely eliminate the matrix problem, because matrix varies between the analyzed samples taken from different patients. It has to be remembered that various immunoassays have different matrix problems. Reference material may be used in reference procedures as well as in routine methods. Reference material may also be used as trueness control.

2.3 Reference methods To assign the concentration value to the reference material, a reference method with the highest possible level in the metrological hierarchy is needed. Such a method must be well characterized not only chemically (what structure is being measured), but also the interaction between the measured antigen and reagent antibodies should be specified. If monoclonal antibody is used in the immunoassay reference method, it should have known epitope specificity. The value for the reference material assigned by reference method represents the best estimate of the “true” value. If reference methods are based on the most up-to-date knowledge, they are considered definitive methods. For

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definitive or reference methods, the uncertainty of the measurement must be always established, simply because there are no methods without errors. In addition, these methods should be insensitive to matrix effect. Definitive methods are time consuming, have low throughput, and are expensive. However, if the description of the method is followed exactly, the result for a given sample should be “true”, which means that it must be within the uncertainty described for the method. Standardization always improves trueness (lack of bias) of the measured analyte concentration, but it does not directly affect immunoassay precision.

2.4 Reference methods for heterogeneous proteins For many heterogeneous molecules, reference measurement procedures are not available, and concentration values are assigned to reference material by consensus. This is the case for many glycoprotein hormone standards consisting of partially purified preparations, and potencies of such standards are determined by bioassay and expressed in arbitrary international units (IUs, defined according to WHO standards) based on bioactivity. This is the case for tumor markers (e.g., carcinoembrionic antigen), some antibodies (e.g., antibody against thyroid peroxidase) and some glycoprotein hormones (e.g., luteinizing hormone [LH], follicle stimulating hormone [FSH]). For some heterogeneous proteins measured by immunoassay, there is no international conventional reference measurement procedure. Also, no reference materials and calibrators are available for many analytes. At present, no reference procedure exists for many heterogeneous proteins measured by immunoassay.

2.5 Traceability Proper standardization of medical laboratory results requires that the established rules of metrology are followed. Metrological traceability in laboratory medicine means reliable transfer of the measurement values from the highest hierarchical level of methods (definitive or reference methods) down to the methods routinely used in the laboratory for analyte concentration measurement in the patient’s sample. According to the International Organization for Standardization (ISO) definition, traceability is the property of the result of a measurement or of the value of a standard whereby it can be related to stated references, usually national or international standards, through a documented unbroken chain of comparisons, all having stated uncertainties (3). Numerically, the traceability of a result can be assessed by its accuracy (no systematic errors, lack of bias). However, distinction should be made between accuracy and trueness. Accuracy is the closeness of the agreement between the measured quantity value and the true quantity value of the measurand, and trueness is the closeness of agreement between the average of an infinite number of replicates of the measured quantity values and a reference quantity value (4). Implementing the traceability in medical laboratory reassures the clinical chemist that analyte concentration in an unknown sample measured by routine measurement system and the result obtained in the same sample by reference or definitive method are equivalent. This is only possible to achieve if it is known what is going to be measured (measurand, quantity intended to be measured), how it will be measured (reference material and reference method must be defined), what will be the

2.6 The uncertainty of measurement

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uncertainty of the measurement, and whether the unbroken chain of calibrations and value assignments were maintained (5,6). Reference methods and reference materials should always be distinguished, although they always coexist and are linked together. If no reference material is available, then the most sophisticated definitive method will be useless in terms of traceability. Also, if reference material is available but there is no reference method to assign the concentration value, such material cannot be used. Such a situation is frequently seen in the case of analytes measured by immunoassay systems, and therefore the concentration of analyte in the patient’s sample is extrapolated from the manufacturer’s selected procedure and the corresponding calibrator. This concerns mainly heterogeneous protein analytes, like tumor markers or troponin (1). Regardless of the lack of reference material or reference method, if the patient’s result from any particular measurement cannot be traceable to internationally accepted reference material, then there is a big probability of inappropriate interpretation with all medical consequences for the patient. For many analytes measured by immunoassay, lack of traceability of the result is the major cause of poor standardization. Assay manufacturers should include the certificate proving the traceability chain for calibrators used in routine immunoassay with clinically acceptable uncertainty. As long as the laboratory is using the commercially available immunoassay reagents and follows the analytical procedure recommended by the manufacturer, no validation process for traceability is needed (7). The hierarchical tree of definitive, reference, and routine methods and appropriate reference materials and calibrators is presented in fFig. 2.1. With regard to the implementation of traceability, it is again important to differentiate between analytes being chemically well defined and analytes in human samples, which are heterogeneous. For the first group of molecules, the results obtained for the patient’s sample are well traceable to the SI units. For heterogeneous analytes, the implementation of standardization is much more difficult. Since they are heterogeneous and their composition in human body fluids varies depending on disease state, time of measurement, and activity of proteases, all reference materials for these compounds are (by definition) only surrogates for the analytes measured in the patient’s sample. Because standard materials may resemble to some extent the typical heterogeneous mixture of the analyte present in the human fluids, they often may represent only the average condition found in blood serum. For these compounds, reference measurement procedures independent of routinely employed analytical principles are currently lacking in the majority of cases. The disagreement between the results obtained by different commercial immunoassays is frequently due to the preparation of calibrators by the manufacturer of assay reagents on a mass basis not being available to other immunoassay producers.

2.6 The uncertainty of measurement Uncertainty, according to the ISO Guide to the Expression of Uncertainty in Measurement is “a parameter associated with the result of a measurement that characterizes the dispersion of the values that could reasonably be attributed to the measurand” (8), where measurand is the quantity intended to be measured. In practical terms, total uncertainty arises from assignment of a numerical value to the measurand present in the calibrator material used in the routine method and from errors normally occurring during the analytical procedure (9). As was mentioned before, for many analytes measured

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Fig. 2.1: Hierarchical link between definitive, reference, and routine methods as well as between reference materials and calibrators. Uncertainty of measurement is increasing from definitive method to routine method. Traceability assessed by accuracy and trueness is depicted. Requirements of commutability between secondary reference material, calibrators, quality control samples, and the patient’s samples are indicated.

by immunoassays, there are no reference materials or reference methods; therefore the assignment of value to the calibrator material poses a high level of uncertainty. The measurement of each analyte is influenced by both systematic error, defined as bias, and random error, defined as coefficient of variation (CV ); thus uncertainty of measurement in immunoassay may be high. When all of the components of the total error can be appropriately corrected, there still remains an uncertainty of the measurement of a given quantity. For each measurement, it is possible to define the interval within which the laboratory result can be found with certain probability. It is obvious that uncertainty of final laboratory results depends on uncertainty of analyte measurement in each step of the traceability chain. The lowest uncertainty of measurement is always attributed to definitive methods, and the highest is characteristic for routine methods. This is especially true for the immunoassay measurement system. It should be pointed out that the term error is not equivalent to the term uncertainty. Medical laboratory analysts are very familiar with the terms random error, systematic error, and total error,

2.7 Epitope as a solution for better standardization

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but the uncertainty that arises from random effects and from imperfect correction of systematic errors is less known. In the case of immunochemistry, incomplete definition of the measurand for many analytes being measured, lack of comparability of standard and sample matrices, and lack of knowledge of how many unknown environmental factors affect each step of analytical procedure are the main reasons for uncertainty. The concept of uncertainty is quite new for medical laboratories, and much discussion on how uncertainty should be measured (if it is really necessary) by laboratory and how (or whether at all) uncertainty should be reported to the clinicians has been published recently, but conflicting opinions can still be found (10,11).

2.7 Epitope as a solution for better standardization Difficulty in establishing traceability for standardization of heterogeneous proteins brought an impulse for looking for new solutions in the field of immunoassay standardization and for new definition of the analyte intended to be measured. From an immunochemistry point of view, the characteristics of the unique analyte structure recognized by the reagent capture antibody are most important, not the whole molecule. Immunoassay is based on the specific reaction between a unique epitope on the analyte and capture antibody. This means that what is really being measured is the structure of protein, which is complementary to the antigen-combing site on the antibody molecule. In the mixture of different forms of protein present in the sample, it is important to find out a unique invariant part of the molecule that can be present in each form, or is present only in one form. If the unique structure is present in all forms of heterogeneous protein, then the total concentration of protein will be measured. However, when measuring the sum of the concentrations of different protein isoforms, there is no information on the individual forms, which may change in the disease process in opposite directions, bringing no change in total protein level. In other words, the information on the relative amount of different protein forms might be lost. If the unique structure can be attributed only to one form, then the standardization of a single protein isoform may be performed. However, to implement such an approach, it is necessary to know exactly the structure and physicochemical properties of the protein form as well as the unique structure of a molecule recognized by antibody. Such an approach has been exploited in the case of glycated hemoglobin form HbA1c (12). It is known that the amino-terminal group of hemoglobin forms a Schiff base with the aldehyde group of glucose, and a spontaneous irreversible Amadori rearrangement produces a ketone derivative. Hydrolysis of both hemoglobin HbA1c and hemoglobin HbA1 by trypsin produces an amino-terminal hexapeptide of a known sequence; however, tryptic digest products of HbA1c differ from those obtained from HbA1 because amino-terminal hexapeptide of HbA1c has an additional 1-deoxyfructose attached to the amino-terminal group. Such a protein fragment is suitable as the primary reference material. Secondary reference measurement procedures have been designed by utilizing tryptic digestion of whole blood followed by quantification by HPLC with mass spectrometry (MS). In immunoassay for HbA1c, the assay reagent antibody is specific for glycated hexapeptide. Having reference method (HPLC/MS) and reference material (glycated hexapeptide of HbA1c ), standardization of immunoassay for HbA1c can be performed, and traceability of the reference system is possible. Using such an approach for standardization of other high-molecular-weight proteins measured by immunoassay requires, however, the knowledge of a unique

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structure of protein fragment, its physical and chemical characteristics, as well as its chemical structure, against which assay reagent antibody is directed. Such an approach may not be possible for all heterogeneous proteins, but even if it would be possible to perform epitope-based standardization for every glycoprotein present in human blood, still no information is available on the most clinically useful protein forms.

2.8 Harmonization of immunoassays If standardization of the analyte measured by immunoassay is not possible, as in the case of heterogeneous proteins, harmonization can be used to improve comparability of methods. It has to be remembered that harmonization of methods is not equivalent to method standardization. The purpose of the harmonization process is to obtain similar results for the analyte measured by different, “harmonized” immunoassays calibrated with the use of the same calibrators. Harmonization of the sample results does not always require a standardized reference method. Two different things must be pointed out. If calibrators used in immunoassays designed for the measurement of a particular analyte have the concentrations assigned on the basis of reference material with an attributed value, not necessarily a “true value”, and in all assays the reaction conditions and the specificity of assay reagent antibodies will be the same, then the results will be harmonized even if traceability is not kept. On the other hand, two well-standardized immunoassays may not be harmonized if there is a difference in the immunoreactivity of the epitope toward the antibody used in the assays, or if different antibodies against the same epitope are used in two assays. In both cases, there will be lack of harmonization. Usually, the results obtained for calibrators in different immunoassays are very well harmonized, but no harmonization can be achieved for the patient’s sample when there are commutability problems (13). This is because the same protein can behave differently in the calibrator and in the patient’s sample with respect to binding to antibody, especially when a different spectrum of heterogeneous proteins is present in the biological sample. Comparability of the immunoassay results may be achieved only if a well-standardized and well-harmonized immunoassay is used, with all problems with commutability solved. At present, many problems connected with improving the intermethod comparability at every step await solution.

2.9 Immunoassay standardization and the patient’s safety The main goal of standardization and harmonization of immunoassays is to obtain results that are comparable not only between two immunoassays (or immunoassay platforms) used in the same laboratory but also worldwide, regardless of the immunoassay or immunochemistry platform used. This would ensure the interchangeability of results over time and space, not only for a single individual but also for clinical and research studies performed in different countries and for different populations, markedly improving patient care everywhere (14). Standardization is not an isolated problem for a laboratory. Rather, it is an international problem to be solved by scientists, laboratory professionals, and assay manufacturers. Much work should be done to improve the comparability and transferability of patients’ results between assays. The benefits of this hard work will be patients’ safety and a decreased cost of health care.

References

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References 1. Jeffcoate SL. Role of reference materials in immunoassay standardization. Scand J Clin Lab Invest Suppl 1991;205: 131–3. 2. Whicher JT. Secondary reference materials. Clin Biochem 1998;31: 441–6. 3. International Organization for Standardization (ISO). In Vitro Diagnostic Medical Devices – Measurement of Quantities in Biological Samples – Metrological Traceability of Values Assigned to Calibrators and Control Materials. ISO 17511:2003. Geneva: ISO; 2003. 4. Stöckl D, D’Hondt H, Thienpont LM. Method validation across the disciplines – Critical investigation of major validation criteria and associated experimental protocols. J Chromatography B 2009;877: 2180–90. 5. International Organization for Standardization (ISO). International Vocabulary of Basic and General Terms in Metrology (VIM). ISO/IEC Guide 99:2007. Geneva: ISO; 2007. 6. Vesper HW, Thienpont LM. Traceability in laboratory medicine. Clin Chem 2009;55: 1067–75. 7. Vesper HW, Miller WG, Myers GL. Reference materials and commutability. Clin Biochem Rev 2007;28: 139–47. 8. International Organization for Standardization (ISO). Guide to the Expression of Uncertainty Measurement. Geneva: ISO; 1993. 9. White GH, Farrance I, on behalf of the AACB Uncertainty of Measurement Working Group. Uncertainty of measurement in quantitative medical testing – A laboratory implementation guide. Clin Biochem Rev 2004;25: S1–24. 10. Plebani M. Evaluating laboratory diagnostic tests and translational research. Clin Chem Lab Med 2010;48: 983–8. 11. Westgard JO. Managing quality vs measuring uncertainty in the medical laboratory. Clin Chem Lab Med 2010;48: 31–40. 12. Goodall I. HbA1c standardization destination – Global IFCC standardization. How, why, where, and when – A tortuous pathway from kit manufacturers, via interlaboratory lyophilized and whole blood comparisons to designated national comparison schemes. Clin Biochem Rev 2005;26: 5–19. 13. Miller WG, Myers GL, Rej R. Why commutability matters. Clin Chem 2006;52: 553–4. 14. Müller MM. Implementation of reference systems in laboratory medicine. Clin Chem 2000;46: 1907–9.

3 Immunoassay calibration and calibration curve fitting

3.1 Calibration process For every routinely performed immunoassay, the relationship between the measured signal (radioactivity, enzyme activity, chemiluminescence, fluorescence, etc., indicating the course of reaction between antigen and antibody) and the concentration of the measured analyte must be established (calibration curve), the process known as assay calibration. Each immunoassay requires a set of calibrators, usually five to eight, with the properly assigned values based on appropriate reference material. Usually, calibrators contain the pure analyte in a solution (ready-to-use), or they are in the form of lyophilized material, which must be dissolved in assay diluent according to the instruction manual. The calibration curve is plotted, now usually by computer, and the concentration of the unknown sample is determined by extrapolation. Calibration curves for analytes measured by immunoassays, especially by competitive methods, differ from the shape of calibration curves obtained for other analytical methods. Immunochemistry calibration curves are rarely linear, and mathematical modeling must be used to calculate the analyte concentration in unknown samples. If immunoassay measurement is performed on automatic immunochemistry platforms, for every relationship between the signal and analyte concentration assigned for a calibrator, the mathematical function describing the calibration curve is calculated by built-in computer, and concentration of the analyte in an unknown sample is directly calculated from the signal and reported in appropriate units. Also, gamma scintillation counters and bench-top plate readers usually have such a built-in computer calculation program. It is necessary that all the curve-fitting mathematical programs match the calibration curve as closely as possible across the whole concentration range. Depending on the assay and/or immunochemistry platform, linear interpolation, spline line fits, polynomial regression, logit-log transformation, or four- or five-parameter log-logistic methods are used (1). Laboratory staff members are not always familiar with mathematics used in a given immunoassay or on immunochemistry platform. So sometimes wrong types of mathematical method are used giving erroneous results especially near both ends of the curve. This usually happens if only one mathematical transformation is applied for all tests performed with the use of a given equipment. Good laboratory practice requires not only a critical look at the computer calculations, but also recording the back-calculated values of the calibrator signal level as well as the original course of the calibration curve, especially for competitive methods. Some immunoassay platforms using immunometric (noncompetitive) assays with linear relationship between the measured signal and analyte concentration require only one or two calibrators to be run for a series of unknown samples. This analytical practice is allowable, because stable, fully automated analyzers have a stored calibration curve (master curve) established by the manufacturer, and such a curve requires only periodical checking. Typical stored immunoassay curve is stable on average for two weeks, and random access analyses can be easily performed. However, one should be aware of possible errors in assay calibration using only one or two calibrators, especially at

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the end of the expiration date for the master curve established by the manufacturer. For many immunoassay tests performed on automatic platforms (e.g., for drugs abuse or for infectious diseases), it is enough to include with the unknown sample only a single calibrator or control to determine a cutoff level between negative and positive patient results. Usually, positive and negative controls are also run as unknowns to check the assay calibration.

3.2 Commutability problem All calibrators used in routine immunoassays for all the measured parameters that are clinically important must behave in a similar way to “all unknown samples” in different assays, and “all unknown samples” must behave consistently, though differently, in the methods. The concept of similar analyte behavior and consistent relationship between the methods is expressed by the term commutability (2). Commutability represents the equivalence of the mathematical relationship between the results of different measurement procedures for a reference material and for representative samples from healthy and diseased individuals (3). Practically, if the reference material will be measured by both the reference method and routine method, the numerical ratio of the obtained results must be the same as the ratio of results obtained by both methods for the patient sample (3,4,5). It is not possible to eliminate the noncommutability problem completely, because biological samples may vary significantly not only between different patients but also in the same patient in relation to clinical condition. Similar behavior of the analyte should be not only in calibrators and patient samples but also in samples used for quality control assurance. It is well known for all laboratory professionals that if control samples measured by different laboratories are noncommutable, then the quality control results must be analyzed for peer groups of methods; otherwise, the control results might be totally incomparable. Thus, a significant difference in method-specific target values for the control sample measured by different immunoassays cannot be attributed only to differences in assay format or characteristics of reagent antibodies employed in the assay, but the commutability problem should always be considered. Again, immunoassay users usually observe very good agreement between the nominal and the measured values when the manufacturer’s control samples are assayed, but for serum-based controls, such agreement is not always obtained. Therefore, the assessment of immunoassay calibration cannot be performed based on only a few quality control samples (6). Thus, it is very important to be aware that the proper calibration process with perfect relationship between assay signal and analyte concentration, and agreement between nominal and measured values of the manufacturer’s assay control samples do not equal good performance of the assay if patients’ samples are measured.

3.3 Matrix effect The main cause of differences between the reference material and the native biological samples is the effect of matrix. The matrix effect is defined as the influence of a property of the sample, independent of the presence of the analyte, on the measurement and thereby on the value of the measurable quantity (7). The sample matrix includes all components of a material system except the analyte itself (7). The matrix

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of the calibrators differs significantly from the matrix of biological specimens, most often serum or plasma. Immunoassay calibrators are frequently prepared in aqueous or organic matrix that is not commutable with that of biological samples. Such calibrators should not be used. It is well known to everyone using immunoassay techniques that once the method calibration is performed, there are usually no objections to the assay performance, but when the native biological samples are assayed, the method performance is questioned. The most desirable materials for immunoassay calibration are the matrix-based materials, but this is not always achievable, especially for synthetic proteins, which may differ from native protein in folding, or when the analyte in the patient’s blood sample taken under serious clinical condition is intended to be measured. To avoid lack of commutability and assure the unbroken traceability chain of the measured value, the preparation and use of secondary reference materials is recommended (5). When transferring the values from one reference material to another, it is also essential to check for commutability. Measurement calibration procedure with reference materials that are not commutable is one of the most important causes of poor comparability of results between immunoassays. It is important to distinguish the bias coming from real differences between the concentrations of the analyte measured by different immunoassays from the bias resulting from lack of commutability. Matrix effect should be taken into account in each step of the standardization/calibration process, starting from definitive methods with primary standard via reference method with reference material and ending on calibrators used to calibrate the routine assay and to assign the value to the measurement in the patient’s sample. Because each step in standardization/calibration may be influenced by matrix effect, consistency from assay to assay in respect to matrix for calibrators should be maintained. If the analyte behaves similarly in reference material, calibrators, and patient samples, they are commutable, and the traceability of the patient results can be fulfilled. It should be remembered, however, that in every immunoassay the antigen-antibody reaction that occurs during the measurement in the patient’s sample is influenced by many factors, like pH, ionic strength, presence of various proteins (specific binding proteins, immunoglobulins, autoantibodies), and different level of hydrophobic molecules; thus the final result will always depend on interference, the sources of which are unique for the patient’s sample.

3.4 Calibration and patient safety Proper calibration of routinely performed immunoassay directly influences the final measurement result in the patient’s sample and thus, if not performed correctly, may affect patient’s safety. Although systematic error due to improper calibration is easy to correct on the basis of a quality control assurance program, the problem concerns mainly traceability and commutability. The laboratory should know the properties of calibrators, their matrix, stability, and final concentration. However, it is the manufacturer’s responsibility to provide a document on the commutability of the working calibrators. Noncommutable, impure, and inconsistent behavior of the immunochemical reaction of the analyte calibrator samples contributes strongly to between-assay variation and thus strongly affect the results obtained in the patient’s sample if the measurement of analyte concentration is repeated over some period of time or the analyte is retested by different immunochemistry method.

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References 1. Nix B, Wild D. Calibration and curve-fitting. In: Wild D, ed. The Immunoassay Handbook. New York: Nature Publishing Group; 2001: 198–210. 2. Moss DW, Whitcher JT. Reference material and reference measurement system in laboratory medicine. Commutability and the problem of method-dependent results. Eur J Clin Chem Clin Biochem 1995;33: 1003–7. 3. International Organization for Standardization (ISO). In Vitro Diagnostic Systems – Measurement of Quantities in Samples of Biological Origin – Description of Reference Materials. ISO 15194:2002. Geneva, Switzerland: ISO, 2002. 4. Vesper HW, Miller WG, Myers GL. Reference materials and commutability. Clin Biochem Rev 2007;28: 139–47. 5. Miller WG, Myers GL, Rej R. Why commutability matters. Clin Chem 2006;52: 553–4. 6. Panteghini M. Traceability, reference systems and result comparability. Clin Biochem Rev 2007;28: 97–104. 7. Stenman UH. Immunoassay standardization: it is possible, who is responsible, who is capable. Clin Chem 2001;47: 815–20.

4 Reference intervals and immunoassay

4.1 General problems with reference intervals for analytes measured by immunoassay The results of laboratory tests are only numerical values without real meaning unless referred to the proper reference interval system. Thus, one may compare several results in the same patient, the results in healthy and diseased subjects, the results in women and in men, those obtained in children and in adults, and so forth. Such comparisons serve only to satisfy curiosity about knowing the difference. For medical purposes, however, it is not enough to know that there is difference; it is also important to know the true values of quantities being compared. Laboratory tests are the indispensable part of patient care, and they are necessary for diagnosis, monitoring treatment, prognosis, and prevention of the disease. Based on the laboratory results, one can make a judgment about the presence or absence of the disease and its severity if adequate reference intervals are available. As was pointed out in chapter 2, the proper standardization of the immunoassay measurement system is necessary to obtain true and reliable results. If a well-standardized and harmonized immunoassay with antibodies of the same specificity will be used for a particular analyte by all laboratories, then the traceable measurement values would allow for worldwide reference intervals to be universally introduced. At present, however, for many heterogeneous analytes the definitions of the molecules intended to be measured by reference procedure are lacking, most of the immunoassay measurements do not have proper standardization, no traceability is followed, and as a consequence, method-specific reference intervals are used for many analytes measured by immunochemistry. Such an approach generates many problems concerning the comparability of reference intervals between laboratories and is one of the main misunderstandings between laboratory professionals and physicians or sometimes also patients, who cannot understand why the analyte concentration result measured by one immunoassay indicates disease but when measured by another immunoassay is normal. Although physicians and patients usually have confidence that the results of laboratory test are reliable, any discrepancy between the immunoassay results is the main reason for requesting repeats of the analyte measurements not only in the same laboratory but frequently in some other laboratory. Endless lack of comparability of test results between different immunoassays used for the measurement of the same analyte causes confusion among physicians and patients due to imperfections in the methodology and is the source of unnecessary frustration among laboratory professionals who are placed in a very uncomfortable position. Also, unreasonable, multiple analyte measurements grossly increase the cost of health care without improving patient care. For many common clinical chemistry analytes measured in biological specimens (e.g., potassium ion), there are well-accepted reference intervals, but for many others, the proper reference intervals or clinical decision cutoff points are lacking. This is true especially for heterogeneous analytes measured by immunoassays. Correctness of establishing reference intervals for every clinically important molecule measured by different

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chemical methods depends strongly on preanalytical procedures, intraindividual and interindividual variability of the analyte, measurement systems, assay standardization, and overall assay quality. In the case of immunochemistry, heterophilic antibody interference, matrix effect, unsatisfactory sensitivity and specificity of some assays, as well as lack of standardization, increase the problems with proper defining of reference intervals. Thus, all these factors may not only directly influence the measurement of analyte in the patient and in the individuals selected from the population as a reference group, but also indirectly influence patient safety, because good reference intervals are necessary for proper interpretation of test results and proper clinical decisions.

4.2 Limitation of different approaches to reference intervals Considering what is normal and what is abnormal, it has to be remembered that the reference intervals can be population based (most analytes), subject based (individual), outcome based (e.g., for HbA1c), based on clinical or medical decisions (e.g., prostate specific antigen, 17-hydroxyprogesterone in newborns), or based on healthy subjects and method reproducibility (e.g., troponin). Population-based reference intervals rely upon a strictly defined selection of reference individuals from the reference population. Reference values are measured in the selected sample group from which the reference distribution is obtained, and reference limits including inner 95% of the measured values are calculated. The reference interval is defined by the reference limits and includes them (1,2). When using population-based reference intervals, laboratories should be conscious that 5% of healthy subjects will remain outside the interval and that some individuals with subclinical disease may be included in reference group as “normals”. Besides, at the moment the individuals are selected for the reference group, many common diseases may be undetected in apparently healthy persons (e.g., alcohol or drug abuse, hepatitis C). Usefulness of population-based reference intervals for analytes measured by immunochemistry depends on the particular analyte. Subject-based reference intervals depend on the index of individuality, defined as the ratio of within-subject variation to between-subjects variation. The index of individuality is usually very low for many molecules measured by immunochemistry, and sometimes the calculation of this index is impossible because for many analytes intraindividual or interindividual variability is not exactly known. However, for some analytes, like thyroid-stimulating hormone, population-based reference intervals are not always useful for the interpretation of results obtained for the individual patient, and rather subject-based reference intervals should be more appropriate. In many laboratories, reference intervals for analytes measured by immunochemistry are usually adapted from the manufacturer’s recommendation or taken from the literature without any reservation, the practice that should be discouraged. The method for establishing reference intervals as given in the manufacturer’s insert is usually not specified. Also, the number of subjects selected for establishing reference intervals does not follow the International Federation of Clinical Chemistry recommendation and usually is too low; frequently, sex and age are not specified, especially for reference intervals of new biochemical markers. For some analytes measured on immunochemistry platforms or with well-specified, frequently used immunoassays, common reference intervals based on multicenter collaborative experiments are published in the medical literature.

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Such reference intervals should not, however, be accepted by laboratories without reservation. Laboratories have to document that the population they serve does not differ and has the same characteristics as the population used for establishing the reference intervals. Also, all preanalytical and analytical procedures should be similar (3,4). It is difficult to meet such requirements by many laboratories, especially those providing service for small communities, because the procedure of establishing or transferring the reference intervals requires both time and money. Even more serious problems with reference intervals may be encountered by laboratories performing immunoassay determinations, especially hormones, for pediatric populations.

4.3 Reference intervals and patient safety Lack of good reference intervals for analytes measured by immunoassay, together with lack of standardization, harmonization, and comparability between the assays both performed manually and using immunochemistry platforms, significantly affects the patient’s safety. This is a really important issue, inasmuch as reference intervals for laboratory tests are one of the most frequently used tools in the clinical decision-making process. But even if good common reference intervals would be achievable for all routine immunoassays, still there is a long way to go to establish specific disease-based reference intervals. From a practical point of view, it is important to realize the purpose of the measurement of a given analyte in the patient: is it for initial diagnosis, monitoring disease, screening, or prognosis of disease outcome? If a tumor marker is being measured and high analyte concentration is expected, then small differences in reference range limits between the assays have practically no meaning. However, sometimes the relapse of the disease is difficult to notice, especially if there are no clinical symptoms and the relapse is accompanied only by a small increase in the concentration of the biochemical marker above the reference interval limit. If the patient is already sick, clinical symptoms are obvious, and the analyte concentration is measured only for confirmation of diagnosis. In such a case, the discussion about the reference interval limit is less important. Good laboratory reference intervals become significant when subjective, biological or very early clinical signs of the disease are observed in the patient, because frequently the concentration of the analyte fluctuates about the clinical decision limit. In many cases, the course of the disease can be described as a continuum of discrete changes in biochemical and physiological processes with gradual increase or decrease in the concentration of a disease marker. Taking into account biological variation of analytes measured by immunoassays, poor intra- and interassay precision, and lack of good reference intervals, it is almost impossible to identify the patient results with a subclinical form of disease before the appearance of clinical symptoms. It is known that it is much better to prevent than to treat disease. If good reference intervals could be established and used for preventing diseases, then the patient would be the most important beneficiary.

References 1. Solberg HE. International Federation of Clinical Chemistry. Scientific committee, Clinical Section. Expert Panel on Theory of Reference Values and International Committee for Standardization in Haematology Standing Committee on Reference Values. Approved

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recommendation (1986) on the theory of reference values. Part 1. The concept of reference values. Clin Chim Acta 1987;165: 111–8. 2. Ceriotti F, Hinzmann R, Panteghini M. Reference intervals: the way forward. Ann Clin Biochem 2009;46: 8–17. 3. Clinical and Laboratory Standard Institute (CLSI). How to Define and Determine Reference Intervals in the Clinical Laboratory; Approved Guideline. 2nd ed. CLSI document C28-A2. Wayne, PA: CLSI; 2000. 4. Solberg HE, PetitClerc C. International Federation of Clinical Chemistry (IFCC). Scientific Committee, Clinical Section. Expert Panel on Theory of Reference Value (EPTRV). Approved recommendation (1988) on the theory of reference values. Part 3. Preparation of individuals and collection of specimen for the production of reference values. Clin Chim Acta 1988;177: S3–11.

5 Laboratory preanalytical and analytical phase of immunoassay

The analytical process is composed of three phases: preanalytical (prelaboratory preanalytical and laboratory preanalytical), analytical, and postanalytical. Each of these phases contributes differently to the total error of the analyte measurements, influencing the interpretation of the results of laboratory tests and in consequence the safety of the patient. The analytical phase used to have the biggest impact on the final results. With the introduction of the fully automated analytical process, the contribution of the analytical phase to the total error of any automatized method, including immunoassays performed on immunochemistry platforms, became much smaller. If the analytical procedure is performed automatically, errors occurring during the preanalytical phase become of major importance for the total error estimation, because there is a general lack of standardized prelaboratory and laboratory preanalytical procedures. The percentage of preanalytical errors in the total error of the determinations is similar irrespective of the analytical method if performed on analytical platforms, including immunoassay platforms. Thus, the same aspects of preanalytical procedures must be taken into consideration while interpreting patient results obtained by immunoassay method. Besides, not everyone understands which steps of the preanalytical process influence immunochemical determinations and how this phase of the analytical process affects patient safety. The knowledge of this matter among physicians is rather vague, and only the elements concerning any analytical determination, like fasting or hemolysis, are usually taken into account. In other words, it is usually known which preanalytical factor may influence the result of immunochemical measurement, but the exact mechanism of interference and how the final results will be changed – whether they will be false positive or false negative, or when the result will be clinically useless – is not always well understood .

5.1 Laboratory preanalytical factors and immunoassay The details of the effects of prelaboratory preanalytical factors on immunochemical determinations, such as sex and age of the patient, stress, diet, body mass, time of blood sampling, drug therapy, body position during the blood drawing, conditions of sampling, and transport of samples to laboratory, are beyond the scope of this book. However, some examples, especially those concerning hormonal determinations, should be mentioned here. The concentration of many analytes measured by immunochemistry, including hormones, are age and sex dependent. Most problems pertain to the interpretation of the concentrations of hormones in newborns, infants, and children. In newborns, the physiological and biochemical changes connected with the adaptation to extrauterine life, the presence of maternal hormones in newborn blood, and immaturity of many enzymatic systems mean that for many hormones the reference intervals are lacking. In later periods of life, the problems with the interpretation pertain to the period of sexual maturation and intensive growth (especially for growth hormone, IGF-I, LH, FSH, estrogens, and testosterone). Another difficulty is connected with hormonal

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measurements and their interpretation in elderly patients, when hormonal changes are connected with the period of menopause or andropause. With aging, many interfering substances, including autoantibodies, may be present more frequently in the patient’s sample. The effect of stress should be taken into account when interpreting the results of the determination of such hormones as growth hormone, prolactin, and cortisol. Longlasting stress may be the cause of changes in the concentration of hormone-binding proteins, reflected in the changes of the concentrations of these hormones for which the measurement of free fraction concentration is important. Diet has a considerable effect on the concentration of these hormones, the level of which depends on the composition of meals, frequency of eating, and time of eating. Though it is known in what direction and when the changes in the concentrations of hormones take place after a meal, it is always necessary to consider the effect of pharmaceuticals (e.g., those delaying emptying of the stomach) received by the patient. On the other hand, the ingested meal may delay the intestinal absorption of drugs causing a decrease in blood concentration. As an example of such effects, caffeine causes an increase in the concentration of cortisol even by 50% after drinking coffee or ingestion of a meal containing this substance. Body mass index (BMI) is an important factor affecting the interpretation of the results of determination of many hormones. Although the information about the concentration of many hormones in individuals with a high BMI can be found in the literature, no separate reference intervals for overweight and obese individuals are used. Besides, there are many substances present in the sample that are characteristic for obese patients (like high levels of lipids) that may influence the immunoassay measurement. The time of the day the blood is drawn for the measurement of analytes is very important, as many hormones display diurnal variation (cortisol, ACTH, growth hormone). Also, pulsatile secretion of some hormones with the extremes ranging up to 25% is common. The exact day of the menstrual cycle, as well as taking birth control pills or hormonal replacement therapy, are important for sex hormone measurement. Also, estrogen-induced increase in binding protein concentration (e.g., thyroxin-binding globulin) may influence the total and free thyroxin measurement. Among the most important laboratory preanalytical factors that may influence the final result of immunochemical measurement, attention should be paid to the type of blood collection devices and anticoagulants used. Other important factors are interference from hemolysis, lipemia, hyperbilirubinemia, and paraproteinemia. Stability of molecules in freshly drawn blood as compared to fresh and frozen serum or plasma, as well as the effect of the freeze-thaw cycle are also very important laboratory preanalytical factors. These factors may affect not only the analytes intended to be measured by immunochemistry methods (e.g., degradation of analyte may occur), but also substances present in the sample that may be, for example, degraded since degradation products might potentially interfere in the immunoassay measurement after proteolytic degradation.

5.2 Blood collection devices The additives used in blood collection devices may influence the analyte concentration. To avoid nonspecific adsorption of protein, red blood cells, and platelets to the tube walls, different surfactants are used (1,2,3). Most frequently, silicone surfactants used interfere with avidin-biotin binding in immunoassay by masking the reagent antibody

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(4,5). Organosilane surfactant (SilvetTM L-720) in Becton Dickinson SSTTM blood collection tubes caused false elevation of triiodothyronine (T3) assay in a dose-dependent manner in ImmuliteTM 2000/2005 system (1,2). The mechanism of SilvetTM L-720 on other immunoassay measurements, like B12 and cancer antigen 15–3, performed on the Advia CentaurTM is not fully understood. There are also reports on the influence of silicone from blood collection tubes on C-reactive protein and hepatitis B surface antigen (6,7). Drawing blood to the tubes containing the gel separating plasma from blood cells was often the cause of interference affecting the determination of proteins and peptides with the use of some immunochemical analyzers (5,8,9). The tubes with separating gels caused interference through, among others, influence on the chemiluminescence signal (10). Also, other chemicals added to the sampling tubes in order to facilitate the flow of blood or serum down the tube wall may be a potential source of preanalytical error in immunoassays.

5.3 Anticoagulants The determination of various analytes by using immunochemical methods are usually performed in blood serum or plasma and also in urine. If immunoassay is designed to measure the analyte in blood plasma, use of EDTA or heparin as anticoagulants is usually recommended. It has to be remembered that the proper ratio of blood volume to the amount of anticoagulant is critical for the measurement of some analytes. An excess of EDTA concentration in relation to blood volume may chelate, in addition to calcium, also magnesium and zinc, which in turn alters the activity of alkaline phosphatase, the enzyme used in chemiluminescence assay (11,12). If heparin is used as anticoagulant, interference with the antigen-antibody reaction may occur, as is the case of troponin measurement, and both falsely low or falsely high results, depending on the assay, may be obtained (8). Although the concentration of some proteins may differ in serum and plasma, the difference being sometimes as high as a few percent, it is usually assumed for the purpose of medical interpretation of the results of immunochemical determinations that the difference does not exist. In the determination of most proteins and peptides, differences between serum and plasma arise mainly from the matrix effect.

5.4 Hemolysis, lipemia, hyperbilirubinemia, paraproteinemia, and immunoassay Typical analytical interference affecting the measurement of many analytes performed in everyday routine work in clinical laboratories like those caused by hemolysis, lipemia, bilirubinemia, and paraproteinemia are usually known by laboratory workers. The measurement of a simple analyte like potassium ion in a hemolyzed sample gives a falsely elevated value, and the only conclusion that can be drawn is that potassium concentration is not higher than the measured value. For analytes measured by immunochemistry, no such simple conclusion is possible. In some assays (enzyme immunoassays, ELISA), the assay signal is measured by spectrophotometry. If the measurement is done at 415, 540, or 570 nm wavelength of light, where hemoglobin shows absorbance peaks, then the spectrophotometric reading may be erroneous. Consequently, a false assay signal is read, causing the inaccuracy of the patient result. Also, the concentration of some analytes (e.g., folates) is much higher in erythrocytes than in serum, and hemolyzed

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samples are useless for the measurement of such molecules. Hemoglobin present in the patient sample due to hemolysis is not the only molecule that can be released from broken erythrocytes. Other substances of cellular origin, not usually present in serum or plasma, may also interfere in the measurement of different analytes by immunoassay. Besides, proteolytic enzymes released from the cells broken during hemolysis may cause additional degradation of proteins and peptides. If proteolysis concerns the epitope of the analyte that is intended to be measured, then a falsely low result may be obtained (13). Proteolysis can also concern specific hormone-binding proteins causing a change in the equilibrium between bound and free hormone and affecting the free hormone fraction measurement. As to whether it is possible to measure the analyte in the hemolyzed sample by immunoassay, there will never be a simple answer for all immunoassay measurements. According to the literature, for some analytes hemolysis does not affect the measurement, but generally it is suggested not to use hemolyzed samples for immunochemical determinations. Interference in immunoassay measurement systems caused by lipemia or hyperbilirubinemia is not frequently reported in the literature. For double-antibody competitive methods where the second antibody with PEG is used for the separation of bound antigen fraction from free (unbound) antigen, lipemia may cause interference by disturbing the precipitation of the antigen-antibody complex leading to falsely elevated results. High serum bilirubin concentration (hyperbilirubinemia) can cause spectral interference in immunoassays with spectrophotometric signal measurement (immunoenzymatic assays), when absorbance is measured at a wavelength near to the bilirubin absorbance peak of about 456 nm. Paraproteins are monoclonal immunoglobulins produced by a single clone of plasma cells. They might be present in blood as monomers, polymers, or protein molecule fragments, usually light chains (Bence Jones proteins) or, rarely, heavy chains or half immunoglobulin molecules. Paraproteins can interfere with all types of manual and automated immunoassays, which are based on spectrophotometric, immuno-nephelometric, and turbidimetric signal measurements. In the presence of paraproteinemia, interference in the assay’s reaction of the antigen with antibody may occur. One of the examples is a negative interference in thyroid-stimulating hormone (TSH) assay in the presence of IgGK paraprotein (14). Taking into account that the prevalence of paraproteinemia in serum samples increases with the patient’s age, it is always worth considering the presence of interference from paraproteins if there is no agreement between the laboratory result and the clinical picture of the patient, especially in geriatric patients with monoclonal gammapathy of unknown significance (MGUS). It may also be worth considering the opposite situation: if the results of analyte (e.g., TSH) measurement do not correlate with the disease in the geriatric patient and interference in the reaction between the antigen and assay antibody is suspected, the presence of MGUS might be considered.

5.5 Analyte stability in fresh and frozen serum samples When proteins and peptides are determined by using immunochemical methods, a few fundamental questions must be asked: (a) What is the stability of a substance measured in biological material? (b) What are the conditions that samples should be kept under after blood is taken? (c) At what temperature should samples be frozen and stored?

5.5 Analyte stability in fresh and frozen serum samples

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(d) And most importantly, can the samples containing the analyte for immunochemical measurement be frozen and thawed without loss of stability of the analyte? It is very difficult to give clear-cut answers to all of these simple questions in the case of many molecules measured by immunochemistry, especially for proteins and peptides present in samples in various molecular forms. The heterogeneity of many proteins in biological samples is not only the inherent property of proteins but also may be a consequence of the disease process, inasmuch as the clinical state of the patient influences the molar ratio of different molecular forms. Different stability of the molecular forms of proteins in vitro may be the cause of a distinct course of the immunochemical reaction or unusual cross-reactivity leading to the changes in the measured final concentration of the analyte. Also, the proportion of different protein forms in a fresh sample may differ from that in a sample that was stored frozen. Not all proteins measured by immunoassay technique are unstable; there are some, like IGF-I, that show good stability at room temperature or resistance to degradation while stored at – 20°C and after several freeze/ thaw cycles (15). The laboratory procedure followed from the moment the sample arrives at laboratory to the start of the measurement procedure, manually or on the immunochemistry platform, varies between laboratories. Also, no standardized procedure exists for proceeding with the sample within the laboratory. For unstable proteins and peptides, blood is drawn usually into the EDTA tube, centrifuged (refrigerated centrifuge is recommended) as soon as possible, and frozen immediately. If a very unstable peptide or protein is going to be measured, then frequently blood must be drawn to the EDTA tubes with aprotinin or other protease inhibitors. Sometimes, aprotinin is added to the plasma after centrifugation of the blood, which is not recommended because the stability of the analyte during blood processing can also be affected. Addition of protease inhibitors is recommended for many peptides, like brain natriuretic peptide or gastrointestinal regulatory peptides (secretin, glucagon-like peptides) (16,17). If samples for unstable protein measurement cannot be assayed immediately, they must be frozen as soon as possible. The shorter the time between taking the blood sample and freezing the plasma, and the lower the temperature during the sample processing, the lower is the probability of peptide degradation to occur. It should, however, be remembered that protein degradation takes place also in frozen samples, regardless the storage temperature, and that some peptides have tendency to adsorb to the tube walls. Although nowadays many immunoassay analyses are processed at once on automatic immunochemistry platforms, in small laboratories samples are assayed after a certain number of samples are collected. In laboratories performing analyses for scientific purposes, considerable variation in length of time that samples are kept frozen is observed. As a general principle, freezing/ thawing of the patient’s serum or plasma sample only once is strongly recommended, but different procedures for thawing the sample are applied and big differences can be observed: from very fast warming of the frozen sample to a much more proper, stepwise procedure: from – 70°C through – 20°C to 4°C and finally to room temperature. Such stepwise procedure helps to protect many analytes from degradation. One of the basic principles of the laboratory preanalytical phase is centrifugation of blood taken from the patient within 60 minutes. Blood taken on anticoagulant should be centrifuged as quickly as possible. In everyday routine, practitioners (nurses, physicians, laboratory technicians) are sometimes not aware of importance of this preanalytical step. Frequently, laboratory technicians do not know how long and at

40


what conditions a sample was kept before it was delivered to the laboratory. Quite often, laboratories are asked to do an additional test several days after blood sampling without following the principles recommended for the test. Another problem appears when, after the requested analysis has been performed, some amount of plasma sample remains, and additional analysis is requested for which the immunochemical method requires serum not plasma. Such a problem is not very frequent, because in the case of some proteins (e.g., for immunoglobulins; A1-antitrypsin; complement constituents C3, C4, and C5 proteins; albumin; and prealbumin) determined by using nephelometric methods, the results obtained either with a serum sample or with plasma obtained from blood taken on lithium heparin are comparable (18).

5.6 Analytical phase in immunoassay measurement Manual and semimanual immunoassays require very precise and timed pipetting of assay reagents and the patient’s sample (competitive methods) or only the patient’s sample (noncompetitive methods). If strictly determined amounts of assay reagents in each sample cannot be assured or the volume of the patient sample is inaccurately pipetted, then the assay performance cannot be accepted because of possibilities of erroneous results that may affect patient safety. Nowadays, most immunoassays are routinely performed on fully automated immunochemistry platforms, so pipetting is no longer the analytical step requiring care. However, many specialized immunochemistry analyses still require manual methods; thus accuracy of pipetting is still an issue. There is a common belief that if everything is done by the automatic analyzer, then nothing remains to be afraid of because all steps of the analytical procedure, such as dispensing the sample and reagents, mixing, washing, and reading the signal, are fully repeatable. Also, physicians (and sometimes patients) are convinced of the simplicity of automatic immunoassay measurement procedures, and they do not appreciate the real complexity of such “simple determinations”. Performing immunochemical determinations by using an automatic analyzer is very efficient, quick, and, from a nonprofessional point of view, very simple. Unfortunately, immunochemical methods, unlike other chemistry methods, are susceptible to various sources of interference inherently connected with the method as such and depending on the presence of various constituents in the patient’s sample. The main reagents used in immunochemical methods are antibodies (capture and signal antibodies) reacting with the antigen, and any substance present in the sample influencing the antigenantibody reaction may potentially have an adverse effect on the final result. The term interference is very well known for clinical chemists, but they think usually about typical interference from hemolysis or drugs present in the sample. Meanwhile, the most difficult to detect, unpredictable, and dangerous interference from the point of view of patient safety is caused by heterophilic antibodies, interference absent in other clinical chemistry methods. The problem of interference is seemingly familiar to many laboratory workers and practically unknown to most physicians. To understand at least some aspects of interference in immunochemistry methods caused by heterophilic antibodies, a background knowledge on the human immune system and natural antibodies present in blood samples is necessary (see the next chapter).

References

41

References 1. Bowen RAR, Chan Y, Cohen J, et al. Effect of blood collection tubes on total triiodothyronine and other laboratory assays. Clin Chem 2005;51: 424–33. 2. Bowen RAR, Chan Y, Ruddel ME, et al. Immunoassay interference by a commonly used blood collection tube additives, the organosilicone surfactant Silwet L-720. Clin Chem 2005;51: 1874–82. 3. Stankovic AK, Parmar G. Assay interferences from blood collection tubes: a cautionary note. Clin Chem 2006;52: 1627–8. 4. Kilinç AS, Düzoylum A, Uncugil CF, Yücel D. Falsely increased free triiodothyronine in sera stored in serum separator tubes. Clin Chem 2002;48: 2296–7. 5. Wickus GG, Mordan RJ, Mathews EA. Interference in the Allgro immunoassay system when blood is collected in silicone-coated tubes. Clin Chem 1992;38: 2347–8. 6. Chang C, Lu J, Chien T, et al. Interference caused by the contents of serum separator tubes in the Vitros CRP assay. Ann Clin Biochem 2003;40: 249–51. 7. Shefield JS, Laibl VR, Roberts SW, Wendel GD. False positive results for the AUSZYME monoclonal test. Obstet Gynecol 2005;105: 449–50. 8. Gerhardt W, Nordin G, Herbert AK, et al. Troponin T and I assays show decreased concentration in heparin plasma compared with serum: lower recoveries in early than in late phase of myocardial injury. Clin Chem 2000;46: 817–21. 9. Galligan J, Ward G, Jacobi J, McMaugh C. Preanalytical variation in samples collected for assay of adrenocorticotrophin. Clin Biochem Rev1996;17: 100. 10. Tate J, Ward G. Interferences in immunoassay. Clin Biochem Rev 2004;25: 105–20. 11. Jones AM, Honour JW. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol 2006;64: 234–44. 12. Glendenning P, Laffer LL, Weber HK, Musk AA, Vasikaran SD. Parathyroid hormone is more stable in EDTA plasma than in serum. Clin Chem 2002;48: 766–7. 13. Wenk RE. Mechanism of interference by hemolysis in immunoassays and requirements for sample quality. Clin Chem 1998;44: 2554. 14. Luzzi VI, Scott MG, Gronowski AM. Negative thyrotropin assay interference associated with an IgGK paraprotein. Clin Chem 2003;49: 709–10. 15. Frystyk J, Freda P, Clemmons DR. The current status of IGF-I assays – a 2009 update. Growth Horm IGF Res 2010;20: 8–18. 16. Evans MJ, Livesey JH, Ellis MJ, Yandle TG. Effect of anticoagulants and storage temperatures on stability of plasma and serum hormones. Clin Biochem 2001;34: 107–12. 17. Yi J, Kim C, Gelfand CA. Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma. J Proteome Res 2007;6: 1768–81. 18. Develter M, Blanckaert N, Komarek A, Bossuyt X. Can heparin plasma be used instead of serum for nephelometric analysis of serum proteins? Clin Chem 2006;52: 1609–10.

6 Human natural antibodies and immunoassay

Basic information on human immunoglobulins (antibodies), which are present in the patient’s sample, is prerequisite for understanding heterophilic interference in immunochemical methods. Laboratory professionals performing the immunochemistry measurements and those who are involved in the interpretation of immunoassay results should be aware of the complexity of the immune system pertaining in particular to the number and types of antibodies occurring in the human organism, not only in the disease state but also in the physiological condition of apparently healthy individuals. Knowledge of similarities and differences between the naturally occurring antibodies should be of paramount importance for every laboratory using any analytical system based on the antigen-antibody reaction in vitro for the measurement of various analytes. It has to be kept in mind that all natural antibodies circulating in human blood are also present in serum/plasma samples, the specimens most frequently used for the determination of the concentration of various molecules by immunochemistry measurement systems. Since the antigen-antibody reaction is the basic principle of immunochemical methods, then in view of the great variety and variable amount (from trace amounts to very high concentrations of the order of grams per liter) of antibodies present in blood serum, the probability of interference from natural antibodies in the reaction between free antigen and capture antibody, in the reaction between the antigen bound to capture antibody and labeled antibody, or interference with the signal measurement system, especially with enzyme labels, is very high and totally unpredictable. Frequency of occurrence, titer, and concentration of various natural antibodies in the sample are strongly dependent on the presence of disease and are patient related. In individuals, the plasma concentration of natural antibodies may be unchanged for years or may considerably fluctuate during the course of the disease. In apparently healthy individuals, the sudden appearance and sudden disappearance of natural antibodies due to various reasons is also possible. No serum or plasma sample is free from the presence of natural antibodies; thus it seems almost impossible to perform immunochemistry measurements, especially for proteins and peptides, without errors caused by the presence of these antibodies. Of course, no laboratory measurement with zero error exists, but only the immunochemistry methods are so vulnerable to the possibility of interference from natural antibodies. It does not mean that every immunoassay measurement is affected by natural antibodies present in the patient’s serum or plasma sample. However, it does mean that when performing the immunoassay measurement of any analyte there is always the possibility that natural antibodies may affect the final result of the determination and thus affect the patient’s safety.

6.1 The human immune system The immune system protects the human body from various pathogens (bacteria, viruses, fungi, and parasites), malignancies, and other harmful agents, including even many chemicals. This function is fulfilled by the orchestration of a variety of effector cells

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and molecules. The recognition of foreign antigens is the hallmark of the specific adaptive immune response. B lymphocytes with their highly specialized antigen receptors, surface immunoglobulins, are responsible for the specific adaptive immune response. These receptors can recognize and respond to almost any harmful agent a person is likely to be exposed to during his or her life span. The immunity mediated by B lymphocytes is known as adaptive humoral immunity, as opposed to that mediated by T lymphocytes and referred to as an adaptive cellular immunity. B cells are specialized to recognize the surface antigens on pathogens living outside the cells (extracellular pathogens) and to differentiate into effector plasma cells. Such effector cells secrete antibodies to target these pathogens in three main ways: neutralization (binding pathogens and blocking their access to cells), opsonization (coating the microorganisms with complement intermediates), and/or complement activation (1). It has to be clarified that B-cell membrane-bound immunoglobulin (antigen receptor) is identical to secreted immunoglobulin (antibody) present in serum except for a small portion of the carboxy terminus of the heavy chain of the constant region. The carboxy terminus of the B-cell receptor is a hydrophobic sequence that anchors the molecule to the membrane, and in the antibody, it is a hydrophilic sequence that facilitates secretion. Thus, the secreted immunoglobulins (antibodies) are the molecules that are present in the circulation and after blood is drawn and serum or plasma are separated; they are present in the patient’s sample used for various analyses, including immunoassay determinations. The same natural antibodies present in human serum or plasma may interfere with the immunoassay reaction between antigen and assay antibody. However, it has to be stressed again that many antibodies present in blood samples have no effect on immunoassay determinations.

6.2 Chemical structure of immunoglobulins The immunoglobulins in blood serum constitute a highly heterogeneous spectrum of glycoproteins consisting of three equal-sized portions connected by a flexible tether containing 3%–13% of carbohydrates depending on the class of the antibody. They have two identical heavy (H) chains and two identical light (L) chains organized into a roughly Y-shaped structure. Each chain consists of a series of similar, but not identical, amino acid sequences. Each sequence of about 110 amino acids long corresponds to a discrete region of protein structure known as a protein domain. Each heavy chain contains four domains consisting of two antiparallel B-sheets, while the light chain contains two such domains. Structurally similar B-sheet domains are linked by a disulfide bridge. Both light and heavy chains consist of variable (V) and constant (C) domains. The amino-terminal sequence of both heavy and light chain varies greatly between different antibodies, but the sequence variability is limited to the first domain (approximately to the first 110 amino acids). The amino-terminal variable domains of heavy and light chains make up together the variable region of the antibody and are responsible for the ability of the antibody to bind a specific antigen. The constant region is made up of constant domains of heavy and light chains. The amino acid sequence of constant regions of heavy (CH) and light (CL) domains is relatively constant, but the domains differ with respect to a degree of glycosylation. These domains bind complement and different cells of immune response (effector function).

6.3 Antigen-combining site and complementarity-determining regions

45

Fig. 6.1: Basic structure of immunoglobulin. V, variable region; C, constant region.

Light chains contain one variable (VL) and one constant (CL) domain. There are two types of light chains: K (kappa) and L (lambda). A given immunoglobulin molecule always contains two K or two L chains, never a mixture of K and L. The ratio of the two types of light chains varies from species to species, and the average ratio in humans is 2:1. Five different classes of human heavy chains – A (alfa) for IgA, G (gamma) for IgG, μ (mu) for IgM, D (delta) for IgD, and E (epsilon) for IgE – with slightly different structures determine the class of immunoglobulin. IgG is by far the most abundant immunoglobulin with an average serum concentration of 12 g/L. Based on structural differences of heavy chains, four subclasses of IgG (IgG1, IgG2, IgG3, IgG4) can be distinguished in humans. The human IgG subclasses are numbered in order of the abundance in serum, with IgG1 being the most abundant. For IgA, two subclasses can be distinguished. All immunoglobulin classes and subclasses differ in molecular structure, biological activity, and biological half-life, and all these characteristics are important if sources of immunoassay interference from serum antibodies are considered. Immunoglobulins can be in monomeric (IgG), dimeric (IgA), or pentameric (IgM) forms. Heavy chains consist of one variable (VH) and three (in the case of IgG and IgA) or four (in the case of IgM and IgE) constant (CH) domains. The schematic structure of human immunoglobulins is presented in fFig. 6.1.

6.3 Antigen-combining site and complementarity-determining regions The number of specific antibodies produced by various species, including humans, is highly diverse (~1014 for humans), and the specificity of a given antibody is determined by the amino acid sequence of variable regions of heavy and light chains. The N-terminal domains of heavy and light chains are important, because each of the three segments forming loops that link the B sheets have highly variable length and sequence.

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Three hypervariable regions of the VL domain and three hypervariable regions in the VH domain on each arm of the Y-shaped immunoglobulin form the antigen-combining site (ACS). The six hypervariable loops of the antigen-combining site determine antigen specificity by forming a surface complementary to the antigen and are most commonly termed the complementarity-determining regions (CDRs). In the antibody-antigen interaction, the three-dimensional structure of six hypervariable loops, or CDRs, of antibody molecule recognizes and binds a specific, also three-dimensional, structure of antigen surface. Because CDRs from both VH and VL domains make the antigen-binding site, the final antigen specificity is determined by the unique combination of heavy and light chains. Because each of the heavy chain variable domains can form a pair with each of the light chain variable domains, in effect a very large number of antigen-binding sites can be formed. Different antibodies have different amino acid sequences in CDRs, so the shapes of the surfaces of CDRs are also different. Antibodies bind only chemical structures (ligands) with surface complementary to that of the antigen-binding site. Antigens that are bound by antibodies have different sizes, and most of them are much larger than the size of CDRs. The fragment of antigen that binds to CDR is known as epitope (antigenic determinant). Many proteins contain more than one epitope and thus may bind to different antibodies. Such binding occurs both in vivo, in the case of membrane immunoglobulin and secreted immunoglobulin (antibody), and in vitro, in the case of the antibody present in serum or plasma. The presence of more than one epitope on a protein molecule is useful in the case of many protein measurements using noncompetitive immunoassays. The binding of small antigens takes place usually in a pocket or groove lying between heavy chain and light chain domains (1). The structure of the antigen epitope surface is usually composed of amino acids originating from different parts of the polypeptide chain that have been brought together by protein folding. Such antigenic determinants are known as discontinuous epitopes because the structure recognized is composed of segments of the protein that are discontinuous in the amino acid sequence of the antigen. Continuous epitope denotes an epitope composed of a single segment of polypeptide chain. Although most antibodies raised against intact, fully folded proteins recognize discontinuous epitopes, some will bind the continuous peptide fragment of the protein. On the other hand, antibodies raised against a peptide that is part of a protein or against a synthetic peptide corresponding to part of a protein sequence, sometimes bind the naturally folded protein.

6.4 Genes for immunoglobulin variable regions The genes for immunoglobulin variable regions are inherited as sets of gene segments, each encoding a part of the variable region of one of the immunoglobulin polypeptide chains. Because there are many different gene segments in each set, and different gene segments are joined together in different cells, each cell generates unique genes for the variable regions of heavy and light chains of the immunoglobulin molecule. Just a few hundred different gene segments can combine in many ways to generate thousands of different receptor chains. In this way, a small amount of genetic material can encode a truly staggering diversity of receptors. The light chain variable domain is encoded by two separate genes: V (variable) and J (joining). The heavy chain variable

6.5 Diversification of immunoglobulins in vivo

47

region is encoded by three separate genes: V, D (diversity), and J. The chains are synthesized independently and then assembled within the cytoplasm. These genes are randomly rearranged to form a continuous gene product (2). This process allows for a combination of gene products giving rise to a huge number of different antibodies with different variable combining sites at the N terminus. As a consequence, a very large number of antibodies is present in the serum or plasma of both healthy and diseased individuals.

6.5 Diversification of immunoglobulins in vivo The primary repertoire of human immunoglobulins is already very diverse. Further diversification enhances both the ability of immunoglobulins to recognize and bind to foreign antigens, and the effector capacities of the expressed antibodies. This diversification is achieved by somatic hypermutation and class switching, which alters the amino acid sequence of the secreted immunoglobulin in a distinct way. Somatic mutations occur at a high rate, creating an opportunity for greater antibody diversity. Additional factors increasing the diversity of antibodies are the mutations taking place within the recombined genes VJ and VDJ. These are mainly nonspecific point mutations and, less frequently, deletions, insertions, or conversions. They usually lead to the change of one amino acid in the variable fragment of the heavy chain. Immunoglobulins synthesized first in a primary response have no traces of mutation. The effects of mutation are observed in antibodies formed in the secondary immune response. The exchange of one amino acid in the variable fragment of the heavy chain may lead to dramatic and adverse situations: total loss of the ability to bind antigen or increase in the affinity to antigen, sometimes by even more than 100-fold. After the exposure of the human organism to the antigen, a series of events in the immune system takes place, yielding at first a highly specific antibody usually of low affinity, then after somatic mutation, a highly specific antibody of greater affinity is produced. In general, the first antigen receptors expressed by B cells are immunoglobulins of classes IgM and IgD, and the first antibody produced during the immune response is always IgM. In secondary (or subsequent) antigenic challenges, the same assembled V region may be expressed in IgG, IgA, or IgE antibodies. Each B cell produces immunoglobulin of a single specificity. The class switching results in production of a high-affinity antibody that has undergone antigen-driven somatic mutation and therefore is thought to be monospecific. However, even these high-affinity “monospecific” antibodies are able to recognize more than one epitope. Switching from IgM to other immunoglobulin classes occurs only after B cells have been stimulated by the antigen. Besides all forms of immunoglobulin class produced normally by human beings, each individual also produces one set of immunoglobulin allotypes derived from only one of the parental genetic forms (3). Some antigenic determinants lie within the antigen-combining site of the antibody, while others are outside. As the process of antibody diversity proceeds, somatic mutations may cause some modification of idiotopes. In effect, two types of idiotopes have been recognized: private and cross-reactive ones (common or public) (1). Private idiotopes represent clonally derived, structurally distinctive variable region epitopes, while public or cross-reacting idiotopes are epitopes shared by genetically similar antibodies. Private idiotopes are not found on antibodies with different

48


antigen-combining-site specificities. Cross-reactive idiotopes are found on antibodies that contain different antigen-combining sites but exhibit similar antigenic properties within idiotype. This means that each individual exhibits antibodies with unique markers. From an immunochemistry point of view, it is important to realize that plasma or serum samples contain a variety of antibodies and that some antibodies are patient specific. Thus, not only cross-reactivity of these antibodies with immunoassay reagent antibodies may be seen, but also different types of interference characteristic only for a given patient can appear. In other words, interference in the immunoassay reaction from human natural antibodies is patient specific. Because the antigenic determinants on antibodies can themselves be antigenic, eliciting the secondary immune response against them results in generation of anti-idiotypic antibodies. These can, in turn, elicit further responses to a third (e.g., anti-anti-idiotypic antibodies) or fourth immune reaction or may cascade ever further. Such phenomena are known as the “idiotypic network” (1,4) and represent a part of the immune response. The idiotypic network has the potential to produce antibodies against numerous heterogeneous idiotopes, thus significantly increasing the array of circulating immunoglobulins. Some of these antibodies have wider specificity and present potentially high cross-reactivity with immunoassay reagent antibodies.

6.6 Natural antibody against exogenous (nonself ) and endogenous (self ) antigens The human immune system inherently produces antibodies that bind to a variety of exogenous (nonself ) antigens as well as endogenous (self ) antigens. These antibodies, called natural, are synthesized mostly against bacterial flora of the gut (e.g., isohemaglutinins), although some of them arise independently of known immunization. Natural antibodies play a crucial role by preventing pathogen dissemination to vital organs and by improving immunogenicity through enhanced antigen-trapping secondary lymphoid organs. The wide variety of reactivity toward microbial components justifies the role of natural antibodies in the primary line of defense against infection (5). The major source of natural antibodies are CD5B-1 cells, which differ from the conventional B cells (B-2 cells), firstly because they are thought to require contact with the antigen for expansion and maintenance, and secondly because in general they do not appear to undergo somatic hypermutation (6). Natural antibodies present in human serum constitute a highly heterogeneous spectrum of proteins differing with respect to chemical structure and specificity. These antibodies occurring in the blood in varying concentrations belong to IgM, IgG, and IgA classes and are directed against the immense variety of antigens (7). They commonly recognize antigens of low affinities. They are also able to react with a high number of apparently antigenically unrelated antigens. The presence of natural antibodies against self antigens in human plasma is largely independent of age, which contrasts with agedependent diversification of reactivities against bacterial antigens (8,9). Their amount and type can continuously change in the same individual, and the changes may be either of short or of very long duration. For instance, after infection or immunization, natural antibodies can remain in the blood for weeks or months. In patients with autoimmune diseases or in persons having steady contact with animals, as well as in those with food allergy, the presence of antibodies may be observed for many years. Thus, the spectrum

6.6 Natural antibody against exogenous (nonself ) and endogenous (self ) antigens

49

of natural antibodies present in the patient’s blood samples varies in time, which has very important consequence if antigen or antibody is measured by immunoassay not only for diagnostic purposes but especially for monitoring the patient. The persistence of an antibody in the plasma and extracellular fluid is determined not only by a stimulatory action of the antigen but also by its isotype, because each isotype has a different halflife in vivo. The types of antibodies present in serum or plasma samples together with molecular weight, mean serum concentration, and half-life are presented in fTab. 6.1. Immunoglobulins, like other proteins, are immunogenic when used to immunize individuals of another species. Anti-immunoglobulin antibodies can recognize the amino acid sequence characterizing the isotype of the injected immunoglobulin. Such antiisotypic antibodies recognize all immunoglobulins of the same isotype in all members of the species from which the injected immunoglobulin comes. It is also possible to raise antibodies that recognize differences in immunoglobulins from members of the same species that are due to the presence of multiple alleles of the individual C genes in the population. Such allelic variants are called allotypes. In contrast to anti-isotypic antibodies, anti-allotypic antibodies will recognize immunoglobulin of a particular isotype only in some members of a species. Finally, as individual antibodies differ in their variable regions, one can raise antibodies against unique features of the antigenbinding site, which are called idiotypes. Such antibodies (anti-idiotypic antibodies) contain an antigenic determinant in the Fab region to which other antibodies can bind. Anti-idiotypic antibodies can also elicit the immune response, and anti-anti-idiotypic antibodies are produced. From an immunochemistry point of view, all types of natural antibodies – anti-isotypic, anti-idiotypic, and anti-anti-idiotypic – present in the patient’s sample may cause interference by either reacting directly with immunoassay antibodies or by causing steric hindrance, thus blocking the access of antigen to capture or signal antibody.

Tab. 6.1: Characteristics of human immunoglobulins. Immunoglobulin

Molecular weight (kDa)

Mean adult serum concentration, (g/L)

Half-life (days)

IgG1

146

9

21

IgG2

146

3

20

IgG3

165

1

7

IgG4

146

0.5

21

IgM

970

1.5

10

IgA1

160

3.0

6

IgA2

160

0.5

6

IgD

184

0.03

3

IgE

188

5 10–5

2

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Human antianimal antibodies arise after exposure to well-characterized antigens of animal origin. They have a high avidity and belong to the IgG, IgA, IgM, and, less frequently, IgE classes. Antianimal antibodies present in the blood can be both of iatrogenic and noniatrogenic origin (10). Iatrogenic origin is when a foreign protein antigen is introduced into the circulation causing the natural response of the human immune system. Various pharmaceuticals currently used for diagnostic or therapeutic purposes (e.g., antibodies transporting therapeutic drugs, antibodies transporting labels to target cells, insulin of animal origin, antiviral or antibacterial sera) are important causes of the formation of antianimal antibodies. Mouse antibodies when administered for therapeutic or imaging purposes may cause the formation of human antimouse antibodies in patients receiving such a treatment (11,12). Prevalence of antianimal antibodies is higher in patients receiving blood transfusions. Also, various nonconventional therapies should be pointed out as a source of immunization by animal proteins. Among noniatrogenic causes of the presence in blood of antianimal antibodies are transfer across the placenta of antianimal antibodies from the mother’s blood to the blood of the fetus, steady contact with animals (socially), occupational exposure of farmworkers, and transport of diet antigens across the intestinal wall (as in celiac disease patients). Sometimes there is no history of previous exposure to animal protein, but if an adult patient in his or her childhood was exposed to diverse animal species, heterophilic, highaffinity antibodies can be present in plasma (13). The reported connection between the occurrence of antianimal antibodies and such diseases as idiopathic cardiomyopathy is interesting (14). In human serum, antibodies that react with self-molecules (autoantibodies) are also present. They are not only associated with autoimmune or chronic diseases but can also be found in healthy individuals. For example, rheumatoid factors (RFs), antibodies to single-stranded (ss) DNA and antinuclear antibodies, are detected in healthy individuals. RFs are autoantibodies that bind to multiple antigenic determinants on the Fc portion of IgG. The Fc binding of RF is not polyspecific in a strict sense, since it is binding to specific epitopes. Subsets of RFs have themselves been shown to be polyspecific, binding to ssDNA, thyroglobulin, insulin, tetanus toxin, lipopolysaccharide, or DNA histone. Most patients who are positive for IgM-RF also exhibit RFs of the IgG and/or IgA classes. Although a majority of patients with seronegative rheumatoid arthritis do not exhibit IgM-RF, they do possess IgG-RF. It is known that RF is produced in response to many bacterial and viral infections and is a part of the normal secondary immune response. The properties of low-affinity, mainly unmutated IgM autoantibodies present in healthy individuals differ fundamentally from those of high-affinity, somatically mutated IgG autoantibodies produced in different disease states. Taking into account the fact that natural antibodies are produced in response to a variety of exogenous and endogenous antigens, as well as to some imperceptible immunizations, generally two types of antibodies can be found in normal blood serum: polyspecific and monospecific. The polyspecific natural antibodies react with more than two apparently unrelated antigens, and binding to the antigen occurs via the antigen-combining site usually of low affinity. Although the bound antigens are unrelated and have different chemical composition, they have some structural homology. Polyspecific natural antibodies are those most abundantly occurring in normal sera (7). The monoclonal antibodies react with one defined antigen and usually have high affinity. Whereas the production in vivo of monoclonal antibodies is usually due

6.7 Natural antibodies and patient safety

51

to exposure to animals and animal products, diet, purposeful immunization (e.g., for imaging techniques and tumor localization) (15,16), and sometimes unknown causes (17), the reasons for the occurrence of heterophilic antibodies in healthy individuals are poorly understood (18). Endogenous human natural antibodies having the ability to bind immunoglobulins of other species, like animal capture and signal antibodies used in immunoassays, are present in serum or plasma of more than 10% of population. Thus, it seems that the natural antibodies are the major source of heterophilic interference in normal individuals (19).

6.7 Natural antibodies and patient safety Natural antibodies can be regarded as a part of the normal immune system, and their presence in health and disease states is nothing unusual. The subject of biological significance and the role of natural antibodies in the human organism is the domain of immunologists, but the role of natural antibodies as endogenous interfering factors in immunochemical methods is the interest of clinical chemists. Thus, knowledge of the type, structure, and biological half-life of natural antibodies present in serum or plasma, and the unusual clinical condition of patients when these antibodies occur in abnormal concentration or avidity, is necessary to understand heterophilic interference. Such knowledge may help to protect patient safety.

References 1. Murphy K, Travers P, Walport M. Janeway’s Immunobiology. 7th ed.: Garland Science, Taylor & Francis Group; New York and London 2008. 2. Nossal GJV. The basic components of the immune system. N Engl J Med 1987;316: 1320–5. 3. Ismail AAA, Walker PL, Cawood ML, Barth JH. Interference in immunoassay is an underestimated problem. Ann Clin Biochem 2002;39: 366–73. 4. Weir DM, Stewart J. Antigen and antigen recognition. In: Immunology. Edinburgh: Churchill Livingstone; 1997. 5. Carroll MC. The role of complement and complement receptors in induction and regulation of immunity. Ann Rev Immunol 1998;16: 545–68. 6. Casali P, Burastero SE, Nakamura M, Inghirami G, Notkins AL. Human lymphocytes making rheumatoid factor and antibody to ssDNA belong to Leu-1 () B-cell subset. Science 1987;236: 77–81. 7. Avrameas S, Ternynck T. Natural autoantibodies: the other side of the immune system. Res Immunol 1995;146: 235–48. 8. Hurez V, Kaveri SV, Kazatchkine MD. Expression and control of the natural autoreactive IgG repertoire in normal human serum. Eur J Immunol 1993;23: 783–9. 9. Lacroix-Desmazes S, Mouthon L, Coutinho A, Kazatchkine MD. Analysis of the natural human IgG antibody repertoire: life-long stability of reactivities towards self antigens contrasts with age-dependent diversification of reactivities against bacterial antigens. Eur J Immnuol 1995;25: 2598–604. 10. Kricka LJ. Human anti-animal antibody interferences in immunological assays. Clin Chem 1999;45: 942–56. 11. Janssen JA, Blankestijn PJ, Docter R, et al. Effects of immunoscintigraphy with monoclonal antibodies in assays of hormones and tumour markers. BMJ 1989;298: 1511–13.

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12. Van Toorenenbergen AW, Hooijkaas H, Heerenbrink GK, Dufour-van den Goorbergh DM. Heterophilic antibody interference in a tryptase immunoassay. Clin Biochem 2008;41: 331–4. 13. Brugts MP, Luermans JG, Lentjes EG, et al. Heterophilic antibodies may be a cause of falsely low total IGF1 levels. Eur J Endrocrinol 2009;161: 561–5. 14. Fukuta S, Yamakawa K, Hayashi Y, et al. Immunological study of heart diseases with special reference to the cytotoxicity of the heterophile antibody against cultured myocardial cells. Jpn Circ J 1984;48: 1354–7. 15. Ballow M., Nelson R. Immunopharmacology: immunomodulation and immunotherapy. JAMA 1997;278: 2008–17. 16. Wilder RB, DeNardo GL, DeNardo SJ. Radioimmunotherapy: recent results and future directions. J Clin Oncol 1996;14: 1383–400. 17. Sosolik RC, Hitchcock CL, Becker WJ. Heterophilic antibodies produce spuriously elevated concentrations of the MB isoenzyme of creatine kinase in a selected patient population. Am J Clin Pathol 1997;107: 506–10. 18. Hennig C, Rink L, Fagin U, Jabs WJ, Kirchner H. The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs. J Immunol Methods 2000;235: 71–80. 19. Levinson SS, Miller JJ. Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. Clin Chim Acta 2002;325: 1–15.

7 Immunoassay interference – How to recognize, eliminate, or reduce it

7.1 Definition and prevalence of interference in immunoassay The incidence of immunoassay interference in the measurement of clinically useful molecules is variable and strongly depends on the type of interfering substance present in the patient’s sample, affinity/avidity of assay reagent antibodies, and assay format. Although most frequently interference from heterophilic antibodies is considered because of its adverse impact on the clinical outcome of the patient, erroneous immunoassay measurements may result also from other causes. The additional factors to consider are preanalytical prelaboratory and laboratory sources of error, the presence in the patient’s sample of autoantibodies against the analyte intended to be measured, and inappropriate molecular form or concentration of specific and nonspecific serum binding proteins for the measured analyte. In some immunochemical measurements, the errors may also be due to a high-dose hook effect. The occurrence of immunoassay interference differs in relation to the selected group of patients. There are no detailed studies concerning the incidence of interference from any possible sources on the measurement of clinically useful molecules by immunoassay methods. Also, no detailed information about the immunoassay interference in relation to the patient’s age, clinical status, assay format, and reagent antibody can be found in the literature. Such studies are almost impossible to perform, as there are many immunoassay formats that can be applied on immunochemistry platforms or in manual assays offered by different manufacturers, and the measurements of the analyte concentration are performed for quite a number of patients, from newborns to centenarians, and at various clinical physiological and pathological conditions. According to Ismail (1), the suggested incidence of immunoassay interference from all known factors of about 0.5% should be accepted as an average value. However, reported cases in the literature usually concern heterophilic interference as a cause of laboratory results leading to drastic consequences for the patient. However, no data on interference from heterophilic antibodies causing lack of accuracy of measurement and affecting the patient’s safety are available. Even so, laboratory professionals should always be aware that interference can appear in any serum or plasma sample, regardless of the clinical condition of the patient, at any time of measurement, with unpredictable influence on the final result of the measurement. If such erroneous results serve as the basis for clinical diagnosis, interference may directly influence the patient’s safety. Analytical interference is defined as an effect of any substance present in the patient’s sample that alters the correct value of the result obtained for the measured analyte, usually expressed as concentration or activity (2). Regardless of the method used in the medical laboratory for the measurement of clinically useful analytes, interference in the analytical phase of measurement must always be considered as an unwanted phenomenon that, as a consequence, may seriously affect the patient’s safety. No clinical chemistry method is free from interference, but immunochemistry methods are more prone to interference due to the principle of measurement – the reaction between antigen and

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specific assay antibody. Besides, immunochemical methods are used not only for the measurement of a variety of endogenous molecules (e.g., proteins, peptides, steroids, biogenic amines, and nucleotides), but also for the determination of exogenous substances (e.g., therapeutic drugs, addiction drugs, or substances getting into the organism unintentionally) against which antibodies can be produced. Immunochemical methods are also used for the measurement of the concentration of antibodies in biological samples. Thus, in talking about immunochemistry, not only one method for one analyte, but numerous methods for a huge number of analytes must be considered. Plasma or serum of any diseased patient or healthy subject may contain many different substances that can be of exogenous (e.g., drugs, anticoagulants) or endogenous origin (e.g., hemoglobin, bilirubin, lipids, paraproteins), and all of them may exert, under certain conditions, influence on the analytical phase of the immunoassay measurement. Also, laboratory preanalytical factors, such as sample processing and sample storage, can cause degradation to varying degrees of different molecules, especially proteins and peptides, and the degradation products normally not present in the sample may interfere in the measurement system. In the case of immunochemistry methods, interfering substances may influence the result of the measured concentration by affecting the analyte directly or may alter binding of reagent antibodies (capture and/or signal antibodies) with the analyte. Interference in immunoassay can arise from analyte-binding proteins (e.g., thyroxin-binding globulin in the case of free thyroxin measurement) and/or autoantibodies against the measured analyte (e.g., anti-insulin antibody in the case of insulin or antithyroglobulin antibody in thyroglobulin measurement). In contrast, other analytical methods used in medical laboratories are much less susceptible to interference, because they are normally used for the measurement of simple chemical structures by chemical methods. An example may be the determination of glucose present in serum or plasma only in free form and with no antibodies against such a structure present. The interference occurring in the immunoassay that directly affects the measurement of analytes is classified as interference independent on analyte concentration. Interference that alters the assay reagent antibody binding with the analyte and refers to the interaction between interfering substances present in the sample with one or more assay reagent antibodies is classified as analyte concentration dependent. Interference of the latter category includes compounds that either cross-react with the reagent antibodies or bind to different parts of the assay capture or signal antibody. The high-dose hook effect is another phenomenon affecting the course of the immunochemical reaction and can be categorized as analyte-concentration-dependent interference.

7.2 Cross-reactivity versus interference in immunoassay Reagent antibodies used in immunochemical methods are highly specific, but taking into account a huge variety of molecules present in biological samples, there is always the possibility that two (or more) substances will have a similar chemical structure and will “cross-react” with assay antibodies. Cross-reactivity should be distinguished from sample-related interference by the nature of the interfering molecules. If the interfering molecule binds to the analyte-binding site of the assay antibody because of the structural similarity to the tested analyte, giving rise to false elevation of the final concentration result, it is a cross-reactant. On the other hand, interference in the immunoassay

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can be defined as the action of any substance present in the sample other than the true cross-reacting molecule causing the bias (systematic error) in the assay results. Crossreactivity is often seen in the case of proteins or peptides with coincidental similarity or with homology of amino acid sequence showing micro- or macroheterogeneity, drugs and their metabolites with a similar chemical structure, or steroids and their metabolites. In other words, cross-reactivity can be defined as the ability of the assay antibody, either capture or signal, to bind chemical structures similar to but not identical with the analyte. Cross-reactivity as a source of interference is more frequently encountered in competitive than in noncompetitive immunoassays because the measured analyte, labeled (tracer) antigen and cross-reacting molecules compete for only one assay antibody. Cross-reaction is characteristic for closely related antigens, but sometimes binding occurs with unrelated immunogens. The distinction between antigen and immunogen should be kept in mind: antigen is defined as any substance that can bind to a specific antibody, and immunogen is any substance that can elicit an immune response (3). Although most antigens have the potential to elicit the production of antibodies, some antigens need to be attached to immunogens in order to induce the immune response. Thus, all immunogens are antigens, but not all antigens are immunogens. Some proteins and peptides are present in the blood in many molecular forms (macroheterogeneity) differing with respect to molecular weight, chemical and physical properties, biological half-life, biological activity, and immunoreactivity. In order to accurately measure such heterogeneous proteins by immunochemistry method, it is important to know what the immunoreactivity of each single form is or how the presence of an additional chemical group (microheterogeneity) – for example, sulfate in the case of sulfated and nonsulfated gastrin-17 – changes the immunoreactivity. Biotransformation of proteins/peptides in the human organism may lead to the formation of protein metabolites that display different levels of cross-reactivity with the parent molecule. In other words, in order to characterize cross-reactivity, it is necessary to know whether and how the assay reagent antibodies recognize different cross-reacting molecular forms of proteins or peptides and what is the main molecular form against which the assay antibody is directed. This is especially important when a sample is taken from a diseased patient, because not only differences between the molecular forms of proteins present in serum or plasma samples of healthy and diseased individuals may exist, but also the molar ratio of different molecular forms may change with the progression of disease. This may be due to the elevated activity of proteases in disease states, which can lead to instability of many molecules. Thus, in the case of heterogeneous proteins, the reagent antibody is sometimes directed against epitope, which is highly conserved between different forms, so both the intact and proteolytically degraded molecules are recognized and measured. However, the measurement of all protein forms does not always have clinical application. Frequently, the measurement of only one form is more important. The problem of heterogeneity of the analyte occurs, among others, in the case of the determination of parathyroid hormone (PTH), human chorionic gonadotropin (hCG), gastrin, cholecystokinin, and glycoprotein hormones (e.g., thyroid-stimulating hormone [TSH], FSH, and prolactin). It is, however, important to remember that immunoreactivity and biological activity are two different characteristics of the protein. Different molecular forms may have the same epitopes, and thus show cross-reactivity with the assay reagent antibody, but may express different biological activities in vivo. The same is true for ectopically produced forms of hormones. The form ectopically produced is

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not always identical with the form produced in the proper gland or cell (as in the case of PTH or gastrin) and may differ in respect to immunoreactivity and biological activity. Therefore, if the specificity of the assay antibody for the normally occurring protein and the protein produced ectopically is different and the abnormal protein form is not detected by the assay, then such assay is useless as a tool for measuring the activity of ectopic tumors. Besides abnormal protein forms, also protein precursors, different proteolytic fragments, metabolites, protein dimers, and forms complexed with other proteins may cause cross-reaction in immunoassays. As already mentioned earlier in this book (chapter 1), the antigen-antibody interaction depends on the chemical structure of the antigen and on the weak intermolecular forces involved (hydrogen bonds, van der Waals interaction, and hydrophobic interaction). Small differences in structure between ligands (small differences in electron clouds) may cause the assay antibody to have similar affinity toward the original and the altered ligand. In competitive immunoassays, like RIA, where only one reagent antibody is used, cross-reaction can be measured by the 50% displacement method, the equal displacement method, or the gradient approach (4). Percentage of cross-reactivity may vary across the assay concentration range, especially if polyclonal antibody is used. In competitive methods, cross-reacting substances most frequently give false-positive (if analyte is not present in the patient’s sample) (fFig. 7.1a) or falsely elevated results (if endogenous analyte is present in the patient‘s sample) because these substances behave similarly to the analyte measured in the immunochemical reaction (fFig. 7.1b). However, it has to be stressed that in depending on the binding affinity of the crossreactant molecule, not only positive but also negative bias may be seen (5). For some competitive assay formats, which include a washing step, if the dissociation rate for the cross-reactant is greater than that for primary ligand, more of the unoccupied antibody sites become available for tracer. In noncompetitive methods, in the presence of cross-reactant in the biological sample, the signal measured in the immunoassay system depends on whether the cross-reacting molecule reacts only with capture antibody, only with signal antibody, or with both antibodies. If the analyte is not present in the sample, the cross-reacting substance has no effect on the final result. Binding of the cross-reacting substance to both capture and signal antibody, with typical “sandwich” formation, causes positive interference (i.e., a falsely elevated result is obtained) (fFig. 7.2a). When the crossreactant binds only to capture antibody but no binding to signal antibody occurs or cross-reactant preferentially binds to signal antibody, only negative interference is observed (i.e., falsely decreased results are obtained) (fFigs. 7.2b and 7.2c). Because cross-reacting molecules occupy the binding sites on the antibody, less analyte can be attached. Cross-reactivity is the best-recognized form of interference in immunoassays. Usually, the producers of analytical immunoassay kits provide information on the known endogenous or exogenous substances that cause cross-reaction, but most frequently, such reactions occur when cross-reactant is present in blood at a very high concentration rarely seen in physiological and even in pathological conditions. In serum samples of healthy individuals, cross-reacting substances are usually at low concentration or are absent and do not pose any significant analytical problem. Cross-reactivity in immunoassay is more frequent when the analyte concentration is measured in the pathological sera (e.g., from the dialyzed patients) or in specific physiological serum

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Fig. 7.1: Cross-reactivity in competitive immunoassay. (a) Analyte is present in the patient's sample: cross-reacting molecule, the measured antigen and tracer compete for assay antibody. The higher the concentration of cross-reacting molecule, the smaller amount of tracer is bound to unoccupied sites on antibody and a falsely elevated result is obtained. (b) Analyte is not present in the patient's sample: cross-reacting molecule and tracer compete for assay antibody. Any amount of cross-reacting molecule gives a falsely positive result.

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Fig. 7.2a and b: Cross-reactivity in noncompetitive immunoassay. (a) Cross-reacting molecules are recognized by both capture and signal assay antibodies. Typical “sandwich” is formed between both assay antibodies and analyte as well as cross-reacting molecule. Falsely elevated (analyte present in sample) or falsely positive (analyte not present in sample) result is obtained. (b) Cross-reacting molecules are recognized by capture antibody but not by signal antibody. Because cross-reacting molecules occupy the binding site in capture antibody, less measured analyte can be detected and a falsely low result is obtained. No interference from cross-reacting molecule is observed if antigen is absent from the sample.

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Fig. 7.2c: Cross-reactivity in noncompetitive immunoassay. (c) Cross-reacting molecules are recognized by signal antibody but not by capture antibody. Because cross-reacting molecules occupy signal antibody, not all complexes of capture antibody with antigen can be detected and a falsely low result is obtained. No interference from cross-reacting molecule is observed if antigen is absent from sample.

samples where endogenous cross-reacting substances may be present at much higher concentration (e.g., in newborns or in pregnant women). Cross-reactions may come also from unknown forms of proteins or metabolites, which may be present in samples due to abnormal degradation of the measured protein or in samples of patients under unconventional treatment. The determination of cross-reactivity is usually performed by spiking of known cross-reacting compounds into a plasma sample with known analyte concentration and calculating the percentage of cross-reactivity (6).

7.3 Analyte specific and nonspecific binding proteins as a source of interference in immunoassay Interference in immunoassay measurement systems from various plasma proteins should be considered while taking into account two groups of proteins: (a) non-specific binding proteins such as albumin, prealbumin, rheumatoid factors (RFs), complement proteins, lysozyme, and fibrinogen, which may bind different molecules (e.g., albumin can bind bilirubin, calcium ions, hormones, or drugs); and (b) binding proteins specific for individual substances (like thyroxin-binding globulin [TBG] for thyroid hormones or sex-hormone-binding globulin [SHBG] for estradiol and testosterone). Special attention

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should be paid to albumin because it is present in plasma at very high concentration as compared to that of hormones or peptides measured by immunoassay (g/L vs. ng/L or less), and it can not only bind but also release considerable amounts of different ligands, thus changing the sample matrix. Binding and releasing albumin ligands rely on processes dependent in vivo on the pathological condition of the patient. One of the best examples demonstrating the effect of albumin on the concentration of the measured analyte is the measurement of free fraction of thyroxin (FT4) in serum or plasma. While the affinity of albumin to thyroxin is negligible as compared to that of TBG, and albumin binds only 20% of thyroxin present in the blood, very high concentrations of this protein in blood plasma is one of the causes of poor reliability of the determination of FT4 concentration in the patients with low albumin concentration (e.g., preterm newborns and patients with serious nonthyroidal diseases). Under inflammatory conditions, the concentration of some proteins, including albumin, decreases (negative acute phase protein), but the concentration of some others, like complement, increases (positive acute phase protein). Very low levels of albumin in seriously ill patients can have an impact on all immunochemically measured analytes that circulate in the blood both as a free form and as a form bound not only to specific binding protein but also to albumin. On the other hand, more than 20 proteins forming complement are elevated in many inflammatory conditions. By binding to Fc fragment of immunoglobulins, complement constituents from the patient’s sample may block the analyte-binding site on assay reagent antibodies causing interference in the immunoassay antigen-antibody reaction. To avoid interference from the complement, the antibody fragment (i.e., the papain digest of reagent immunoglobulin) is used in the immunoassays as capture antibody; otherwise analyte determination in EDTA-plasma should be performed. Other ligands, such as therapeutic drugs, are also bound and transported by albumin in the blood; thus the changes in the concentration of this protein will influence the equilibrium between bound and free analyte fraction and thus affect the determination of free fractions of the drugs measured by immunoassay. Many hormones, such as thyroid hormones, growth hormone, sex hormones, insulinlike growth factors, and cortisol, circulate both as complexes with binding proteins and as free forms. Some of nonspecific hormone-binding proteins are present in samples at very high concentrations but have low binding affinity (albumin), while others have high affinity of hormone binding and low concentrations (SHBG, TBG, and cortisolbinding globulin [CBG]). Endogenous hormone-binding proteins are present in plasma or serum samples at different concentrations. Both blood concentrations of these proteins and hormone-binding characteristics are subjected to genetic and acquired influences. By using immunoassay, it is possible to measure serum concentrations of total hormone, free fraction of hormone, or the complex of hormone with binding protein. The measurement of total hormone levels in the patient’s sample requires the addition of chemical reagents, which will dissociate the hormone from binding proteins. Among chemicals used for such purpose are salicylate, 8-anilino-1-naphtalene-sulfonic acid, thiomerosol, and various competitive inhibitors. Although in everyday routine laboratory practice there are no analytical problems with the measurement of total hormone, it should be kept in mind that total hormone concentration does not reflect the amount of biologically active molecules and that the level of total hormone depends strongly on the concentration of binding proteins. For example, an increased level of total thyroxin in the first trimester of pregnancy is due to an increase in TBG concentration, but not to

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thyroid dysfunction. Variation in serum or plasma of TBG concentration affects to a great extent the measurement of thyroid hormones and easily leads to misdiagnosis. In analogy, total calcium concentration, a common clinical chemistry measurement, strongly depends on albumin level and does not reflect ionized (free) calcium level. The plasma levels of free hormone fractions are much lower as compared to the total hormone concentrations. Therefore, free hormone measurement is more difficult, requires more sensitive methods, and depends strongly on the concentration of specific binding proteins. The prerequisite for the proper measurement of free hormone fraction is that the equilibrium between bound and free fractions will not be disturbed upon the addition of analytical reagents. In competitive liquid-phase immunoassay, a fixed amount of labeled antigen (tracer) and variable amount of unlabeled antigen (standard or analyte from the patient’s sample) compete for a fixed amount of assay reagent antibody. Any interference from the analyte-binding proteins depends upon the ratio of labeled to unlabeled antigen, on the concentration of binding proteins, and on the affinity of the assay reagent antibody to antigen. Plasma-binding proteins bind both labeled and unlabeled antigens in a similar way. In competitive assays, even if a small amount of tracer (labeled antigen) is bound by analyte-binding protein, a smaller amount of the tracer will be bound to unoccupied sites on the reagent antibody, and falsely elevated results will be obtained (fFig. 7.3a). This is because in competitive assays there is an inverse relation between signal level and analyte concentration; thus the lower the signal, the higher the analyte level, assuming that the signal coming from the complex of antigen with antibody is measured. In a noncompetitive solid-phase immunoassay, the capture antibody and labeled antibody (both being in excess in relation to the measured analyte) form a “sandwich” with the antigen. Specific binding proteins can bind the analyte from the patient’s sample, thus less antigen is bound to the assay capture antibody, and falsely low results are seen (the lower the signal, the lower the analyte concentration) (fFig. 7.3b). In the case of using an analogue noncompetitive immunoassay, no binding of analogue to endogenous specific binding proteins occurs, and the final measurement result is less dependent on changes in the concentration of binding proteins. It should be mentioned that abnormal forms of endogenous binding proteins present in human plasma may also influence the immunoassay measurement systems used for the determination of free hormone fractions. Similar problems to those encountered in the measurement of free analyte fraction in the presence of binding proteins is seen when the disease-specific pseudoreceptors and endogenous inhibitors are present in the plasma. They can bind to the ligand of interest (the measured analyte) and alter its binding characteristics to immunoassay reagent antibodies. The amounts and proportions of specific and nonspecific plasma binding proteins, soluble receptors, or different endogenous inhibitors may differ in health and disease and may depend on the severity of disease. Also, the proportions of these analyte-binding proteins may change as an effect of storage-related instability due to the action of proteases and/or degradation due to freeze/thaw processes. This means, that not only the stability of the measured analyte has to be considered but also the stability of other sample components, including specific analyte-binding proteins, are important for the course of the immunochemical reaction.

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Fig. 7.3: Binding protein (BP) interference. (a) In competitive immunoassay, BP can bind both free analyte and tracer; thus less tracer is available for saturation of unoccupied binding sites in assay antibody and falsely elevated result is obtained. (b) In noncompetitive immunoassay, BP can bind the measured antigen; thus less antigen is attached to solid-phase capture antibody and less “sandwich” complexes are formed causing always a falsely low result.

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7.4 Autoantibodies as a source of interference Human antibodies that react with self-molecules are referred to as natural antibodies or autoantibodies. In healthy individuals, autoantibodies are mainly nonmutated, polyreactive IgM of low affinity. Occasionally, low-titer IgG may occur as autoantibodies. Taking into account decavalency of IgM, these autoantibodies possess a high overall binding avidity. They differ from somatically mutated autoantibodies of high affinity, like antithyroglobulin antibodies or anti-insulin antibodies, occurring mainly in disease states. The titers of some autoantibodies, such as RFs, are elevated in infections and in a variety of autoimmune disorders. Because autoantibodies could be detectable long before the onset of disease, their measurement in the patient’s sample is very useful for clinical purposes, especially for diagnosing the preclinical stage of autoimmune diseases. Many autoantibodies, like RFs, antinuclear antibodies, antibodies against single-stranded (ss) DNA, antineutrophile cytoplasmic antibodies, antiphospholipid antibodies, antiendothelial antibodies, antibodies against hormones (e.g., insulin antibodies), and antibodies against microsomes, are routinely measured as markers of autoimmune disease. Each type of autoantibody may express a large variation in plasma titer at different periods of time, depending on the patient’s disease stage and/or treatment. They can be measured either by using the immunoassay of antigen format or by using assay antibodies against autoantibodies determined. Both types of assay are susceptible to interference, but in assay using an antigen format, the result of the measurement depends on serum concentration of endogenous antigen already bound to autoantibodies. If antithyroglobulin autoantibodies are measured in the presence of high concentrations of thyroglobulin, then a falsely low result of antithyroglobulin antibody concentration will be obtained. The measurement of proteins and other molecules against which autoantibodies may be present in human plasma samples is a big challenge for laboratories, not only from an analytical point of view but also because there is a possibility of obtaining erroneous results affecting the patient’s safety. Interference in immunoassay measurement arising from autoantibodies occurring in the patient’s sample depends on the assay methodology. In competitive methods, like RIA, there is a competition between the labeled analyte (tracer) and the analyte from the unknown sample for a limited amount of the first antibody. After the reaction reaches equilibrium, the complexes of antibody with analyte (both labeled and unlabeled) are precipitated by the second antibody (the double-antibody competitive method). As the concentration of the analyte increases, the measured signal (radioactivity) decreases. During the competitive immunoassay measurement, autoantibodies present in the patient’s sample can bind the tracer used in the assay (labeled analyte) in the same way as they bind the unlabeled analyte in the patient’s sample. Similar binding of labeled and unlabeled antigen occurs, however, only if the analyte present in the sample and the labeled antigen used in the assay do not differ chemically and show a low level of heterogeneity. The presence of autoantibodies in the patient’s sample may cause overestimation or underestimation of the RIA result, depending on the specificity of the precipitating antibody directed against the complexes of the first antibody with labeled and unlabeled antigens. If the precipitating antibody (second antibody) is specific only for complexes of assay antibody with antigen and assay antibody with tracer, but does not precipitate complexes of human autoantibody bound with analyte and tracer, then the autoantibody-tracer and autoantibody-analyte complexes do not precipitate, and a lower signal level is measured. Thus, the apparent

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analyte concentration in the sample is falsely elevated (fFig. 7.4a). However, if the second antibody cross-reacts with human autoantibodies, then the autoantibody-tracer complex precipitates, and as a consequence, a higher signal is measured and the apparent analyte concentration is falsely decreased. The amount of autoantibody-tracer complex formed in the reaction mixture depends directly on auto-antibody concentration but not necessarily in the proportional way. Low volume of the sample used in the immunoassay helps to overcome the interference from autoantibodies, because the concentration of the formed autoantibody-tracer complex will be lower when the amount of autoantibodies is smaller. In noncompetitive methods, the presence of autoantibodies in the patient’s sample always leads to underestimation of the final results, because the typical sandwich between capture antibody, analyte, and signal antibody cannot be formed if the analyte is bound to autoantibody (fFig. 7.4b). The types of interference from autoantibodies present in the patient’s sample described above are examples of analyte-dependent interference. In human blood samples, there can be present autoantibodies against many molecules. From the point of view of the patient’s safety, however, the most important are those autoantibodies that are directed against molecules commonly measured by immunoassay technology. As typical examples, interference from antithyroglobulin in the measurement of thyroglobulin concentration in patients with differentiated thyroid carcinoma, interference from anti-insulin antibodies in the measurement of insulin concentration, and rare but very important, interference from autoantibodies against thyroxin in the measurement of thyroxin should be mentioned. Recently, interference in immunoassay for troponin measurement from autoantibodies against troponin was reported (7). As was mentioned above, the final concentration of the measured analyte in the presence of its autoantibody depends on the assay format and may have considerable impact on the patient’s safety, as is the case in thyroglobulin and troponin measurements by immunochemistry in the presence of antithyroglobulin and antitroponin autoantibodies, respectively. If the total concentration of the analyte in the sample is being measured with appropriate immunoassay antibodies, then both the unbound and complexed with autoantibody analyte will be measured. The presence of autoantibodies in the patient’s sample is one of the major causes of interference in the measurement of free forms of hormones or other analytes partially bound to various binding proteins (8,9). Many unexpected sources of interference in immunoassay may appear if endogenous autoantibodies are present in the sample, even if their concentration is very low. They may affect binding between antigen and assay antibody in an analyteindependent way and thus can be considered as typical heterophilic antibodies. They also may interfere not only in the measurement of analyte, against which the autoantibodies are directed, but also in the measurement of other analytes to a different extent. In the case of spurious and unexpected immunoassay results obtained for the patient’s sample, simple analytical procedures like dilution test, recovery test, or retesting the measurement with a fresh patient sample are usually recommended. In the case of some immunochemical measurements (e.g., thyroglobulin assay), also the measurement of autoantibody against thyroglobulin, if possible, is recommended. If it is known that an increased concentration of autoantibodies against the measured analyte is present in the patient’s sample, it is only possible to say that the interference in the case of this particular sample may occur, but the magnitude

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Fig. 7.4: Interference from autoantibodies (AutoAb). (a) In competitive assay, AutoAb can bind the measured antigen and tracer forming AutoAb-Ag and AutoAb-Ag* complexes. The second antibody is used to precipitate the AgAb and Ag*Ab complexes. If second antibody precipitates also AutoAb-Ag*, then a falsely decreased result is obtained. If the complex AutoAb-Ag* does not precipitate, a falsely increased result is obtained. (b) In noncompetitive assay, AutoAb binds antigen in serum sample, and less free antigen is available for binding by capture antibody; thus always a falsely decreased result is obtained.

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and direction of changes in the final result of analyte concentration are almost impossible to predict. Thus, the interpretation of the results of analyte measurement in autoantibody-positive and autoantibody-negative patient serum samples must be different, and if the analyte measurement is performed in autoantibody-positive sera, the physician should be informed about the possibility of misinterpretation of the patient’s results. Frequently, the serum concentration of autoantibodies that cause interference in the measurement of a particular analyte is initially very low but may increase slowly over a time, and if interference is not suspected, it may remain undetected. The routine procedure of delta checking may even convince laboratory professionals that the measurement is accurate. Usually, laboratory professionals and physicians say that “this result is characteristic for this patient”, especially if the patient is under clinical observation for a longer period of time. It seems, however, that even “typical” results for the patient immunoassay should be verified for the presence of interfering substances. Although laboratory professionals are frequently involved in the interpretation of the patient results concerning the diagnosis of autoimmune diseases based on the concentration of autoantibodies, the discussion between laboratory professionals and physicians about the possibility of interference due to the presence of autoantibodies does not frequently take place. Because more and more unexpected autoantibodies against different analytes measured routinely by immunoassay are proved to occur in patient samples, both clinicians and laboratory professionals should be aware that such types of interference may be present more frequently than normally expected.

7.5 Nonimmune IgG complexes as a source of interference Immunoglobulin G (IgG) belongs to a group of highly abundant serum proteins. Its average concentration in serum amounts to about 12 g/L, and it has a long half-life of about 23 days. The C1 domain of immunoglobulins has an enormous capacity to react with various peptides and proteins, forming complexes in a nonimmune manner. These complexes can be found not only in pathological conditions (e.g., IgG-prolactin complexes in patients with prolactinoma) but also in healthy subjects (e.g., anaphylatoxins C3a, C4a, C5a linked to IgG). Serum proteins with a molecular weight below the kidney cutoff value (molecular weight of about 45 kDa) cannot persist in the circulation for a long time, but when complexed with serum IgG or other proteins of high molecular weight, they can escape rapid clearance by proteolytic degradation and glomerular filtration. In vivo, the nonimmune IgG complexes may have properties that are absent in IgG or in associated proteins considered separately. In many cases, the formation of nonimmune IgG complexes is very important. For instance, such complexes eliminate anaphylatoxins, and in this way, many inflammatory and anaphylactic reactions can be prevented (10). The nonimmune complexes of immunoglobulins with their target proteins or peptides, known as protein macroforms, when present in patient serum samples may create problems in immunochemical measurement systems used for the determination of the concentration of peptides and proteins. Erroneous results of the determination of protein concentrations may be obtained when such complexes occur, grossly affecting the patient’s safety. Macroforms of proteins are metabolized more slowly as

7.5 Nonimmune IgG complexes as a source of interference

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compared to native proteins and accumulate in the blood. Frequently, they are devoid of biological activity but may express immunoreactivity toward reagent antibodies used in immunoassay measurement systems. To understand the interference from protein macroforms in immunochemical measurements of proteins, the fundamental question should be asked as to whether the assay capture antibody binds only the free form of protein or peptide (native form), only the IgG-complexed protein form, or both free and complexed forms of protein. The recognition of each form by assay antibody depends on how the complex of IgG with protein, in relation to protein epitopes, is formed. In many immunoassays, free and complexed forms of proteins are not distinguishable by assay capture and signal antibodies, and both molecules are measured. This situation takes place when antianalyte antibody present in the sample binds with the analyte epitope, which is distant from epitopes recognized by immunoassay reagent antibodies. Then regardless of the form of analyte (free or complexed), capture and signal antibodies have unhindered access to epitopes present on both molecules (free form and complexed form) (fFigs. 7.5a and 7.5c). Due to the longer biological half-life of the macroform of protein and its accumulation in the blood, the measured analyte concentration is falsely elevated and does not represent the true concentration of the

Fig. 7.5: Measurement of a macroform of protein by immunoassay. (a) Measurement of normal protein by noncompetitive method. (b) If macroprotein is formed by binding of IgG to protein in such a way that the epitope recognized by assay antibodies is blocked, then the macroform is not detected. (c) If IgG is bound to protein at different epitopes on protein leaving the epitopes for assay antibodies unblocked, then the protein macroform is measured and a falsely elevated result is obtained.

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noncomplexed protein (fFig. 7.5c). The extent to which the immunoassay reagent antibodies bind macroform depends, however, on specificities of capture and signal antibodies used by immunochemistry systems. As an example, macroprolactin, the complex of IgG and prolactin, interferes with the measurement of serum prolactin concentration by most immunochemical methods. If the immunoassay antibody recognizes only the free protein/peptide form but not the IgG-complexed form, the measurement (in the absence of any other interference) is correct from the analytical point of view and a “true” result is obtained (fFig. 7.5b). This is because autoantibodies block an epitope against which the assay capture and/ or signal antibodies are directed. However, as various protein molecules could be associated with the most abundant serum proteins blocking the analyte epitopes and causing them to escape detection by immunoassay, a low level of the measured protein/peptide can be due not only to the diminished synthesis or increased degradation but also to the fact that more protein is complexed. In some cases, the situation when only a complexed protein/peptide is measured by immunoassay may be advantageous, because the complexed protein is usually considered as a marker indicating the presence of a disease. The presence of macroforms in the patient’s sample may be one of the reasons for poor comparability of different immunochemistry systems used for some protein measurements, especially at their higher concentrations. The problems with macroforms are not new and concern not just immunochemistry measurement systems. Circulating immunoglobulins complexed with enzymes and other proteins that are measured in medical laboratories have been observed for many years. The best-known example is the recognition of the existence of macroamylase (11). Pitfalls during the measurement of proteins or peptides in the presence of protein macroforms are also well known for laboratories performing the service for cardiology emergency departments. Sometimes, disagreement between troponin concentration and isoenzyme MB of creatine kinase (CK-MB) activity or CK-MBmass level measurements can be observed. Enzymatic activity of CK-MB is measured by immunoinhibition method using antibody against the M subunit of CK. By blocking the M subunit, it is possible to measure the activity of CK related only to the B subunit, which corresponds to half of the total CK-MB activity. The inhibition test is accurate if a small amount of isoenzyme BB of creatine kinase (CK-BB) is present. However, if a macroform of CK-BB is present in the sample, then the activity of CK-MB is falsely elevated. Such a result does not correspond to the magnitude of change in CK-MBmass concentration measured by immunochemistry, because assay capture antibody is directed against the B subunit and labeled antibody is directed against the M subunit. Lack of agreement between CK-MB activity, CK-MBmass, and/or troponin concentration results must be interpreted not only as an erroneous measurement of troponin concentration, but also as possible interference from the CK-BB macroform. However, as was already mentioned, complexes of troponin with autoantibodies against troponin are also proved to be present in the patient samples (7). This classical example shows again how a good knowledge of methodology used in different measurement systems could be useful for proper interpretation of the results of laboratory tests and thus help to improve patient health care and patient safety.

7.6 Interference from heterophilic antibodies

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7.6 Interference from heterophilic antibodies 7.6.1 Nomenclature and prevalence of heterophilic antibodies in patient samples For decades, endogenous antibodies present in human plasma or serum samples, which have the ability to bind to immunoassay reagent antibodies (capture or signal antibodies, or both) or to enzymes used as labels, have been called heterophiles or heterophilic antibodies. According to Taber’s Cyclopedic Medical Dictionary, heterophilic antibody is an antibody responding to an “antigen other than the specific one” (12). Heterophilic antibodies, as a product of the natural process of antibody diversification, show a large degree of multispecificity with a broad reactivity for many different types of molecules (13,14,15). It has to be stressed that human “so-called heterophilic antibody” can be any inducible antibody, but the term heterophilic has been introduced only for analytical purposes. This term is not the best description for all antibodies present in a human sample that can interfere with the antigen-antibody reaction in vitro. Kaplan and Levinson (16) defined interference by heterophilic antibodies as interference caused by any subclass of human antibodies directed against any part of a murine antibody. These human antibodies must be of sufficient titer and affinity to have an analytically significant effect. The immunogen that induces production of heterophilic antibodies may be known, but frequently it is unknown. According to the classification proposed by these authors: 1. Interfering antibodies should be called heterophile when there is no history of medical treatment with animal immunoglobulins or other well-defined immunogens and the interfering antibodies can be shown to be multispecific or exhibit natural RF activity. The antibodies used in competitive and noncompetitive immunoassays as reagent antibodies are often monoclonal antibodies, and there is a possibility that they will react with heterophilic antibodies present in human plasma because of their multispecificity. When there is no well-defined immunogen, the suggestion is to call the interfering antibodies heterophilic antibodies. 2. Interfering antibodies should be called specific human anti-animal antibodies (HAAAs) when there is a history of treatment with animal immunoglobulin, and immunoglobulin from the same species is used in the immunoassay as capture or signal antibody. Because in noncompetitive immunoassays the murine antibodies are used, the human antimouse antibodies (HAMAs) can interfere with antigen-antibody reaction. When there is a well-defined animal immunogen, interfering antibodies are called antianimal antibodies. However, there are situations when heterophilic antibodies coexist with specific HAAAs. Usually, “heterophilic” antibodies are produced in the natural process of antibody formation against a broad variety of microbial components and play the major role in the primary line of immune defense. They may also be produced as a result of a known autoimmunization. Their presence in healthy individuals is quite common. It can be said that heterophilic antibodies are any inducible antibodies in the human organism that are polyreactive and have the ability to bind heterogeneous, frequently poorly defined antigens. They may recognize identical epitopes, which can be present on two different antigens (cross-reactivity), or may recognize two different epitopes on two different antigens. Frequently, heterophilic antibodies are of the IgM class (15). The

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best-known heterophilic antibodies are RFs, which are antibodies that bind to many antigenic determinants on the Fc fragment of IgG. They occur in 70% of patients with rheumatoid arthritis and in 4%–5% of healthy persons. Sometimes they appear also in other autoimmune diseases. Properties of heterophilic antibodies in respect to binding to assay reagent antibodies may be similar to properties of autoantibodies (induced by self-antigen) or antianimal antibodies (induced by antigen of animal origin) and may be anti-isotypic, anti-idiotypic, or anti-anti-idiotypic antibodies (17). Among antianimal antibodies that could be present in human plasma or serum samples, the most frequently occurring are HAMAs produced as an effect of using mouse immunoglobulins for imaging or therapeutic purposes (radioimmunoscintigraphy, radioimmunotherapy). The presence of HAMAs can be shown in the patient’s blood even three years after immunoscintigraphy, and their serum concentrations may range from several micrograms per liter to even grams per liter (17). To minimize the interference from HAMAs, the patients should be given immunosuppressive drugs before and during the administration of mouse monoclonal antibodies (19). According to the literature reports, HAMAs of both IgG type and IgM type can be present in human blood (20,21,22,23). Besides HAMAs, other types of antianimal antibodies may be present in blood samples. Etiology of origin of such immunoassay interfering antibodies is poorly understood. From an analytical point of view, the presence in blood samples of antianimal antibodies of the same species from which originate immunoassay reagent antibodies (either capture or signal antibody) is important if analytical interference is considered. If rabbit globulins directed against thymocytes are used as an immunosuppressant, then in the patients receiving such a therapy, antirabbit antibodies (HARAs) may appear and substantially interfere in immunochemical methods that use assay antibodies originating from rabbit. Another cause of the presence of HARAs in serum samples can be keeping rabbits by patient at home as a social animal. In the literature, clinical case reports concerning the interference from HARAs in different formats of immunoassay (e.g., in C-reactive protein (CRP) and erytropoietin measurement) have been published (24,25). Another report describes the case of a woman patient for whom during 10 years the reason of very high concentrations of gastrointestinal hormones (somatostatin, vasoactive intestinal peptide, pancreatic polypeptide, gastrin, glucagon, and neurotensin) could not be found. Only after blocking the interfering HARAs with nonimmunogenic rabbit serum was the existence of interference from antianimal antibodies proved (26). In immunochemical methods based on nephelometry or turbidimetry, polyclonal antibodies originating from rabbit are very frequently used. When such methods are used, the frequency of interference due to the presence of HARAs may be as high as 5%. These antibodies interfere with the determination of, among others, transthyretin and haptoglobin, which suggests the necessity of verification of doubtful results by using the immunochemistry methods with assay antibodies (capture, signal, or both) originating from another species (27). Besides HAMAs and HARAs, the patients’ plasma or serum samples may contain human antigoat, antisheep, antirat, or anti–guinea pig antibodies, but the literature reports on interference caused by these antibodies are not frequently seen because antibodies obtained from these species are rarely used in immunoassays as reagent antibodies. However, falsely elevated thyroid hormone levels due to antisheep antibody interference in automated chemiluminescence immunoassay have been reported (28).


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The occurrence of heterophilic antibodies in samples of human serum or plasma in the population was estimated at 30%–40% (29,30). Their concentration in the blood and detectability by using different methods depend on the type of antibody. Some sources estimate the prevalence of HAMAs in the population at more than 6%, depending on the assay format used for HAMA determination and on the type of HAMAs (31). According to one report (32), the prevalence of HAMAs and other antianimal antibodies was estimated as being as high as 80%. In a recent study by Koshida et al. (33), the reported prevalence of HAMAs equals 11.7%, with a similar percentage of IgG-type and IgM-type HAMAs. Generally, it is difficult to estimate the frequency of occurrence of HAMAs because there are several HAMA detection systems (in-house or commercial) using different standards for calibration and different assay procedures (34). Some methods are designed for detection of anti-isotypic antibodies, and when using such methods, anti-idiotypic antibodies may escape detection. The patients with diagnosed cancer tend to acquire HAMAs more frequently than those without cancer. From an immunochemistry point of view, the prevalence of heterophilic antibodies in a sample of the general human population is less important than is the incidence of immunoassay interference. When immunoassay without any blocking substances added to the assay buffer is used, heterophilic antibodies are found in a very large number of serum or plasma samples (35). In most of the currently used immunochemical methods, various blocking factors are added to the assay buffer to overcome the problem of interference from heterophilic antibodies; thus the incidence of immunoassay interference is rare, ranging from 0.4% to 4% (36,37). However, Ismail (1) pointed out that though the interference from endogenous antibodies is statistically low (about 0.5% according to his estimates), the distinction should be made between analytical error rate and clinical error rate. Taking into account analytical error due to immunoassay interference and prevalence of the diseases characterized by more frequent presence of interfering substances in blood plasma, the number of false results leading to wrong diagnosis can be much higher. It was also reported that the frequency of immunoassay interference is usually higher for newly released assays (38). It is, however, necessary to distinguish between overall prevalence of heterophilic antibodies in routine specimens, which is high, from the prevalence of heterophilic antibodies causing interference in immunoassays, because most of antibodies do not cause interference (39,40). However, it may happen that when heterophilic antibodies are initially absent in the patient’s sample (no interference is present), they appear after a time (interference is seen) and then they may disappear again (41,42). Looking from an analytical point of view, the real percentage of routine samples containing interfering antibodies is hard to estimate because the types of interference usually reported in the literature are those that cause extreme disagreement between laboratory results and the clinical picture of the patient. Small systematic errors in measurement accuracy or results that are erroneous but do not worry the physician and do not cause doubt, escape the attention of laboratory professionals, and frequently, the measurement is performed with a fresh patient sample on other occasions. Such types of interference are not easy to detect, and they are not included in any statistics concerning the occurrence of heterophilic antibodies in plasma samples and incidence of interference in immunoassays. Thus, no data concerning patient safety are available if such types of interference are present. It would be nice to have information about

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the prevalence of interfering antibodies in plasma samples for different groups of patients, not only for the general population. However, immunoassays are the moving targets – the assay format, assay antibodies, and blocking agents are being changed very frequently, and even if a very well-designed study on prevalence of heterophilic antibodies was possible to perform, probably in the meantime a new assay with different susceptibility to interference would be introduced and other information on the prevalence of heterophilic antibodies would be published. 7.6.2 Heterophilic antibodies versus assay reagent antibodies The characteristic property of heterophilic antibodies present in serum or plasma samples is the binding to assay antibodies of animal origin used as reagents in immunoassay and no binding to human immunoglobulins. The concentration of the capture antibody used in the immunochemical method is strictly defined, no matter whether the method is competitive (limited concentration of capture antibody) or noncompetitive (excess concentration of capture antibody in relation to antigen concentration). Similarly, the concentration of labeled antibodies (noncompetitive methods) used for the detection of the antigen-antibody reaction is also strictly defined for a given method. Thus, regardless of the format of the immunoassay, the exactly defined amount of animal antibodies (capture and signal antibodies), most commonly mouse monoclonal antibodies, may be exposed to a huge amount of human immunoglobulins and nonimmunoglobulin proteins of a broad spectrum of activity when serum or plasma is added to the reaction mixture. Immunoglobulin concentration in the patient’s sample depends on isotype, and amounts are on the average of 7–15 g/L in the case of IgG. The amount, concentration, type, and affinity of heterophilic antibodies in patient samples are largely unknown and variable. Also, the mode of interaction between interfering heterophilic antibodies and assay reagent antibodies, due to their complex stereochemistry and differences in reaction kinetics, is unpredictable. Although antibodies in humans are produced against single endogenous or exogenous immunogens, the heterophilic antibodies may interact with assay reagent antibodies originating from another species (mouse, rabbit, and goat) because of a possible interspecies cross-reaction among similar epitopes on immunoglobulins. As was already mentioned, the presence of heterophilic antibodies in the patient’s sample does not always mean that they will interact with assay antibodies, and that interference in immunoassay measurement must occur. The interference from heterophilic antibodies in immunochemical measurement may concern one or more analytes determined in the patient’s sample, one or more immunoassay systems being used for the measurement of the same analyte (e.g., competitive RIA method or noncompetitive chemiluminescence method for TSH measurement), and one or many patient samples taken at different time intervals. The magnitude of the effect depends on the concentration and/or titer of the interfering heterophilic antibodies, but not necessarily in a directly proportional way. It is possible that high concentrations of heterophilic antibodies will cause insignificant interference, while very low concentrations of anti-idiotypic heterophilic antibodies will cause the measurement accuracy problem. Also, the direction of changes in the measured analyte concentration is not always easy to predict. The degree of interference depends on the method format and the differences in affinity, avidity, or titer between the assay reagent antibodies and interfering antibodies present in the patient’s sample. The avidity of interfering antibodies


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may be similar, higher, or lower than that of the capture antibodies and signal antibodies. During the interaction between antibody and antigen, conformational changes of antigen induced by antibody may also alter the specificity of antibody. Also, a direct immobilization of assay monoclonal antibodies on solid phase may change binding characteristics; thus susceptibility to heterophilic interference could be different for the same antibody attached to solid phase from that used in liquid-phase assay. It means that interfering antibodies react with antibodies used in one assay but may not react with antibodies use in another assay, or in one assay they may give false-positive results, while in another the result will be false negative. Reagent antibodies of animal origin (capture and label antibodies) are usually bivalent and belong to a high-affinity IgG class, whereas endogenous interfering antibodies may belong to any immunoglobulin class (IgM, IgG, or IgA). Heterophilic antibodies of the IgM class dominate as interfering agents in immunochemical methods, and heterophilic antibodies of IgG and IgA classes occur mostly together with the IgM class (43). It was shown that heterophilic antibodies of the IgM and IgG classes interfere in the immunoassay measurement system by binding to Fab fragments of the assay antibody, since the substitution of Fab fragments for the whole antibody does not eliminate the interference (44,45). IgM anti-immunoglobulin antibodies (RFs), present in many serum samples from patients with autoimmune disease, are frequently the cause of interference in noncompetitive methods. Interference from heterophilic antibodies may cause both falsely elevated and falsely lowered analyte concentrations when measured in the patient’s sample. It depends on the site of interference in the immunochemical reaction, that is, on the site of binding of interfering antibodies to the assay reagent antibodies and on the nature of interfering antibodies. The interfering antibodies can be directed toward any combination of solid-phase capture antibody, linker of antibody to solid phase and label antibody, as well as toward enzyme proteins used to label antibody in immunoenzymatic methods. Nephelometric and turbidimetric methods designed for the measurement of proteins present in blood serum or plasma at high concentrations are less prone to interference from heterophilic antibodies as compared to assays designed for the measurement of very low analyte concentration. 7.6.3 Interference from heterophilic antibodies and assay format Assay format is one of the important characteristics of immunoassay determining whether the assay is more or less prone to the interference from heterophilic antibodies. In immunochemical methods, it is necessary to take into account the way the assay capture antibody reacts with the antigen. It is desirable that binding between these two molecules takes place with a high association constant; thus the antibody should have a high affinity to the antigen. However, if one takes into account antibody that has low affinity but many binding places, it is necessary to consider the total binding strength that is its avidity. In consequence, despite low affinity, the reaction of binding antigen with antibody is moved in the direction of antigen-antibody complex formation, and thus is characterized by a low dissociation constant. The kinetics of the immunoassay reaction is described by the ratio of two rate constants (association rate constant and dissociation rate constant) and is termed as equilibrium constant or affinity constant (Ka). There is, however, a difference between competitive (equilibrium immunoassay) and noncompetitive (nonequilibrium immunoassay) in relation to assay

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kinetics. The thermodynamic model for many immunoassay reactions is often very difficult to define because many antigens possess multiple epitopes and are heterogenous. Sometimes binding of antigen with antibody causes allosteric effects, the assay reagent antibodies are not always homogenous, and current immunometric assays are generally nonequilibrium assays and as such depend strongly on some characteristics of the sample in which the analyte is measured. All this determines whether interference from heterophilic antibodies will occur. 7.6.3.1 Interference from heterophilic antibodies in competitive methods In early competitive RIA methods, high-affinity antibodies were used, and in order to improve the sensitivity of the method, frequently sequential addition of assay reagents and a long incubation time were applied. Heterophilic interference in these methods was rarely reported in the literature, and it was shown that in RIA, heterophilic antibodies could interfere only at unusually high concentrations (46). One explanation of a low incidence of false-positive or false-negative results in competitive assays is that in competitive methods only one antibody is used and in comparison to noncompetitive methods, no bridging between capture, heterophilic, and labeled antibodies takes place. However, this is true only for those competitive methods in which the separation of the antigen-antibody complex (bound fraction) from free, unbound antigen is performed by using, instead of the double-antibody method, other precipitation methods. If the precipitation of bound fraction is accomplished by the addition of second antibody directed against the complex of antigen with the first assay antibody, then two different antibodies have to be taken into account if interference from heterophilic antibodies in competitive methods is considered. Another reason for lower incidence of heterophilic interference in competitive methods is that in these methods the reagent antibody of very high affinity is used, and low-affinity heterophilic antibodies have much lower opportunity to interfere with the assay reagent. In addition, if heterophilic antibodies have a property of autoantibodies directed against the analyte intended to be measured, and then radiolabeled antigen (tracer) will react with heterophilic antibodies in a similar way as nonlabeled antigen, so both antigens, free and bound to heterophilic antibody, compete with the tracer for assay reagent antibody. The latter was especially true for RIA using for the separation step dextran-coated charcoal. Low incidence of heterophilic interference in early competitive methods was also noted because laboratories were not aware of the possibility of such problems and interference was simply not noticed. The interference from heterophilic antibodies in competitive methods depends on the nature of interfering antibodies, especially on to what region of the assay antibody molecule they bind. In serum samples anti-isotypic, anti-idiotypic, or anti-anti-idiotypic heterophilic antibodies may be present. If anti-isotypic heterophilic antibodies are present, then binding at the antigenic determinants on the constant (Fc) region of murine antibody occurs causing steric hindrance. If this happens, both labeled and unlabeled antigens have no access to the assay reagent antibody and falsely elevated results are obtained, regardless of the method used for the separation of bound (antigen-antibody complex) from free (unbound) antigen. This is because smaller amounts of the complex of labeled antigen with assay antibody is formed; thus the measured signal is low, which means that the measured analyte concentration is higher (reverse relation between signal and analyte concentration in competitive methods). However, the magnitude of the


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effect of such heterophilic interference is hard to predict. If anti-idiotypic heterophile antibodies present in serum or plasma samples block the binding site on the first assay antibody, then less labeled antigen (tracer) and unlabeled antigen is attached to the assay antibody, less complex antigen-antibody is generated, and falsely elevated results are obtained, regardless of the separation method used. If anti-anti-idiotypic heterophilic antibodies directed against the specific idiotype of the assay reagent antibody are present in serum samples, either falsely low or falsely high results may be observed. This type of heterophilic antibody may bind antigen (both labeled and unlabeled) as a ligand, and the final result depends on the separation step used in the assay. In liquid-phase competitive double-antibody methods, the second antibody can or cannot recognize the antigen/ heterophilic antibody complex. If the antigen/heterophilic antibody complex precipitates, then no interference occurs. If the antigen/heterophilic antibody complex does not precipitate, the result is falsely elevated (low signal). A similar mechanism of interference caused by autoantibody against the measured analyte has already been described earlier in the text. In competitive methods designed for the measurement of antibody concentration in serum samples (immunoassay using the antigen format), binding of anti-idiotypic heterophilic antibodies to the labeled assay antibody can lead to falsely elevated results. In addition to interference caused by heterophilic antibodies described above, modern competitive immunoassay with signaling system other than using radioactive isotopes may be susceptible to interference due to possible binding of polyspecific heterophilic antibodies to the conjugate, enzyme, or other parts of the detection system, accounting for false results (47,48,49). 7.6.3.2 Interference from heterophilic antibodies in noncompetitive methods Noncompetitive immunochemistry methods are used for the determination of antigens that have at least two antigenic determinants on the molecule. Despite the initial very enthusiastic opinions on the value of these methods for the measurement of many substances of endogenous and exogenous origin, it came out that the assays using mouse monoclonal antibodies, with both simultaneous and two-step addition of capture and detection assay antibodies, are susceptible to heterophilic interference like the assays using polyclonal antibodies (43,44). In a two-stage noncompetitive method, the washing step does not completely eliminate the interference, but compromises between the elimination of nonspecific binding substances and disturbing physical interaction between the antigen epitope and the binding site on the antibody molecule. In the immunochemistry methods using automatic equipment, the washing step during the analytical procedure is often a source of error because of incomplete removal of interfering substances. The effect of heterophilic interference in noncompetitive methods should be regarded according to whether the method is homogeneous (without the separation of the antigenantibody complex from other sample constituents) or heterogeneous (the separation of the antigen-antibody complex obligatory), and whether the method is designed for the determination of antigen or antibody concentration in serum samples. Overestimation of the analyte concentration (false-positive assay outcome) due to the heterophilic antibody interference in noncompetitive methods is most frequently reported (50). However, false-negative assay results have also been noted. Interference in two-site noncompetitive methods is usually connected with the presence of natural anti-idiotypic heterophilic

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antibodies, but also interference from anti-isotypic and anti-anti-idiotypic heterophilic antibodies may occur. The possible mechanisms of interaction of heterophilic antibody with the assay reagent antibodies in noncompetitive assay have been proposed by Klee (51). The outline of this proposition is given below in more detail. 1. Nonspecific heterophilic antibody cross-links the capture and labeled antibody by binding to isotypic determinants expressed on the Fc portion of both antibodies. Two possible situations should be regarded: (a) interference if the analyte is absent from the sample, and (b) interference if the analyte is present in the sample. When the analyte is absent, a typical “bridge” is formed between antibodies, and a falsepositive result is obtained. When the analyte is present, false elevation of the result is seen. 2. Nonspecific heterophilic antibody binds to capture antibody in such a way that the binding of antigen by capture antibody is sterically inhibited (steric hindrance), and the complex of antigen with antibody cannot be formed; thus the label antibody cannot be attached. As a consequence, the result is falsely low, because the measured signal is low. 3. Anti-idiotypic heterophilic antibody binds to antigen-combining site on capture antibody. If this happens, the smaller number of binding sites is accessible for the measured antigen, few “sandwiches” are formed, and the result of the analyte measurement is falsely low (low signal). 4. Anti-anti-idiotypic heterophilic antibodies present in the sample behave like capture immunoassay antibody, and both antibodies compete for the measured antigen. In such a case, binding of analyte to heterophilic antibody means that less analyte binds to capture antibody, and falsely low result values are obtained. It has to be kept in mind that heterophilic antibodies can bind and/or block the solidphase capture antibody (homogenous and heterogenous assays) or can bind and/or block the assay-labeled antibody (homogenous assays) as well as the analyte in the case of anti-anti-idiotypic heterophilic antibodies. The final result of analyte concentration measured in the sample depends on characteristics of the disturbing molecules. If the presence in serum or plasma samples of heterophilic antibodies directed against different fragments of assay antibody molecules cannot be excluded, their influences on final measurement results may not be even possible to predict. As was already mentioned above, the effect of heterophilic antibodies present in the sample on final result of immunoassay measurement depends on assay format. The simplified comparison of heterophilic interference in competitive and noncompetitive methods is presented in fTab. 7.1. It is obvious that the measurement of the analyte concentration in the presence of heterophilic antibodies may give totally incomparable results on two different immunochemistry platforms with different assay formats. The discrepancy between results is not easy to explain, and decisions as to which result is correct must take into consideration the clinical status of the patient. However, comparison of two methods having the same assay format may give comparable, although erroneous, results if heterophilic antibodies affect the assay antibody and thus assay reaction in similar way. Such a situation is very dangerous for the patient, because in the


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Tab. 7.1: Mechanism of heterophilic antibody interference in competitive and noncompetitive immunoassays. Type of heterophilic antibody

Mechanism of heterophilic antibody interference

Result of competitive method

Result of noncompetitive method

Anti-isotypic

Bridging capture and signal antibody

Not applicable

Falsely elevated (antigen present in sample) Falsely positive (antigen not present in sample)

Anti-isotypic

Binding to the assay antibody and blocking the access of the measured antigen to the antigen-combining site on the assay antibody by steric hindrance

Falsely elevated, regardless of the method used for the separation of bound and free fractions

Falsely decreased

Anti-idiotypic

Binding to the antigencombining site on the assay antibody and blocking the binding of antigen

Falsely elevated

Falsely decreased

Anti-antiidiotypic

Competition between the heterophilic antibody and the assay antibody for the measured antigen

Falsely decreased, if second antibody precipitates antigenheterophilic antibody complex Falsely elevated, if second antibody does not precipitate antigen-heterophilic antibody complex

Falsely decreased

case of lack of clinical symptoms or when nonspecific clinical symptoms are present, misdiagnosis and mistreatment may affect the patient’s safety. 7.6.4 Measurement of HAMA in serum sample Reagent kits designed for the determination of HAMA by immunofluorescence, immunometric ELISA methods, or dot blotting are commercially available. Both isotypic and idiotypic HAMA can be determined. Because HAMA may differ significantly among patients in specificity (idiotype or isotype) and class of immunoglobulin (IgA, IgG, IgM), the comparability of the available methods is poor, and frequently the final result of HAMA measurement depends on the assay format. One cannot be sure whether the

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method measures IgG, IgG subclasses, or IgM. Lack of standardization of the methods for HAMA determination is also the reason for poor comparability of different assay results (32). Despite the accessibility of commercial reagent kits, the determination of HAMA is also difficult because of heterogeneity of these circulating antibodies related to idiotypic configuration of the mouse antibody. As for many immunoassays designed for the measurement of heterogeneous antibodies, also in the case of HAMA measurement, the binding characteristics depend on both circulating antibody concentration and its affinity. Therefore, it has to be realized that similar signals are obtained for lowaffinity antibodies present at high concentration and for high-affinity antibodies present at low concentration. Interpretation of HAMA titers requires caution as the titer does not always correlate with the degree of interference in various immunoassays. From a practical point of view, the measurement of HAMA in the patient’s sample is not always useful in predicting the interference in analyte measurement by any kind of assay format.

7.7 Detection of heterophilic antibodies in the patient’s sample For anyone using immunochemistry methods for the determination of different plasma proteins/peptides or other analytes, it is important to be aware of and to suspect the possibility of interference coming from various sources, which may cause falsely low or falsely high results to be obtained in every patient sample. The more laboratory professionals are conscious of the fallibility of immunoassay techniques and suspect the possibility of interference, the more frequently and unexpectedly different types of interference in everyday work are found. In practice, always the mismatch between the laboratory result and clinical situation concerning the patient status strongly indicates, after excluding other preanalytical and postanalytical errors, the possibility of interference, which should never be ignored. Extremely low or extremely high results of analyte concentration are very easy to find out, and such results are usually retested by a laboratory before acceptance and reporting to physician, but no one does any additional analytical work if the results of analyte concentration are within the reference interval, slightly below or slightly above the normal. Finding the presence of heterophilic antibodies in the sample is not simple, because there are so many different assay formats of the competitive and noncompetitive types used by laboratories all over the world, and no analytical algorithm for the detection of interference applicable to all assays and all analytes can be introduced. For each assay, the affinity, specificity, and species origin of reagent antibodies, detection systems, as well as the conditions under which the reaction between antigen and antibody is performed are different. It has been reported that a priori screening for the detection of interference does not improve the quality of the obtained results and is not recommended (52). There is no simple laboratory preanalytical procedure that could improve the quality of immunochemical determination through the detection of the possible sources of interference from heterophilic antibodies. However, if there is a high probability of the presence of interference, the laboratory has several uncomplicated practical solutions that may help to confirm or disprove the possibility of interference from heterophilic antibodies. These solutions are as follows: (a) retesting of the same specimen by the laboratory; (b) obtaining a new specimen from the patient and retesting; (c) making the

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dilution test; (d) loading the specimen with the standard and testing recovery; (e) testing the specimen by using a different immunochemistry method or alternate technology; (f ) treating the specimen with PEG or with heterophile blockers and retesting; (g) checking the relationship between hormones, if one does exist; (h) extracting the analyte from the sample, redissolving in zero diluent, and retesting. All of the above procedures have, however, some limitations. The reagents used to test the patient sample for the presence of heterophilic antibody causing interference can themselves be a source of variable interference in some assay systems. Also, no standardization for each analytical procedure is available. Therefore, always the validity of the method used to look for interference as well as the expected range of values or direction of result changes should be checked for each assay (50). The details of the abovementioned procedures are described below. 7.7.1 Retesting the same specimen Repeating the immunoassay measurement of the analyte using the same patient’s sample is almost always performed by laboratories in the case of a questionable result of analyte measurement, but unless there were some evident errors during the preanalytical phase of the procedure, like mislabeled patient specimen or improper sample storage, it does not help much for proving the presence of interference. Retesting the same specimen is reasonable only in cases when the sample tube was contaminated or the used coated assay tubes/wells were of poor quality. Repeating should also be performed when in the course of the determination there was a sudden change of incubation temperature or intentional shortening of incubation time, even a short break of electric current supply or disclosed error of sample identification. However, the majority of the results obtained during retesting have rather similar values, especially with good quality of immunoassay reagents and good quality assurance system. Frequently, in such a situation laboratory is erroneously convinced of correctness and accuracy of the measurement system for a given analyte. Of course, this concerns the situation when the repeated determination is performed using fresh, unfrozen samples of serum with a short period of time elapsing from the first measurement. Repeating the analyte determination using the same patient sample that had been frozen may lead to the discordance of results between the two measurements. Freezing/thawing of serum samples frequently results in structural changes of proteins or peptides present in the samples due to the proteolytic degradation. This concerns not only the measured protein or peptide, but also other proteins present in the serum or plasma samples. The freeze/thaw process may be one of the reasons causing interfering heterophilic antibodies present in the sample to be precipitated or partially degraded. Sometimes the immunoassay determinations of the same analyte in the centrifuged and noncentrifuged thawed serum sample may give discordant results. One of the recommendations of good laboratory practice is working out univocal proceedings for every immunochemistry test that requires retesting. However, the author’s own experience with the measurement of A-fetoprotein (AFP) in pediatric populations is against repeating the measurement of the analyte in the same sample when immunoassay platforms are used, unless there is a strong suspicion of laboratory preanalytical error. Retesting the same sample before it is frozen usually gives a similar result, unless the measured analyte concentration is extremely low or extremely high, but even then repeats give the same order of analyte concentration and do not change

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the reported result by much. Repeating the measurement of analyte concentration using samples that were thawed at least once may cause frustration for clinical chemists because the results obtained for the same sample may differ significantly each time the determination is performed. The general problem is that there is no consensus concerning the methodology and criteria used to evaluate the level of agreement between the original and repeated results obtained by immunoassay measurement. For manual immunoassays, like RIA, retesting the specimen with the suspicious result is necessary if the determinations are performed as singles because pipetting, manual mixing, and washing steps are very prone to analytical errors. 7.7.2 Obtaining a new specimen and retesting Getting a new specimen from the patient may be sometimes impossible, especially for nonhospitalized patients, and even if it is possible, it causes delays in final reporting of the result to the physician and, in consequence, delays in diagnostic procedures and introduction of proper patient treatment. Nevertheless, repeating the measurement of the analyte in a fresh sample is often necessary for confirmation of the questionable immunoassay result. Frequently, results obtained for two samples taken from the same patient at different times show almost perfect agreement; however, this does not prove the reliability of the analytical measurement. Usually, similarity of patient results are observed when the presumed concentration of interfering heterophilic antibodies in the blood does not change during the period of time separating the drawing of the two blood samples. When the concentration of heterophilic antibodies or other interfering substances (like drugs or other substances taken intentionally) changes between drawing of the two blood samples, the results may differ significantly. In such a situation, it is difficult (or sometimes almost impossible) for laboratory professionals to decide which result is correct and which one should be reported to the physician. Then the third measurement in a subsequent sample is usually performed. The time-related change in the concentration of some analytes (e.g., protein hormones or steroid hormones) may be related to many preanalytical factors (diurnal rhythm, stress, diet, therapeutic drugs) and not to interference. However, based on the practical experience of many laboratories, it is recommended to repeat the measurement of the analyte always in a freshly taken blood sample when there is discrepancy between the result of the determination of analyte concentration and the clinical condition of the patient. But is has to be kept in mind that either discrepancy or agreement between the results does not indicate nor prove the presence or absence of interference from heterophilic antibodies. 7.7.3 Dilution test Dilution test (doubling dilution or fractional dilution) using appropriate sample diluent or “analyte free” serum is frequently used to check the presence of interfering substances in a biological specimen. Patient samples can be automatically diluted by the measurement system or can be prepared manually. The final concentrations of the analyte in diluted samples are calculated by applying the appropriate dilution factor. In order to visualize the results, the measured analyte concentration is plotted versus the expected result (“true result”). Nonlinearity obtained in the dilution test is suggestive for the presence of interfering substances in serum samples (fFig. 7.6). The dilution test is


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Fig. 7.6: Dilution test results obtained for sample containing interfering antibodies and for normal sample.

very simple and relatively inexpensive, but sometimes the performance of the dilution test is incorrect due to a low precision of pipetting, wrong calculation of dilution factor, or when alcoholic solution is diluted with water, as is the case in dilution procedures of some standards (e.g., steroids). The dilution test is frequently used in searching for interference from heterophilic antibodies present in patient samples in immunochemistry measurements. However, if the interfering antibodies are present in the sample, the interpretation of the dilution test data for analytes measured by immunochemistry is not always unambiguous. The results of analyte concentration measured by immunoassay obtained for the diluted samples depend on several factors. The most important questions to consider are what is being measured (antigen or antibody concentration), what is the immunoassay method format (competitive or noncompetitive), what is the affinity/avidity of immunoassay capture and signal antibodies, and what is the affinity/avidity of interfering heterophilic antibodies present in the patient sample. With each dilution of the plasma sample, the concentrations of both analyte and natural interfering antibodies (or other interfering molecules) decrease to the same extent. Although the ratio of the concentrations of the analyte and the interfering antibodies is the same for diluted and undiluted samples, the lower concentration of salt and other sample components in the diluted sample causing change in the ionic strength may have an impact on the obtained result of the measurement. It is known that the reaction between antigen and antibody, the formation and dissociation of the antigen-antibody complex depends, among other factors, on ionic strength; thus the course of immunoassay reaction for diluted and undiluted sample may differ. On the other hand, interfering heterophilic

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antibodies may influence the antigen-antibody reaction mostly in a nonspecific (antiisotypic heterophilic antibodies) way or specific (anti-idiotypic or anti-anti-idiotypic heterophilic antibodies) way, and their effect depends not only on affinity and avidity, but also on their concentration. For any immunoassay measurement, the concentrations of capture and signal antibodies in the reagent mixture remain unchanged because they are not being diluted, whereas the titer and the concentration of the interfering natural antibodies are different in diluted and undiluted samples. Therefore, the final results of immunoassay obtained for undiluted and diluted samples may have (after dilution factor correction) either similar or different values. The interfering heterophilic antibody in patient samples, which has comparable or even higher affinity than the assay reagent antibodies, probably will influence the assay antigen-antibody reaction in the diluted sample, and the obtained result will be falsely low if after dilution the titer of low-affinity interfering antibodies remains in excess. If, after sample dilution, the concentration and titer of high-avidity interfering antibodies become insignificant, then no interference in the measurement will take place and an increase in the measured analyte concentration, as compared to undiluted sample, will be observed (53). In a noncompetitive immunoassay, two assay antibodies (capture and signal) are used. In this assay format, nonspecific heterophilic antibodies can bind to capture antibody and block the binding of the antigen partially or completely, and if no binding with signal antibody occurs, a falsely low result can be expected. It is also possible that heterophilic interfering antibodies (anti-idiotypic heterophilic antibodies) cross-link the capture antibody and signal antibody, and a falsely high result is obtained. The direction of changes in analyte concentration after dilution depends on the valence of antibodies (2 for IgG and 10 for IgM) and the concentration of heterophilic antibodies in the diluted sample. It has to be taken into account that the concentration of heterophilic antibodies after dilution may be still very high; thus linearity in the dilution test is likely. The same is true if low-affinity antibodies of IgM class are present in the sample. The dilution test may also be ineffective in the case of a low concentration of high-avidity heterophilic antibody. Linearity in the dilution test does not guarantee accuracy of the immunoassay determination of many analytes and does not indicate for sure, as was discussed earlier, the absence of interfering antibodies. A common practice in the laboratories is subjective assessment of linearity based on only one or two dilutions of sample. Such a practice should be discouraged. The results of the determination of a given substance, both before and after the dilution, are not absolute and might be burdened with errors. If the total error of the measurement is 4%, then when the true value amounts to, for example, 100 ng/mL, each value between 96 and 104 ng/mL will be correct. After repeating the dilution of the sample, the theoretical result equal to 50 ng/mL will be burdened with the same error. Every dilution experiment is subjected to both pipetting and measurement errors. Manual plotting of the relation between the theoretical and the obtained concentration without precise mathematical approach must be burdened with a considerable error. Rarely, the laboratory collects all the results of the dilution test performed by using the same method for a given analyte in samples originating from different patients. Based on a few results obtained for diluted samples, it is possible to obtain an almost ideal relationship between the calculated and the measured values. Generally, visual assessment of the scatter plot is difficult, and statistical tests are recommended. Assessment of linearity in the dilution test based on the calculation of the correlation coefficient (r) should not be used, because at constant standard


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deviation (as it is the case in many immunochemistry measurements) r increases with concentration range. Therefore, each laboratory performing the dilution tests for analytes measured by immunoassays should specify its own acceptance criteria for linearity based on a sufficient number of measurements in diluted samples. This means that all results obtained for the diluted and undiluted samples within the broad range of concentrations of a given analyte should be elaborated by applying proper graphical and statistical methods. The effect of sample dilution on the measurement of antibody concentration by immunochemistry method has to be considered separately, because the situation is different as compared to the measurement of concentration of antigen in the diluted sample. In the case of antibody measurement, the affinity of antibody in the patient’s sample must correspond exactly to that of the antibody present in the assay calibrators. After sample dilution, the concentration of all plasma constituents (including ions) as well as of the measured antibody decreases, but the reactivity of antibody after dilution depends on its affinity. If the patient’s sample contains high-affinity antibody, then after the dilution, a higher concentration value should be expected. If the patient’s sample contains lowaffinity antibody, lower antibody concentration after the dilution will be seen. Such an effect of sample dilution on antibody measurement in the patient’s sample is expected regardless of the presence or absence of interfering heterophilic antibodies. However, the presence of heterophilic antibodies in the patient’s sample may change the results in an unpredictable way, due to differences in affinity/avidity of the measured antibody and heterophilic antibody, as well as to the nature of interfering antibodies. Taking into account all possibilities of interference in immunoassay coming from natural antibodies and changes in interaction between the assay reagent antibodies and antigen after sample dilution due to lower analyte concentration, it should be stressed that the presence of heterophilic interference should not be confirmed solely by the results of a dilution test. If the nonlinearity is observed in the dilution test, there is a strong possibility that the sample contains interfering antibody. However, this is not always the case, because the dilution test has been successful in detecting inaccuracy in only about 60% of samples with natural interfering antibodies (54). Sometimes samples with interfering antibodies show perfect linearity on serial (doubling) or fractional dilution, increasing the laboratory confidence in accuracy of measurement (55). Heterophilic antibody interference causes falsely high results or falsely elevated results, as is the case in noncompetitive methods when anti-isotypic heterophilic antibodies bridge the capture and signal antibodies. Anti-isotypic heterophilic antibodies also cause false result elevation in competitive methods. It has to be remembered, however, that if anti-idiotypic (noncompetitive methods) or anti-anti-idiotypic heterophilic antibodies (competitive and noncompetitive methods) are present, then falsely low results are obtained. Because of that, it is a good laboratory practice to dilute not only samples with extremely high analyte concentration but also those with very low concentration being clinically unexpected or inconsistent with other laboratory correlates or the clinical picture of the patient. It should be finally added that nonlinearity in the dilution test may originate from other causes (e.g., heterogeneity of the analyte). It is necessary to bear in mind all the time that results inconsistent with theoretical values obtained in the dilution test may suggest the presence of interfering antibodies, but this does not mean that the accordant results must be equivalent to the absence of the interfering substances.

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7.7.4 Recovery test The recovery test is used to show the ability of a given analytical method to measure correctly the analyte concentration when a known amount of the measured substance is added to serum, plasma, or calibrator. In another words, the recovery test provides information about whether the method is able to measure the analyte in the presence of all other compounds that are present in the matrix of the tested sample. The measurement of recovery is an effective assessment of testing accuracy. Unfortunately, not all laboratories perform the recovery test properly along with the appropriate mathematical recovery calculation, which limits the usefulness of the test. In order to perform the recovery test correctly, the calculation of the ratio of the measured to predicted increase in the concentration should be performed. Besides assessing the accuracy of the measurement, the recovery test provides also the measure of competitive interference that may be due to known and unknown compounds present in the patient’s sample. The recovery test also has some limitations. One of them is the difference between the pure analyte added to the sample and the analyte present already in the sample. It is known that physical and chemical properties of many pure proteins or peptides in solution (e.g., solubility, secondary structure, and binding characteristics to plasma proteins) added to the sample in order to test recovery differ significantly from the properties of analyte present in any kind of biological sample. If this is the case, immunoassay reagent antibodies may not react in the same way with the analyte present in the patient’s sample and in sample loaded with pure analyte solution. This could be seen even if interfering heterophilic antibodies are not present in the patient’s sample. Also, if interfering heterophilic antibodies are present in the patient’s sample, the addition of pure analyte may change the course of reaction between antigen and antibody and, consequently, change the effect of heterophilic antibody on the final result. At least three or more basic samples spiked with three different concentrations (usually low, normal, and high) of analyte should be performed in order to calculate the recovery. The final recovery value depends on analyte concentration in basic sample matrix, the concentration of the pure analyte added to the sample, and the analytical performance of recovery test. Regardless of the presence of interfering compounds in the samples, the smaller the amount of recovery spike in relation to the analyte concentration in the basic sample, the greater the potential error in the calculated recovery. Due to the analytical measurement errors for both basic and spiked samples, calculating only one recovery value is not enough, and good laboratory practice requires recovery calculation together with 95% confidence intervals. The calculated recovery of less than 90% may indicate not only the presence of interfering substances but also incorrect calibration, as the recovery test is a practical way to assess accuracy. However, this is true for all analytes measured by immunoassay except for the measurement of antibody concentration, because spiking the sample with antibody leads to changes not only in antibody concentration but also in antibody avidity; thus the proper analytical assessment of the final recovery value may not be possible. 7.7.5 Testing the specimen using another immunoassay or with alternative technology The measurement of analyte concentration by using another immunochemical method does not mean sending and repeating the analyte determination in another laboratory.


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This is a common practice by physicians or patients whenever they have any reservation concerning the result or they want to check whether the result will be the same in another laboratory. Sending the sample for retesting in another laboratory, usually the cooperative one is also common practice of laboratory professionals. Unlike other analytical methods used in clinical chemistry, which are usually well standardized and for which analytical performance is similar on almost all biochemistry platforms in different laboratories regardless of the manufacturers, comparability of the results obtained by different immunochemical methods requires good understanding of basic principles of immunoassay. Although most physicians have certain knowledge on immunochemistry, they are usually not familiar with the analytical background of immunoassay. For most patients, immunoassay principles are practically unknown. Checking the results of immunoassay measurement in another laboratory by physicians or patients can be understandable. Repeating the immunoassay measurement on another immunochemistry system in the case of a questionable result on request of laboratory professionals without taking into account the basic immunoassay principles is not acceptable and is not a part of good laboratory practice. Therefore, if there is the necessity of repeating the determination of analyte in the same or another laboratory by using another immunochemical method in order to exclude the interference, it means repeating the determination by immunochemistry method in which the reagent antibodies (capture and/or signal antibody) originate from another species, or the method with the assay antibody from the same species but with a modified structure (e.g., antibody fragment). In other words, the antibodies used in the method being compared and in the method chosen for comparison should have different antigen-combining sites or at least the Fc portion of antibody removed. fFig. 7.7 presents schematically the principle of testing the specimen by using another immunoassay in the case of searching for interference from heterophilic antibodies present in the patient’s sample. In the chosen example, in a noncompetitive immunochemistry method murine capture antibody and signal antibody are used. If HAMAs are present in the patient’s sample, they can either cause steric hindrance by binding to the Fc portion of murine assay antibody (anti-isotypic HAMA) or bind to antigen-combining site of assay antibody (anti-idiotypic HAMA), thus blocking the access of the measured antigen to capture antibody so that the complex of antigen-antibody cannot be formed (fFig. 7.7a). This is especially true when the assay reagent antibodies have similar idiotypes as the mouse immunoglobulin injected into the patient for therapeutic or diagnostic purposes. In such a case, falsely low results can be expected. A similar mechanism of interference will be present in any immunochemical method, if capture antibody with the same antigen-combining site originating from mouse is used. In other words, although the method format used for the measurement of the concentration of a given analyte is important, the primary cause of the interference from heterophilic antibodies of the HAMA type is the interaction of HAMA with the capture antibody originating from mouse but not from other species. Repeating the measurement of analyte concentration by using immunoassay with the capture antibody originating from another species or antibody fragment (fFig. 7.7b) will eliminate the problem of interference caused by the presence of HAMA, because the measured antigen from the patient’s sample has unblocked access to the assay reagent antibody. Blocking signal antibody or both antibodies by heterophilic antibodies may be regarded similarly, and as a consequence, a falsely low analyte concentration will be obtained due to the

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Fig. 7.7: Measurement of analyte by two different assays. (a) In assay with mouse capture antibody, the presence of human antimouse antibody (HAMA) blocks the binding of antigen to capture antibody causing falsely low results. (b) In assay with rabbit capture antibody, the presence of HAMA does not interfere with antigen measurement and accurate results may be obtained.

lack of possibility of forming a typical “sandwich”. Interference from heterophilic antibodies may also concern the enzyme used as label, causing a decrease or complete lack of signal and, consequently, a falsely low result. In such a case, immunochemistry platform with other signal measurement system should be chosen for investigating the interference from heterophilic antibody. If the same assay antibody is used in competitive (method being compared) and noncompetitive methods (used for comparison), the presence of anti-isotypic or anti-idiotypic heterophilic antibodies gives totally different results, both being erroneous. Thus, again, before retesting the immunochemistry measurement in another laboratory or using another immunochemistry system, laboratory professionals should have exact information about the species of origin and specificity of reagent antibodies used in the immunoassay chosen for retesting the questionable immunochemistry results. Unfortunately, the detailed characteristics of capture and signaling antibodies are not always specified in the assay instruction manual, because the information concerning antibodies may be commercially sensitive. Nevertheless, even


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incomplete information about assay reagent antibodies helps to decide which assay should be used for retesting the samples with questionable results. Commonly in everyday routine work, if there is the necessity to compare the analyte measurements by using two different immunoassays, nobody asks questions about the assay format or origin of assay reagent antibodies used. Usually, a common terminology, like “antibody method” (RIA or ELISA), or manufacturer’s name is used by laboratory professionals and physicians to characterize the method used for the analyte determination. It is obvious that RIA and ELISA methods differ in signal measurement system and may also differ in assay format and details in the analytical procedure, but they may use the assay reagents (i.e., capture and label antibodies) originating from the same species. What is more, exactly the same capture and/or signal antibody is frequently used in two methods, as is the case for different assays for the same analyte launched by the same manufacturer. Sending out the patient’s sample for retesting to another laboratory without knowing what are the basic assay reagents in each of the two immunoassays used for comparison is not enough for good laboratory practice. It is necessary to bear in mind that both the method being tested and the method used for comparison may have the same susceptibility to heterophilic antibody interference or that both are interference resistant. In such a case, the comparison of the results obtained by using two methods is not helpful in detecting interference, and obtaining similar results by both methods does not prove the accuracy of the measurement and interference cannot be excluded. Unfortunately, similarity of immunoassay results obtained by two different immunochemistry methods in two different laboratories usually convinces the physician that the measurement is accurate, and without taking into account that both results might be erroneous, the patient’s safety could be jeopardized. The determination of the analyte by using another technology, such as liquid chromatography/mass spectrometry (LC/MS), if applicable, usually provides the answer to the question of whether the determination by immunochemical method was burdened with errors due to interfering substances present in the patient’s sample. Most immunochemistry laboratories perform great number of tests everyday for clinical purposes without any interest in a scientific approach to solve the problems connected with inappropriate measurements. A great variety of analytes can be measured by immunoassay, but at present only a few of them can be quantified by using LC/MS. Only specialized laboratories possess such equipment and employ highly qualified personnel able to use it. Furthermore, laboratories having LC/MS equipment at their disposal encounter also many problems with the measurement of different analytes, and the results are not always error free. LC/MS quantification of protein is challenging. Before chromatographic separation, protein cleavage by enzymatic proteolysis into small fragments is performed in order to obtain smaller fragments having amino acids sequence characteristic for the precursor protein. The separation of smaller fragments simplifies the quantification process. However, complexity of serum or plasma samples, the amount of protein needed for complete digestion prior to analysis, and interference from peptides homologous in immunoaffinity procedure are still the obstacles for the proper protein measurement in the patient’s sample (56). Both immunoassay and mass spectrometry are affected by polymorphisms, posttranslational protein modification, and the existence of multiple protein isoforms present in plasma. Although new solutions are needed to improve the measurement of many analytes, for the time being mass spectrometry is difficult to apply

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for routine measurement of huge numbers of samples, and besides, this technology is also not error free. 7.7.6 Checking the relationship between hormones, if one does exist The plasma concentration of some hormones has a direct relationship with that of another hormone, if the two hormones are linked together by negative- or positivefeedback regulation. For example, with low plasma FT4 concentration, an elevated TSH level can be expected. Also, a high level of cortisol should be accompanied by a low concentration of ACTH. Checking the relationship between the measured hormone and the metabolite regulated by the hormone could also be useful. If the expected relationship between the two physiologically linked analytes is not seen, after ruling out poor compliance with a hormone replacement regimen or other possible causes, assay interference should be suspected. However, if heterogeneous proteins are measured, the lack of relationship between the two hormones linked by feedback may be due to an increased level of one of the isoforms of protein that differs from the main form not only in biological activity but also in immunoreactivity. This is especially true for glycoprotein hormones, (e.g., TSH) in many pathological conditions. 7.7.7 Extracting the analyte from the sample, resuspending in zero diluent, and testing In older immunoassays, the extraction of some analytes (e.g., steroids) from blood serum was necessary prior to the measurement by immunoassay. From the analytical point of view, extraction procedure is technically simple, but treatment of the sample with organic solvent, evaporation, and dissolving of dry residue in the assay buffer or diluent all add to the total error of the measurement. Without a fixed and standardized protocol of the extraction procedure, the final result may not be reliable. Many immunoassays nowadays are fully automatic, and no extraction procedure is available for any analyte determined on immunochemistry platform. On the other hand, if the extraction is performed manually prior to the immunoassay, the matrix of the extracted samples differs significantly from the matrix of native samples for which immunoassay was tailored. Thus, any analytical manipulation with the sample before immunochemistry measurement may have an important effect on the interpretation of results. 7.7.8 Effectiveness of detection of heterophilic antibodies No matter how the laboratory will be searching for interference, each detection method will allow only arriving at the conviction that heterophilic antibodies might be present in the patient’s sample, but at present no method exists to prove definitely the absence of interference. In other words, it is the demonstration of the existence and not the exclusion of interference that is looked for. All the tests described above could not provide proof that a given immunoassay is working properly and provides reliable results. One should be aware that neither a normal result of the dilution test nor confirmation of the result of analyte determination by using another immunochemistry method provides the basis for the conclusion that a given immunoassay is free of interference. On the basis of the current knowledge about immunochemical methods, it seems that the minimization of the number of false-positive or false-negative results will be difficult without finding

7.8 Methods used for removal or inactivation of interfering heterophilic antibodies

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solutions making possible standardization of prelaboratory and laboratory preanalytical handling of samples of biological material separately for each determined molecule. It is not at present possible to avoid unexpected and unpredictable interference from heterophilic antibodies affecting immunoassay results. The immunoassay results considerably exceeding the method-related reference interval for a given analyte or those not matching the clinical condition of the patient are usually checked by laboratory, but the results being within normal reference intervals rarely are repeated in the course of everyday routine and are reported as such to the physician. There is no evaluation in the literature of the problems showing how many “normal” results obtained by immunochemistry are within the reference intervals due to interference by heterophilic antibodies and how many of them delayed the proper diagnosis and treatment of patients.

7.8 Methods used for removal or inactivation of interfering heterophilic antibodies The determination of HAMA concentration in the patient’s sample is useful when looking for interference from heterophilic antibodies. However, the problem does not rely on how to measure heterophilic antibodies, but mainly how to eliminate them prior to the measurement of a particular analyte by immunoassay. When any preanalytical procedure for removing interfering heterophilic antibodies is effective, the determination of interfering antibodies is not necessary, especially when the characteristics of heterophilic antibodies in the patient samples is largely unknown. Unfortunately, this is not as simple as it looks, because it is not always clear whether the interference is caused by low-affinity or high-affinity heterophilic antibodies and no information about the type of interfering antibodies (anti-isotypic, anti-idiotypic, or anti-anti-idiotypic) is usually available. There are not many methods available enabling the elimination (or reduction) of interference from heterophilic antibodies. It is possible to remove or to inactivate interfering antibodies by (a) precipitation of the interfering antibodies with PEG, (b) use of agents blocking the heterophilic antibodies, (c) use of antibody fragments instead of intact antibody molecules (in the case of in-house immunoassays), and (d) use of different buffer additives. Each of these approaches increases the cost of the immunoassay determination and causes delays in obtaining the timed results of the measurement without certainty that the interference will be eliminated or reduced. However, as interference from heterophilic antibodies may affect practically every immunoassay measurement, each laboratory using immunochemistry methods should have appropriate protocols for analytical procedures to be applied in the case of suspicion of interference. Some details on the methods available for this purpose are given below. 7.8.1 Precipitation of immunoglobulins with PEG Immunoglobulins present in patient samples can be removed by simple treatment with PEG, which changes the properties of immunoglobulins, including their stability and immunogenicity (58). After the addition of PEG solution to serum, the sparsely soluble complexes of immunoglobulins with PEG are formed. These complexes may be separated from the mixture of serum plus unused PEG excess by centrifugation. Precipitation

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efficiency depends on the class and concentration of immunoglobulins in the sample. PEG at the final concentration of 125 g/L precipitates most immunoglobulins of class IgM and IgG, and approximately 70% of immunoglobulin of class IgA, but besides immunoglobulins, also other sample constituents may be precipitated. Sometimes coprecipitation of the analyte intended to be measured, especially large glycoproteins, may occur (58,59). Although PEG precipitation is very useful for removal of serum immunoglobulins, including heterophilic antibodies, coprecipitation of analytes is one of the main limitations of this method in practice. It has to be also kept in mind that the addition of PEG causes sample dilution; thus the appropriate dilution factor should be used to calculate the final immunoassay result. Use of PEG precipitation as a preanalytical procedure in routine laboratory testing improves the accuracy of immunoassay measurement, but it does not remove the interference from heterophilic antibodies completely. Besides, no automation of PEG precipitation procedure is available on any immunochemistry platform, and the presence of PEG in the sample causes a positive interference to varying degree (60). This obstacle limits the use of this method as a standard procedure for those immunoassay determinations in which heterophilic antibodies or complexes of immunoglobulin with analyte interfere with the measurement. Recently, the use of reference intervals derived by the use of identically PEG-treated sera from healthy subjects for the measurement of prolactin concentration was recommended (61). This may be a good solution if complexes of analyte with immunoglobulins are present in patient samples. However, reference intervals obtained by such an approach cannot be a good solution for the determination of analytes in samples where heterophilic antibodies are present. 7.8.2 Blocking the interfering antibodies Using the heterophilic antibody blocking tubes before the analyte measurement by immunoassay is another simple technique aimed at the reduction or elimination of interfering antibodies present in the patient’s sample. Heterophilic antibody blocking tubes contain immunoglobulins appropriate for binding human heterophilic antibodies; thus using these tubes helps to reduce or remove interference caused by the majority of these interfering antibodies. Heterophilic blocking tubes, as summarized in two comprehensive review articles, have been shown to block between 75% and 100% of interfering antibodies (32,36). Heterophilic antibodies can be blocked by passive blocking methods, that is, by using nonspecific substances (e.g., mouse IgG, aggregated IgG, or nonspecific monoclonal antibodies), and the effectiveness of such blocking depends on the affinity of heterophilic antibodies present in the patient’s sample. Another way to block heterophilic antibodies in serum samples is to use a reagent causing steric hindrance after binding to interfering antibodies. After such blocking, the heterophilic antibody has no access to idiotype of monoclonal assay capture antibody. Heterophilic blocking reagent (HBR) produced by some manufacturers is a specific binder that is directed against human heterophilic antibodies. When using this regent, the blocking is accomplished by steric hindrance. Use of reagents blocking heterophilic antibodies may help to overcome interference, but it is possible to block only such substances which are a known “enemy”. It is difficult to eliminate interference caused by unidentified heterophilic antibodies of unknown characteristics. Changes, either increase or decrease, in the concentration of the measured analyte after blocking the interfering

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antibodies indicate the presence of heterophile antibodies in the sample. Unfortunately, this method does not always allow confirming with certainty the absence of interference. Adding the sample to HBR-containing heterophilic blocking tube is a convenient way of removing the interfering antibodies and verifying the patient’s result obtained for a serum sample not treated by any blocking reagent. HBR is routinely used to block heterophilic interactions with assay’s reagent antibodies in immunochemical methods (400 μg/mL is the recommended concentration for blocking the interfering antibodies). However, an insufficient amount of HBR may cause the enhanced interaction between heterophilic and assay antibodies instead of blocking the effect of heterophilic antibodies. This means that if HAMA concentration in the patient’s sample is high and the amount of HBR is too small, the effect of interference may be stronger and the result may be higher than before blocking (33). It was reported recently that an assay using animal antibody complex (goat/antimouse antibody/mouse monoclonal antibody) could give spurious results when treated with heterophilic blocking tubes. Thus to overcome the problem of heterophilic blocking, nonmurine immunoglobulins are necessary (56). It was suggested that this phenomenon may occur because the assays containing solidphase goat antimouse monoclonal antibody complexes prohibit the use of heterophilic blocking tubes containing murine components (62). Therefore, for those assays that contain an antimouse component, it is advisable to check for false positives using other tests (63). 7.8.3 Use of antibody fragments In order to prevent interference from heterophilic antibodies, antibody fragments instead of whole antibody molecules (intact immunoglobulin) are used by manufacturers (or laboratories) as immunoassay reagent antibodies. As isotypic heterophilic antibodies bind to the Fc fragment of assay antibody, part of assay antibody with removed Fc fragment has a reduced activity toward heterophilic antibodies as compared to native IgG (64,65). This is because no binding to the Fc portion of assay antibody with steric hindrance will occur. If interfering antibodies are directed against the Fc region of assay reagent antibodies, as is the case, for instance, with RF, some other fragment of the immunoglobulin is usually used if immunoassay is intended to measure analyte in patients with autoimmune diseases. If the interfering antibodies interact mainly with the Fab region, then the use of the antibody fragment may lessen the interference but not always eliminate it. Humanization of animal antibody is also used for the reduction of immunoassay interference (66). However, interference may still occur even with immunoassays involving chimeric antibodies (67). Improving immunoassay performance and reliability can also be achieved by antibody engineering (68). Modification of assay antibodies in order to make them less prone to react with heterophilic antibodies is applied in many immunoassays. The combining of assay antibodies from murine subclasses also reduces interference (43). By having the information about the immunoassay reagent antibodies, it is easier to predict whether the assay is more or less prone to interference from heterophilic antibodies.

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7.8.4 Use of buffer additives Buffers do not cause bias in immunoassay, but pH, ionic strength, and different additives may have an effect on the conformational changes of proteins and peptides and influence the course of immunochemical reaction. Even small changes of steric structure may influence the reaction between antigen and antibody. Various immunoassay buffer additives may reduce interference from heterophilic antibodies. The identity and composition of different substances blocking heterophilic antibodies added to the assay buffers remain a sensitive matter for producers of immunochemical reagent kits and is a wellhidden manufacturer’s secret. Practically no information on this matter can be found in the literature. Frequently, small quantities of serum or immunoglobulins from an animal of the same species as used for obtaining the reagent antibodies applied in the assay are added to overcome heterophilic interference (29,36,69). Nonimmune mouse IgG is used to overcome nonspecific anti-isotypic forms of HAMA (binding to Fc portion of assay antibodies), but it does not protect from anti-idiotypic or anti-anti-idiotypic forms of HAMA. It is obvious that the amount of nonimmune animal serum necessary to neutralize HAMA present in the patient’s sample depends not only on the concentration but also on the affinity of HAMA. It has been recommended to use between 0.1 and 0.7 g/L of polyclonal mouse IgG (70,71). Nonimmunogenic globulins cannot block all interference, and sometimes immunoglobulins originating from one species are blocked more efficiently by immunoglobulins from other species (45). Although mouse IgG is most frequently used in the currently accessible noncompetitive immunoassays, bovine serum has been shown to be more efficient than murine serum in blocking heterophilic antibodies (72). It has been suggested that heterophilic antibodies in most patients are bovine IgGs originating from the ingestion of bovine meat and cow’s milk (29). This indicates the existence of specific complementary interaction between idiotypic antibodies being the main cause of interference. It has been shown that polymerized IgG, chemically aggregated IgG, or heat-aggregated IgG added to assay buffer has better ability to reduce interference coming from heterophilic antibodies (29,43,73). Unfortunately, immunoglobulins from serum of a single individual cannot block all heterophilic interference. Also pooled immunoglobulins from several species or pooled immunoglobulins from human sera are not fully effective in blocking heterophilic antibodies, because in pooled human sera the majority of IgG molecules occur as dimers with Fab-Fab binding (74).

7.9 High-dose effect (hook effect, prozone effect) The hook effect was first recognized in the mid-1970s in a homogenous single-step immunometric assay (noncompetitive assay with the excess of capture monoclonal antibody, no separation step). For samples with very high or extremely high concentration of the analyte, artificially low results of the measurement were observed (hook effect) (75). In noncompetitive homogenous format of immunoassay, all reagents – capture antibody, analyte, and labeled antibody – are added simultaneously, and the observed hook effect is primarily the effect of analyte concentration, although the characteristics of the antibodies play also a significant role. In the case of a huge excess of antigen, both capture and signal antibodies become fully saturated, and the typical “sandwich” cannot be formed. As a result, a very low signal, apparently indicating low analyte concentration, is obtained. It has to be recalled here that in noncompetitive methods, the

7.9 High-dose effect (hook effect, prozone effect)

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signal generated in the assay is proportional to analyte concentration. The plot depicting the relation between the magnitude of the signal and the concentration of analyte shows that to a certain concentration there is proportionality between the signal and analyte concentration, and the calibration curve is linear. At higher concentrations of analyte, the amount of either solid-phase capture antibody or labeled antibody could be inadequate, leading to saturation, and a plateau response is observed. Further increase of concentration above that at which all antigen-combining sites of capture antibody are occupied by analyte, cause that the proper “sandwich” cannot be formed because the excess of free analyte starts to bind to signal antibody. As a consequence, assay signal is decreasing, and the curve depicting the relation between signal and analyte concentration is descending (fFig. 7.8). The proportional relationship between signal and analyte concentration does no longer exist, and the signal related to very high concentration of analyte is the same as the signal related to low analyte concentration

Fig. 7.8: The hook effect in immunoassay: until the concentrations of assay capture and label antibodies are in excess in relation to the measured analyte, then the relation between assay signal and antigen concentration is proportional (a and b). If all binding sites on assay antibodies are saturated, then the plateau is reached (c). Further increase of analyte concentration can cause simultaneous saturation of both assay antibodies and the curve is descending (d), and the signal for very high analyte concentration is the same as for low or normal concentration (hook).

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(hook). Theoretically, such a situation is only applicable to a noncompetitive assay without the washing step before the addition of labeled antibody (homogenous assays with simultaneous addition of all reagents). However, it has to be clarified, that although in all noncompetitive methods the amount of both capture and labeled antibodies is unlimited and in excess in relation to antigen, their amount is anyway constant and fixed. Thus, in all noncompetitive assays (homogenous and heterogeneous), the assay signal no longer increases when plateau at higher concentrations of analyte is reached, because of limited amounts of reagent antibodies. Competitive immunoassay methods with limited amount of antibody and noncompetitive methods with limited but excessive in relation to antigen (unlimited) amount of capture antibody should not be confused. In all two-step immunoassays, the incidence of the hook effect is less frequent as compared to one-step assay; however, the washing step after the formation of the complex of assay capture antibody with analyte but before the addition of assay signal antibody does not completely eliminate the problem. If an extremely high analyte concentration is present in the patient’s sample, the washing step removes all analyte molecules for which there were not enough antigen-combining sites on capture antibody, and the high concentration result is obtained but without the “hook”. In such cases, most laboratories repeat the measurement in the diluted sample in order to check the possibility of appearance of the hook effect. The dilution of the sample, either with assay diluent or with serum of normal analyte concentration, should be performed until the stable quantitative response is achieved. It means that the point has to be reached at which both antibodies are in excess in relation to antigen. However, unexpected analytical pitfalls can be observed, as the dilution always changes the sample matrix. There is no simple preanalytical laboratory procedure for all assays and for all analytes measured by immunochemistry to avoid the hook effect. Frequently, the laboratory is not aware that the concentration of analyte can be extremely high and even after diluting the patient’s sample 10 or 20 times, the measured concentration is still within the normal interval. Only after the dilution 100–1,000 times, the presence of a huge analyte concentration in the sample can be disclosed. This is true especially for the determination of tumor markers. One such example described in the literature concerns the determination of AFP in a child with hepatoblastoma (76). This liver tumor, appearing most frequently between the third month and the third year of life, secretes huge amounts of AFP. In the reported case, the first measured concentration of plasma AFP was barely 60% above the upper normal range. After the dilution 100 times (manually) and consecutive 100 times (by automatic diluter), the obtained value was 2,500,000 ng/mL. This example shows the scale of the problem. Therefore, when the substances that may have extremely high concentrations (e.g., tumor markers: PSA, carcinoembrionic antigen, AFP, cancer antigen-125, thyroglobulin, prolactin) are determined, it is necessary to know the value of the concentration above which the hook effect may occur (usually provided by the immunoassay kit producer). In the case of any discrepancy between the result of the determination and clinical diagnosis, the measurement should be repeated after diluting the sample more than usually. Assay manufacturers increase the amount of reagent antibodies to avoid the hook effect without informing the assay users, but this does not always help. Higher concentration of capture antibody in the assay only raises the threshold of analyte concentration at which it is possible for the hook effect to occur. However, if in the two-site noncompetitive immunoassay with both the capture and label antibody being added

7.10 Low-dose hook effect

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simultaneously, label antibody linked to the analyte and free analyte compete for a limited number of antigen-combining sites on capture antibody. It has to be reiterated that in noncompetitive assay, the amount of capture antibody is unlimited for the analyte concentration when the analyte concentration is within working range of the assay but limited for a huge excess of analyte. In the presence of very high analyte concentration, the amount of analyte bound to signal antibody is much smaller as compared to free analyte, and more analyte tends to bind to solid-phase capture antibody. If this is the case, the assay signal (generated after the complex of capture antibody with analyte and signal antibody is formed) decreases, and a falsely low result is obtained. It means that an increase in the amount of capture antibody does not always help to overcome the hook effect. It is not possible to predict the possibility of appearance of the hook effect prior to the assay, especially if tumor markers are measured. To avoid reporting falsely low patient results in samples where the analyte concentration is extremely high, and thus to improve the patient safety, two approaches have been applied by laboratories. Some laboratories measure the analyte in a pool of patients’ samples, usually 10 samples, and compare the obtained result with the expected average result of a batch of individual samples (77). If the concentration of analyte in a pooled specimen is higher than expected, one (or more) of the samples in the pool must have higher analyte concentration. Thus, only one extra sample being the mixture of equal volumes of batch patient samples is measured. However, if one of the samples used to make a pooled specimen contains interfering antibodies or other interfering substances, the final results may be difficult to interpret. Also, if the analyte concentration in one sample is extremely high, then the dilution with a small pool of samples could not be enough to make the assay capture and/or signal antibodies in excess in relation to the measured antigen. Other laboratories use a different approach. They dilute every patient specimen with expected very high analyte concentration and compare the result with the result obtained for the undiluted sample. When the measured concentration in the diluted sample is higher than the concentration in the undiluted sample, the hook effect in analyte measurement is possible. Such an approach doubles the cost of analysis. However, taking into account the cost of all forms of medical treatment as well as patient safety in the case of an erroneous result reported due to the hook effect, the extra cost of one additional test is meaningless and might be profitable. Depending on the type of the analyte measured by immunochemistry and its susceptibility to the hook effect, each laboratory should have adequate control procedures concerning the possibility of the hook effect in the patient’s sample, because it may grossly affect the patient’s safety.

7.10 Low-dose hook effect The low-dose hook effect is seen in competitive binding assays, and it means a paradoxical increase in the signal level generated in the assay with increasing analyte concentration. Normally in competitive assays, a decrease in signal level with an increase in analyte concentration is observed. The low-dose hook effect in the competitive binding assays is not an analyte (antigen) concentration effect as it is in the case of the high-dose hook effect in noncompetitive assays. The phenomenon of the low-dose hook effect is frequently seen in the measurement of gastrointestinal peptides as well as in ACTH determination. The low-dose hook effect, carefully studied by Fernando et al.

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(78), concerns the low concentration end of calibration curve. Currently, for the measurement of very low concentrations of analytes, like troponin, noncompetitive assays are used, and the low-dose effect is no longer the problem for the laboratory.

7.11 Interference from heterophilic antibodies and patient safety Unfortunately, there is no single universal method for detection and reduction/elimination of human heterophilic antibodies present in the patient’s sample. Depending on the type, concentration, and affinity of heterophilic interfering antibodies present in the sample, assay format, type, and affinity of immunoassay reagent antibodies, considerable discrepancies in efficiency of different methodologies used to protect the patient from getting erroneous results are observed. Besides, the reagents used to detect or eliminate heterophilic antibodies can themselves be a source of variable interference, depending on assay. Therefore, the validity of each assay and method for heterophilic antibody detection should be checked (50). The interference from heterophilic antibodies are unpredictable, and because they are related to the patient’s sample, even the best immunochemistry measurement systems, the best assay standardization, and perfect quality control assessment program do not protect from unexpected erroneous immunoassay results and thus from misdiagnosis and mistreatment of the patient. Although immunochemistry has been used for more than 50 years, an increasing amount of information on the imperfection of immunochemical methods creates unjust conviction that laboratories do not perform their work correctly. Immunochemistry is not perfect, but without immunochemistry, astonishing progress in many fields of medicine would not be possible. Moreover, no other such simple and fast alternate technology is available at present. Therefore, laboratory professionals responsible for approving and reporting the immunoassay results and physicians making the diagnosis based on these results must be aware of the fallibility of immunochemical methods. As the users of immunochemistry platforms, laboratory professionals cannot do the changes in assay format or assay reagents, but they can work out laboratory analytical procedures enabling appropriate steps to be taken with samples being the source of doubtful results due to any kind of interference. Flagging the abnormal laboratory results is a common laboratory procedure easy to perform by any kind of chemistry, hematology, or immunochemistry platforms. No such simple flagging can be introduced for samples, for which the probability of interference can influence the analyte measurement and erroneous results can be expected. Looking from the patient safety point of view, objective statistics and a scientific approach are urgently needed to minimize the number of false-positive or false-negative immunoassay results. Before this happens, as almost every scientific paper on immunoassay interference or case report on false immunoassay results point out, the best way to proper interpretation of the immunoassay results is the partnership or discussion between laboratory professionals and physicians ordering immunochemical determinations. Unfortunately, even this is not always enough to protect the patient from the negative effects of unpredictable and often undetectable interference.

7.12 Interpretation of immunoassay results is an art

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7.12 Interpretation of immunoassay results is an art In the contemporary world, more and more sophisticated equipment is used by physicians and laboratories for faster and better health care. Shortage of time and high costs of health care are the main reasons for patients’ distress – somewhere the tiny, mutual connection between physicians and patients disappears, and everyone is acting according to continuously changing algorithms and guidelines. The same is true for laboratory measurements – less and less attention is paid to the individual patient’s sample, and only the patient number ascribed for automatic analytical procedure is important for laboratory. In practice, a clinical chemist frequently does not know what the sample looks like. As Ismail (1) perfectly wrote in his review, a clinical chemist is no more the analyst but is a highly skilled “machine operator”. But no machine can be built that could replace the laboratory professional responsible not only for reporting the immunochemistry results to a physician but also for interpreting the laboratory data. It is said that medicine is an art. But can we say that the interpretation of immunoassay results is an art? Yes, in some way it is. Every patient is different; everyone has a unique biochemical blood composition in the sense of chemical constituents and their concentration. Although the majority of the blood constituents in healthy individuals are very similar, always the concentration differences between the patients related to biological variability exist. Considerable differences are seen between diseased and healthy individuals not only for molecules normally present in blood but also for unusual molecules, both of endogenous and exogenous origin. Among endogenous substances, molecules of cellular origin if damage to cells was done, and molecules synthesized and secreted by tumors should be mentioned. To this category belong also natural antibodies arising against many exogenous antigens that the human organism is exposed to during a lifetime. The interpretation of immunoassay results cannot be done without taking into account the presence of interfering substances, which may be present in every patient sample and never be identical from sample to sample. Thus, medicine is an art, but also the interpretation of immunoassay results is an art, as laboratory professionals should look at the patient’s sample as at the unique part of the patient – so when the patients differ, in the same way the patients’ samples also differ. And no legislative regulations or sophisticated analytical and technical procedures can be introduced to protect patients’ safety jeopardized by erroneous laboratory results. For the time being, there is no better alternative to the proper interpretation of possible erroneous results as good understanding of weak points of immunochemical methods and a quantitative approach to the interpretation of the immunoassay results. Otherwise, laboratories will never meet medical needs, and patients’ expectations related to proper health care.

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68. Warren DJ, Bjerner J, Paus E, BØrmer OP, Nustad K. Use of in vivo biotinylated singlechain antibody as capture reagent in an immunometric assay to decrease the incidence of interference from heterophilic antibodies. Clin Chem 2005;51: 830–8. 69. Sosolik RC, Hitchcock CL, Becker WJ. Heterophilic antibodies produce spuriously elevated concentrations of the MB isoenzyme of creatine kinase in a selected patient population. Am J Clin Pathol 1997;107: 506–10. 70. Reinsberg J, Nocke W. Falsely low results in CA 125 determination due to anti-idiotypic antibodies induced by infusion of [131]F(ab’)2 fragments of the OC125 antibody. Eur J Clin Chem Clin Biochem 1993;31: 323–7. 71. Reinsberg J. Different efficacy of various blocking reagents to eliminate interferences by human antimouse antibodies with a two-site immunoassay. Clin Biochem 1996;29: 145–8. 72. Weber TH, Kapyaho KI, Tanner P. Endogenous interference in immunoassays in clinical chemistry. A review. Scand J Clin Lab Invest 1990;201: 77–82. 73. Bjerner J. Human anti-immunoglobulin antibodies interfering in immunometric assays. Scand J Clin Lab Invest 2005;65: 349–64. 74. Roux KH, Tankersley DL. A view of the human idiotypic repertoire. Electron microscopic and immunologic analyses of spontaneous idiotype- and anti-idiotype dimers in pooled human IgG. J Immunol 1990;144: 1387–95. 75. Fernando SA, Wilson GS. Studies on the ‘hook’ effect in the one-step sandwich immunoassay. J Immunol Methods 1992;151: 47–66. 76. Jassam N, Jones CM, Briscoe T, Horner JH. The hook effect: a need for constant vigilance. Ann J Clin Biochem 2006;43: 314–17. 77. Butch AW. Dilution protocols for detection of hook effects/prozone phenomenon. Clin Chem 2000;46: 1719–20. 78. Fernando SA, Sportsman JR, Wilson GS. Studies of the low dose ‘hook effect’ in a competitive homogenous immunoassay. J Immunol Methods 1992;151: 27–46.

8 Immunoassay and patient safety

8.1 Fallibility of immunoassays Laboratory-based diagnostics could not exist without the immunoassay measurement system, the technique allowing the determination of very low concentrations of a variety of chemical compounds present in biological specimens. Although immunodiagnostics is a specialized laboratory area, it has tremendous impact on many fields of medicine, especially on endocrinology, immunology, oncology, and hematology. Continuous increase in the number of potential biological markers that could be measured by immunoassay and growing demand for already routinely performed immunoassay tests, especially hormones, tumor markers, antigens, antibodies, and drugs, forced manufacturers to develop new, or modernize the existing, fully automated and semiautomated immunochemistry platforms. Simplicity, speed, and reasonable cost of immunoassay tests performed on highly automated instruments together with good precision form the basis of universal conviction among laboratories and physicians that all immunoassays are perfect. The truth is, however, that all immunochemistry measurements are susceptible not only to errors in preanalytical, analytical, and postanalytical phases of the testing process concerning similarly all other clinical chemistry methods, but they are also plagued by errors not encountered in other analytical methods, inherently connected with the principle of the immunochemical method – the antigen-antibody reaction. Laboratory procedures aimed at the elimination or reduction of common laboratory errors are widely known and successfully used. However, there is no universal analytical procedure that could detect and eliminate the specific errors burdening immunoassay methods. The fallibility of the immunochemistry measurement system and its possible dangerous consequences for patient safety is underestimated by laboratories and practically unknown to many physicians. In practice, most laboratory technicians using immunochemistry platforms are trained only to operate the platform, load the assay reagents to the machine, and properly perform calibration, quality control procedures, and periodical maintenance. Sometimes, even laboratory professionals do not have the proper knowledge on fallibility of immunoassay methods, and they act according to strictly established laboratory procedures: check the lot number of the reagent, check the calibration curve, check the quality control system, and if all is correct, approve the patient result. If all analytical procedures are working properly, especially the quality assurance system, then the result is reported to the physician without any feeling of uncertainty. Meanwhile, the fallibility of immunoassay stems not only from the lack of proper standardization and method-dependent reference intervals or lack of comparability of different immunoassay methods, but also from susceptibility of the measurement system to interference from some components of the sample matrix, mainly heterophilic antibodies. In other words, it is not the problem of the method as such, because the whole analytical procedure is working properly. It is a problem of composition of some patient samples, and no assurance system exists to identify such suspicious samples.

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Since the introduction of the first immunoassay into medical laboratories more than 50 years ago, a numerous of scientific papers regarding the performance of manual, semiautomatic, and fully automatic immunoassay systems have been published. Most of these papers described clinical cases with documented immunoassay interference or dealt with such problems as comparison of different immunoassays or reference intervals for the analytes measured by this method. Although immunoassay interferences are relatively rare, poor performance of immunoassays and their fallibility is a very serious laboratory problem, since among many thousands of assays that are performed every day all over the world, more and more results remain in disagreement with the clinical condition of the patient. Erroneous immunoassay results grossly affect clinical decisions regarding diagnosis and treatment and consequently affect the patient’s safety. Some thought has to be devoted to the problem of who is responsible for erroneous results, which ultimately jeopardize the patient’s safety. A very thin line exists between the place where the responsibility of the assay manufacturers ends and where the responsibility of laboratories begins. From the patient safety point of view, it does not matter who is responsible for erroneous laboratory results; finally, the physician is the one who is blamed for wrong clinical decisions based on inaccurate, although several times repeated, laboratory results. The discussion on the responsibility of any single person for erroneous laboratory results obtained by the immunoassay measurement system is meaningless. More important is what to do and how to do it in order to avoid the pitfalls in analyte measurement by immunoassays. When searching the literature concerning erroneous immunoassay results with adverse effects on patient safety, one conclusion emerged: a good relationship between laboratory professionals and clinicians and immediate consultation on any disagreement between the laboratory result and the clinical state of the patient is the only way to think of and investigate the possibility of interference and solve the problem. In theory, it is a very good conclusion, and an interdisciplinary medical team solving the diagnostic problem, especially if it is connected with the patient’s safety, is the most desirable solution for everyone. However, in everyday practice, it does not look that simple. In today’s very busy, fast-moving commercial world, a physician does not have enough time to make a special arrangement for discussion on one single erroneous result. Besides, as everyone working in clinical laboratory knows, laboratory and clinical staff represent two different worlds and talk two different professional languages, frequently not understanding each other. Physicians require and expect from the laboratory reliable results and simple yes or no answers for the question, is the result normal? Such a requirement is almost impossible to meet in the case of most analytes measured by immunochemistry techniques. After many years of laboratory experience, it seems to me that in the case of disagreement between a laboratory result and the clinical picture of the patient, the discussion between laboratory and physician cannot be the only solution. In my opinion, both laboratory workers and physicians should have specific knowledge concerning the immunoassay techniques in order to make the communication on immunoassay results most productive and to protect the patient from misdiagnosis and mistreatment based on wrong results. To diminish the number of erroneous immunochemistry results arising from fallibility of immunoassay technology, the laboratory must know the assay and physician must have basic knowledge on critical points in immunoassay.

8.2 Know your immunoassay

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8.2 Know your immunoassay Laboratory workers performing the immunoassay measurement are obliged to follow the instructions attached to the assay reagent kit manual or instruction manual for the immunochemistry platform. The amount and quality of information contained in the instruction leaflets differ significantly between assay manufacturers. Some instructions have limited information concerning the assay and only the analytical procedure to follow is included. Fortunately, most assay manufacturers provide full information including principles of the method; summary and explanation of the test; list of supplies needed but not provided; the means of reagent preparation; information about specimen collection, handling, and storage conditions; the exact analytical procedure to follow together with typical data for calibration curve and calculation of the results. Information about assay performance and limitations concerning assay specificity, sensitivity, detection limit, intra- and interassay precision, accuracy determined by dilution and recovery test, hook effect (if applicable), and conversion factor for units of measurement is also included. In addition, the leaflets usually include information about the control samples contained in the assay kit and reference interval for the analyte to be measured. Assay manuals for immunochemistry platforms also contain information on onboard reagent stability and calibration intervals as well as requirements concerning master curve calibration. Some information on heterophilic antibody interference can also be found. However, there are a few problems with assay kit instructions. First, they are not always read by laboratory workers, except for the information regarding the reagent preparation and analytical procedure to follow or information about preparing the analytical system and reagent loading. Most of the manual assays contain ready-to-use reagents and require simple analytical steps, like pipetting serum sample and reagents into the appropriate assay tubes and measuring the signal. For many users of immunochemistry platforms, the instruction manual is almost unnecessary, because clinical chemists are usually trained to have knowledge of how to load the reagents into the machine, how to check the calibration curve, how to load the patient sample, and where the results will be shown up. Second, for users in many countries, the translated instruction manual is a nightmare. In most cases, the instructions were translated from English to the user’s language by linguists who do not know the professional laboratory vocabulary, and frequently it is almost impossible to understand all of the information included. The only fully understandable information used by laboratory technicians is that condensed in a table containing the volumes of assay reagents and sample that must be added to the appropriate tube and specifying the incubation time needed for the analytical procedure. Third, although the instruction manual is full of information concerning the limitations in assay performance, it cannot be transferred directly into routine assay performance without reservation. However, using the information on assay characteristics and performance supplied by the manufacturer as a base, it should be mandatory for each laboratory to check all the information on assay limitations under routine work. It is amazing how such knowledge is useful when interpreting unusual patient results. Some remarks concerning the information included in instruction manual that should be known for laboratory professionals involved in the interpretation of immunoassay results are presented below:

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1. Principle of the immunoassay and assay reagents. For good laboratory practice, it is not enough to know that the assay is of RIA, ELISA, or chemiluminescence type. Laboratory professionals should know what is the method format (competitive or noncompetitive assay), which assay reagent is labeled, and what is the basic principle of signal measurement. Species origin of capture and signal antibodies should be known. In the case of immunometric methods, the information about the murine antibody (full molecule, fragment, chimeric) could also be useful if retesting of the determination is intended to be performed by using another manual immunoassay or immunochemistry platform. Information about the species origin of reagent antibodies is indispensable when interference from human antianimal antibodies is suspected. For example, if two monoclonal murine antibodies are used, the probability of interference from human antirabbit antibodies is very low. Antibody specificity against a defined epitope is not always reported in the instructions. Besides the information about the reagent antibodies, laboratory professionals should also have information about the traceability of calibrators to international standard. Such information is necessary when two assays for the measurement of the analyte are compared. Also, the commutability between calibrators and biological specimens in which immunoassay determinations are carried out is one of the most important analytical issues, but information on this subject is rarely provided by manufacturers. In the case of assays for newly discovered biological markers, which are often used for scientific research, kit calibrators and quality control samples are formulated in buffers, causing the problem with commutability. Frequently, kit calibrators differ from primary reference, thus the interpretation of results may lead to erroneous conclusions. If no information on the assay calibration is provided, the laboratory should be doubtful about the assay quality. 2. Specimen collection, preparation, and storage. Usually, kit instructions contain the recommendation as to what kind of biological specimen is suitable for the measurement of a given analyte. Both plasma and serum from morning blood samples are recommended equally. The recommendations based on National Committee for Clinical Laboratory Standards concerning the conditions for handling and storing blood samples are added (1). From the point of view of the patient’s safety, one of the most important preanalytical issues is the stability of the analyte intended to be measured. Always the answer to the question of how quickly after blood collection the sample should be centrifuged, and, if not assayed immediately, how quickly the sample should be frozen must be known. For analytes stable at room temperature for at least 24 hours (e.g., steroids, thyroxin, IGF-I), sample processing before assay does not have a big impact on the result of the final measurement. However, for the majority of analytes, the stability is an important issue, and the laboratory should pay to it as much attention as possible while processing the blood sample. This is a difficult matter, because the laboratory usually does not know when exactly the blood was drawn, what was the temperature during the transport, and so forth. In my opinion, the recommendation to centrifuge the blood sample not later than 60 minutes after blood collection is particularly important for analytes measured by immunoassay but is not always respected. Besides, the stability of analytes can also be affected during the analytical process; therefore, for manual assays the time between the addition of assay reagent and the patient sample should be the same for all samples tested in a series. Another big issue concerning the stability of


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molecules measured by immunoassay is the freeze/thaw treatment of the sample. In my opinion, for the measurement of proteins and peptides, each sample should not be frozen and thawed more than once, even if the analyte is stable during this procedure. This is because the freeze/thaw process may cause changes also in the sample matrix, and degradation products of other molecules may affect the reaction between antigen and assay antibody in unpredictable ways. Thus, the stability of the analyte in the patient’s sample should be distinguished from the stability of potentially interfering substances in the sample matrix. Another issue concerning the analytes measured by immunoassay is long-term sample storage. Sometimes for research purposes, samples are stored for months before immunoassay measurement is performed. It has to be remembered, however, that keeping the sample even at –70°C does not protect proteins and peptides from proteolysis. It is advisable, regardless of the information included in the assay instructions or assay manual for the immunochemistry platform concerning specimen handling and storage, that laboratory professionals should work out a standardized procedure for sample processing in prelaboratory and laboratory preanalytical steps for every analyte measured by immunochemistry. 3. Analytical assay procedure. Laboratories are obliged to follow the manufacturer’s analytical assay procedure because any deviation from the recommended assay protocol is the laboratories’ risk and responsibility. However, even if the analytical procedure is followed strictly, a significant change in environmental conditions (e.g., room temperature) could still occur. Most of the manual and automatic immunoassays work properly at room temperature, but temperature in non-air-conditioned laboratories may vary from 16°C to 35°C. The detailed documentation of a quality control assurance program together with environmental temperature monitoring in order to find out any possible systematic errors, paying special attention to increasing or decreasing trends of control sample concentration, is highly recommended. 4. Calculation of results. For manual or semimanual assays, like RIA, the calculation formula is known, and for the computer-assisted methods, the manufacturers recommend mathematical function for constructing the calibration curve fitting and for calculating the result of the measurement in the patient’s sample. For fully automatic immunochemistry platforms, the mathematical apparatus is built-in and programmed by manufacturers. However, when a new assay is introduced, it is always necessary to check whether the mathematical method used for the calculation of results was properly programmed on the equipment (platforms or gamma scintillator counters). Inconsistency across laboratories in curve fitting of nonlinear immunoassay data is one of the causes of poor comparability of results between the assays. 5. Assay sensitivity and assay range. For those analytes measured by immunoassay, for which low concentrations in serum or plasma are diagnostically important, the instruction manual includes analytical sensitivity and sometimes functional sensitivity. The reagent package insert frequently states that the assay has a dynamic range extending from zero concentration to a certain value in the upper limit, but in practice, an assay is not capable of measuring concentrations down to zero accurately. Usually, analytical sensitivity provided by manufacturers is based on several times zero standard measurements and calculation of the mean value plus 2 or 3 standard deviations, which means that the limit of detection is given. This value, defining only “the ability to measure nothing” (2), is being used by laboratory professionals

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as the lowest analyte concentration the assay can measure, although the lowest detection limit measured in the same way by laboratories in routine practice usually has much higher concentration values. From an analytical point of view, the lowest concentration at which the analyte can not only be reliably measured but at which some predefined goals for bias and imprecision are met (limit of quantification) is definitively more appropriate for the interpretation of clinically important analytes at low concentration (e.g., thyroid-stimulating hormone [TSH], troponin). Functional sensitivity in instruction manuals is given as the analyte concentration with coefficient of variation (CV ) equal to 20%, or less, depending on the analyte determined using multiple patient and quality control samples in the low range of analyte concentrations. Good laboratory practice requires, however, determining the assay limit of detection and assay functional sensitivity under routine conditions; otherwise, the reported values for the patient’s sample could be misinterpreted. 6. Assay specificity. The percentage of cross-reactivity is usually provided by manufacturers in the form of a list of compounds that may cross-react with assay reagent antibodies due to structural similarities to the measured analyte. It is impossible for practical reasons to include the information about all cross-reacting substances tested by manufacturers, and also no information is provided on cross-reactivity for heterogeneous analytes in the patient’s sample under specific clinical conditions. In practice, the laboratory does not perform a cross-reactivity study, unless an unexpected cause of interference is found and the interfering molecule, for example, a drug, is known. 7. Intra- and interassay precision. All assay instruction manuals include intra- and interassay precision at the selected analyte concentrations. Information about the type of sample (standards or patient samples) used to establish the assay precision is not always given, and sometimes the within-run and run-to-run CV values obtained only for high analyte concentrations are given. In some assay manuals, the intra- and interassay precision is given for analyte concentrations covering the entire assay concentration range. However, it is highly recommended that each laboratory should establish intra- and interassay precision at a different concentration range in order to properly interpret the changes in serial measurements of analyte in patient samples, especially if the patient requires monitoring of changes in analyte concentration during the treatment. 8. Assay accuracy. The dilution test and recovery test are used by manufacturers to estimate assay accuracy. Limited information is usually given about the type of sample (calibrator or patient serum) used by the manufacturer for the dilution test, as well as for the recovery test. Although the zero calibrator or assay diluent is usually recommended for the dilution test, generally no information is given concerning the patient’s sample (normal or pathological serum). The laboratory does not always know: (a) to what extent the sample can be diluted; (b) whether the assay diluent provided by the manufacturer is the only solution appropriate for the dilution test; and c) whether the relation between theoretical concentration and the measured concentration is linear, regardless of the dilution factor. For some assays, information like “do not dilute more than four times” is given although higher dilution is needed to estimate the analyte concentration in the patient’s sample. The information about the recovery test (spiking test) as given in the instruction manuals is even more enigmatic than the information for the dilution test. Frequently,


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only the added and recovered amounts of analyte and calculated percentage of recovery are provided. From such data it is difficult even to guess whether the recovery test was performed by adding the known amount of analyte to zero calibrator or other types of sample were applied. If serum samples are used for the recovery test, the data included in assay manual concern the samples with similar, low analyte concentrations; thus it is not known what will be recovery if serum with a high analyte concentration was used. According to good laboratory practice, it is necessary to collect all dilution test and recovery test data for each analyte and to use the proper graphical and mathematical calculation for the diluted patient samples as well as for spiked samples. Having experience with accuracy tests performed not only by using calibrators but, most importantly, using patient samples taken under various clinical conditions is highly recommended. 9. Internal quality control. Each assay kit contains two or three manufacturer’s control samples with the recommendation that all should be assayed on each day that samples are analyzed. It is known to laboratory professionals that the measured concentration should remain always within the concentration range as specified on each control vial, because the range to fit in is so broad that something unusual would have to happen to miss the target. Taking into account the matrix differences between control samples supplied with the assay and patient samples, measuring such controls is not very helpful for assay validation in respect to quality control assurance. In my opinion, each laboratory using immunoassay techniques, especially manual assays, should rely on quality control samples based on serum at three different concentration levels (low, normal, high), kept frozen and thawed only once. It is important to run the control samples also when two-point calibration is performed. 10. Hook effect (if applicable). The concentration value at which the hook effect may occur should be known to laboratory professionals. Frequent checking of the information on this matter in the instruction manual attached to the newly purchased kits is highly recommended as the manufacturers may improve the assay characteristics and change the assay reagent antibody concentration. 11. Reference intervals. For each analyte measured by immunoassay, manufacturers specify the reference intervals. Usually, the following statement is included: these values are given only for guidance; each laboratory should establish its own normal range of values for the diagnostic evaluation of patient results. Sometimes the information on the number of individuals serving as the “reference group”, usually 10 to 20 (for low-quality assays), and their mean age is provided. Although the reference intervals for analytes measured by common immunochemistry platforms can be found in the medical literature, each laboratory must verify the manufacturer’s reference intervals, either by establishing their own reference ranges or by adapting the common reference intervals or manufacturer’s data to their own population. Relying only on the reference interval provided by the manufacturer does not fulfill the requirements of good laboratory practice. Laboratories should document how their reference interval for each analyte was established, even when “validating” their own reference interval. The above most important points concerning the assay manuals and kit instructions for immunoassays should be perfectly known by laboratory professionals. The statement

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“know your assay” means a full knowledge of the assay characteristics and its performance. Every single detail concerning the course of the reaction between antigen and antibody and all aspects of assay limitations may influence the interpretation of the final immunoassay results and thus influence patient safety.

8.3 Basic knowledge on critical points in immunoassay for physicians Physicians have always been very busy with patients, but nowadays they also have to face an enormous number of new or improved laboratory tests and imaging techniques, new drugs and new procedures for more efficient patient treatment, together with steadily growing paper work. In addition, they should continuously improve their medical knowledge, which is growing exponentially. In today’s fast-changing world, it is impossible to follow (and remember) all the new medical and diagnostic information coming each day from different sources. On the other hand, many of the recent innovations in the medical sciences have grossly improved patient care, patient survival, and patient safety; thus physicians should be familiar with almost everything. Continuously improved immunochemistry methods and growing possibility of measurement of new biochemical markers contribute also to the development of medicine. There are many new assays characterized by better specificity, sensitivity, and excellent performance that have been designed for the determination of the “old” analytes, adding a lot of new information to be absorbed not only by laboratories but also by physicians. Such improvements in assay characteristics frequently change the established guidelines for the management of the patient. Laboratory professionals should follow the progress in clinical chemistry, including immunoassay, and should share the new information in the laboratory science with physicians. But for any discussion, the background information on the field is necessary on both sides to bring forth a successful final conclusion for the benefit of the patient. Physicians do not need to have full analytical knowledge of immunoassay performance, but they should have basic information on critical points in immunochemistry. Such knowledge may have an important positive influence on the interpretation of immunoassay results and patient safety. These critical points are as follows: (a) analytical sensitivity, functional sensitivity, and assay specificity; (b) general problems with assay standardization; (c) interference from heterophilic antibodies; (d) interference from autoantibodies and binding proteins; (e) the hook effect issue; and (f ) reference intervals. The details of these critical points are outlined below. a. Analytical sensitivity, functional sensitivity, and specificity. Usually in a discussion between a laboratory and a physician on assay sensitivity, the physician thinks about the diagnostic sensitivity of the test and not about the analytical or functional sensitivity of the method. But willingness of both sides involved in the discussion might not be enough to solve the problem with, for example, troponin concentration measurement, because laboratory professionals and physicians talk different languages and proper understanding of the problem rarely reaches the desired level. Understanding the analytical problems concerning the measurement of very low concentrations of analyte and understanding the functional sensitivity will significantly improve the interpretation of patient results by physicians. Knowing the term analytical sensitivity

8.3 Basic knowledge on critical points in immunoassay for physicians

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of the immunoassay method, no future miscommunication regarding, for example, the measurement of CRP and highly sensitive CRP (hsCRP) thyroid-stimulating hormone (TSH) or hsTSH, troponin and highly sensitive troponin will take place. Currently, many physicians ask for “highly sensitive CRP,” and they do not realize that it is the same C-reactive protein but measured by a highly sensitive method. Also, no misunderstanding in requesting highly sensitive CRP assay for CRP concentration in patients with bacterial infection will be seen in the future. At present, the term functional sensitivity is unknown to many physicians, and most of them do not realize how important functional sensitivity is, for example, in the case of troponin concentration measurement. Specificity of the immunoassay is usually known, but physicians should know more about the heterogeneity of proteins and cross-reaction between different isoforms of proteins, especially when the presence of protein isoforms is disease related. The discussion about isoforms of the analyte, which are not measured by immunoassay but show biological activity, will not be necessary if the physician has the knowledge on heterogeneity of proteins and understands that the assay antibody may have different specificity toward different protein forms or simply that the assay antibody cannot detect the isoforms. b. General problems with immunoassay standardization. Physicians should have information on what is being measured by the assay, for example, which form of hCG or parathyroid hormone is determined by a given immunoassay method. They do not need to have a deep knowledge on traceability, although they should be familiar with the commutability problem while comparing the analyte concentration in serum and plasma samples or when the determination of some molecules in sera obtained from the patient in extreme clinical conditions is performed. In such a situation, not only the concentration of the measured analyte is extremely high but other biochemical parameters may be abnormal (very high or extremely low) and may affect the reaction between the antigen and antibody. Consequently, accuracy of the measurement may be doubtful. This is true especially for the measurement of the free analyte form, when the concentration of binding proteins and their capacity for ligand binding grossly vary in extremely clinical conditions or when high-titer autoantibodies are present in the patient’s sample. Understanding of the lack of comparability of different assays due to general lack of immunoassay standardization, and the necessity of using method-dependent reference intervals should decrease the number of samples unnecessarily reassayed in other laboratories, which definitely will decrease the cost of health care. c. Interference from heterophilic antibodies. One of the most important issues concerning interference from heterophilic antibodies is to bring the physician’s attention to this very serious problem. Physicians must be always conscious of the possibility of erroneous patient results due to interference. They must realize that interference from heterophilic antibodies is the patient sample problem and not an unintentional laboratory error. For medical professionals not involved in laboratory medicine, it is difficult to understand that immunoassay measurement systems work almost perfectly if analysis is performed on a sample taken from a healthy individual, which is free from heterophilic antibodies, but the probability of erroneous results increases when the analyzed sample contains unusual composition and concentration of some molecules, including interfering antibodies. Although laboratories perform different

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procedures to detect and/or eliminate interference from heterophilic antibodies, generally the problem remains unsolved. Therefore, everyone doing the immunoassay determinations and/or using the immunoassay test results for medical purposes (diagnosis, treatment, monitoring, etc.) must be aware of the unpredictability of interference from heterophilic antibodies. If physicians will understand and will be aware of this problem, then the discussion between laboratory professional and clinicians will be advantageous for the patient. d. Interference from autoantibodies and binding proteins. The presence of autoantibodies against the analyte measured by immunoassay and the presence in blood of specific and nonspecific binding proteins for many hormones is usually known to physicians, but they are not aware that autoantibodies or binding proteins may interfere in the immunoassay measurement of a particular analyte. In many clinical conditions, changes in the concentration of binding proteins or the presence of abnormal protein forms influences differently the measurement of total and free hormone concentrations. Similarly, the presence of autoantibodies against the analyte measured in the patient’s sample may affect the measured molecule in an analyte-dependent and analyte-independent way causing both false-positive and false-negative results. Awareness of such clinical situations is necessary to protect the patient’s safety. e. Hook effect (high-dose effect). Physicians should understand the hook effect and they should have information from the laboratory about analyte measurement susceptible to the high-dose effect, and at which level of analyte concentration the hook effect may occur. Knowing about this analyte-dependent interference, which is characteristic only for immunoassay and not for other chemistry methods, will facilitate the discussion on laboratory results not fitting the clinical picture, especially in the case of tumor markers. f. Reference intervals for analytes measured by immunoassays. Reference intervals are the most frequently used tool in making clinical diagnoses. Physician should always remember that due to the lack of immunoassay standardization, only the methoddependent reference intervals, validated for a local population, can be used for a given analyte. They should be informed when the method or immunochemistry platform has been changed and new reference intervals must be introduced. Conversion factors to recalculate the resulting value concentrations between the “old” method and “new” method or between the results obtained by two different platforms should never be introduced. This is especially true when the assay of higher sensitivity is introduced and the cutoff concentration must be changed. The question remains, who should be responsible for teaching medical students or physicians the critical points in immunoassay? It is not a question concerning only immunoassay. Medical student should be taught not only about all new laboratory technologies and methods and about the usefulness of measurement of biochemical markers in different clinical conditions, but they should also understand the advantage of new methods and possible pitfalls affecting medical decisions and patient safety. Knowing the overloaded teaching programs in medical schools, the addition of an elective course on laboratory medicine will be difficult to introduce. However, a 3-hour course to outline the majority of critical aspects of immunoassay technology that relate to the patient’s safety is nowadays necessary to introduce.

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8.4 Immunoassay in research Many biochemical markers, especially newly discovered ones, are measured by immunochemistry for research purposes. Usually, scientists pay attention only for clinical validation of the research performed but analytical validation of biomarker measurement is frequently outside their way of thinking. Such an approach should not be accepted. Scientists using immunoassay kits for research purposes should also “know the assay” in a similar way as laboratory professionals do, and, if multicenter clinical studies are performed, all aspects of assay standardization, uncertainty of measurement, assay validation, reference intervals, and immunoassay interferences should also be taken into account. Many newly introduced assays for biomarkers are not suitable for the measurement of biomarkers in some patient populations, no data exist concerning the effect of drug treatment on biomarker concentration, and almost nothing is known about the interference from different endogenous molecules on final assay results. Many conclusions based on immunoassay measurement using the immunochemistry platform of one company may not be comparable with the results obtained by another assay; thus the research data can be regarded as assay specific. Thus, good research should be based on validated assays, and all assay limitations should be considered before the final scientific conclusions are made.

8.5 Who is responsible for the patient’s safety? Immunoassays are not perfect, but no alternative method enabling simple, convenient, and fast routine measurement of such a large number of analytes, from simple molecules to highly heterogeneous proteins, is available at present. Much effort has been made to understand and overcome immunoassay problems, especially the problem of interference, even if no simple laboratory procedure that allows finding sample-specific interference exists. Awareness that any immunoassay result, which is clinically unexpected or inconsistent with other clinical findings or biochemical correlates, can result from the presence in the sample of interfering substances may help to protect the patient from unnecessary and potentially dangerous clinical intervention. Usually, samples with enormously high or very low analyte concentrations are suspected by laboratory professionals as containing the substances interfering in the immunoassay reaction. These samples are frequently retested in different laboratories using different immunochemistry platforms, and all available analytical procedures are applied in order to find out and eliminate the interfering substances or, at least, to prove that the result might not be clinically useful. Less attention is paid to those results that differ significantly from previous measurement without clinical explanation for such changes. Also, nobody is checking the results that are within the range of reference intervals, although they may be normal due to interfering substances. Such a situation may affect the patent’s safety due to possible delays in the introduction of appropriate treatment. Endless repeating of each result of immunoassay test requested by physicians is very expensive and is not possible for practical reasons. Reporting all immunoassay test results by laboratory professionals without any reservation should not take place. Thus, a reasonable approach must be the solution. The incidence of incorrect results depends on the patient’s group for which immunochemistry measurements are performed. For ambulatory patients, the patients on clinical checkup, and healthy volunteers, discordance

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between the clinical state of the patient and test results is not seen very often. But in the patients with autoimmune disease, abnormal plasma proteins, different types of cancer, receiving monoclonal antibody therapy or blood transfusion, in newborns, as well as vegetarians and individuals having contact with animal, unusual results are seen more frequently, although no detailed studies on incidence are available. However, cautiousness must concern every immunoassay measurement in the patient’s sample, regardless of the clinical state. It has to be once more stressed that erroneous results due to immunoassay fallibility of the immunochemistry measurement system are not necessarily the laboratory’s mistakes and may concern every patient sample. Therefore, both physicians and laboratory professionals should continuously improve their knowledge on immunoassay interference for better communication concerning the laboratory results because they are equally responsible for the patient’s safety.

References 1. National Committee for Clinical Laboratory Standards (NCCLS). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. 2nd ed. NCCLS Document H18A2. Wayne, PA: NCCLS; 1999. 2. Needleman SB, Romberg RW. Limits of linearity and detection of some drugs of abuse. J Anal Toxicol 1990;14: 34–8.

Part II Immunochemistry measurements in practice: Examples of problems in some current immunoassays

Example 1 Parathyroid hormone (PTH) – Heterogeneity as a major problem in PTH measurement by immunochemistry

PTH is the primary hormone regulating bone and mineral metabolism. Acting in concert with 1,25-dihydroxyvitamin D, it participates in both calcium and phosphate homeostasis. Biologically active, intact PTH (amino acids 1–84) and inactive fragments containing the middle and carboxy-terminal amino acid fragments are secreted by the chief cells of the parathyroid glands into the circulation. An additional source of inactive fragments of PTH is the peripheral metabolism of intact PTH mostly in the liver and kidneys. It has been demonstrated that PTH fragment (amino acids 7–84) plays an important role in calcium homeostasis having some inhibitory properties (1). In addition, based on HPLC fractionation, an amino-terminal form of PTH (N-PTH, “amino” PTH, atypical PTH) distinct from PTH (1–84), but having immunologic similarity, was identified in patients with severe primary and secondary hyperparathyroidism (2,3,4). From the very first PTH assay, heterogeneity of circulating PTH was a big challenge for the immunoassay measurement of this peptide with satisfactory accuracy. The first generation of PTH assays comprised the competitive assays using a single antibody recognizing the middle (amino acids 44–68) and C-terminal (amino acid 53–84) regions of PTH. These assays had a poor analytical sensitivity, and it was impossible to distinguish between low and normal PTH concentration values in patient samples. Also, serum PTH concentration measured by first-generation assay was frequently falsely elevated and did not correlate with the clinical state of patients. Competitive immunoassays are no longer used for the measurement of PTH concentration mainly because in addition to intact PTH (amino acids 1–84), numerous fragments of PTH molecules present in patients’ samples were measured; thus final PTH concentration results were overestimated to different extents. Noncompetitive immunoassay format was applied in the second generation of PTH assays (intact PTH assays), which were designed to measure the whole PTH peptide structure (amino acids 1–84), called “intact PTH.” The assay used capture antibody against the C-terminal region of PTH (amino acids 39–84) and labeled antibody against the N-terminal region (amino acids 12–24 or amino acids 26–32, depending on assay manufacturers) (5,6). Although the PTH assays of the second generation were designed to measure intact PTH and not to measure C-terminal and midfragments, these assays detect a shorter PTH fragment (amino acids 7–84), called also “non-1–84” or “N-terminaltruncated” PTH. Recently, it was shown that “non-1–84” PTH is not a single peptide but represents the family of fragments of different lengths with the longest fragment starting at position 4 and the shortest one starting at position 15 (7). The ability of detecting of N-PTH by the second generation of PTH assay depended on the epitope of the anti-Nterminal antibody (8). Although the determination of PTH concentration by using the immunoassays of the second generation greatly improved the correlation between the laboratory results and the clinical condition of the patients, it appeared very quickly that the determination of the “intact PTH” also did not meet the clinical requirements.

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Example 1 Parathyroid hormone (PTH)

The reason was that the antibodies used in those assays recognized not only the whole PTH molecule but also other forms of the peptide, especially PTH (amino acids 7–84), which is probably produced both in the parathyroid glands and in blood (9). Since PTH (amino acids 7–84) acts as an agonist of PTH (amino acids 1–84) concerning calcium homeostasis, it is eliminated entirely through the kidneys and has a longer half-life as compared to intact PTH (amino acids 1–84). The scientific literature has questioned the usefulness of second-generation PTH assays, especially because they are not suitable for the evaluation of parathyroid function in patients with kidney dysfunction and in those with the disturbances of calcium metabolism (10). The third-generation PTH assays, also noncompetitive assays, similar to second-generation assays, use capture antibody directed toward the C-terminal region of PTH, but the labeled antibody is directed against the very first (N-terminal) amino acids (amino acids 1–4) (11). The third generation is called “Whole PTH assay” or “BioIntact PTH” (12,13). These assays do not measure the shorter PTH fragments and C-terminal fragments, but they measure, besides the main form full-length PTH (amino acids 1–84, intact PTH), the N-PTH (“atypical” PTH) form. Comparisons of second- and third-generation noncompetitive PTH assays in relation to the PTH forms they measure are presented in fFig. E1.1. At present, the third-generation assays do not show any superiority over the second-generation assays in clinical practice. There is some confusion about the assay generations for PTH measurement in the literature; thus the nomenclature of the assay generations needs clarification. In some sources, competitive methods are called the first generation of assay, and noncompetitive assays are called the second and the third generation. Other sources use different nomenclature: competitive methods are called first generation of assay; and noncompetitive methods, first- or second-generation assays instead of second and third generation.

Fig. E1.1: Comparison of second and third generation of parathyroid hormone (PTH) assays in relation to different PTH forms measured by these assays. Shadowed boxes represent the PTH forms measured by appropriate generation of assay.


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This could be confusing to laboratory professionals and physicians searching the literature for scientific information about the usefulness of PTH in clinical settings. Both second and third generation (noncompetitive immunoassays) are characterized by good analytical quality. However, although the values obtained by these two assays are highly correlated, the differences of 10%–25% between the values of the measured concentrations have been documented (14,15,16) because third-generation assays do not detect the non-(amino acids 1–84) PTH fragments. It has to be kept in mind that PTH assays, as many other immunochemistry measurements, do not have proper standardization and that second- and third-generation assays are calibrated against synthetic PTH (amino acids 1–84) from various sources. In addition, there is currently no recognized international standard made of synthetic peptide. PTH assays not only suffer from lack of good standardization, but also harmonization has not yet been achieved among assays. Comparison of 15 different assays (13 second- and third-generation assays) revealed median bias ranging from 44% to 123% using the Nichols Allegro Intact PTH assay as reference method (17). As for other analytes measured by immunochemistry, method-specific cutoff values, validated for the population, for which laboratories provide the service, should be used by laboratories for the interpretation of PTH results. In selecting the individuals for validating the PTH reference intervals, it is important to consider other factors influencing the PTH determination. It is well known that serum PTH level depends on vitamin D concentration, regardless of the assay used for the measurement of PTH peptide. It was shown that when subjects with vitamin D deficiency were excluded from the selected reference groups of patients from the apparently healthy general population, the upper limit of the reference interval was lowered by 25%–35% (8). Also, age and race should be taken into account when establishing or validating the reference intervals for PTH. In the elderly, there is an age-dependent increase in PTH concentration that correlates roughly with the physiological decline of renal function (18). Higher PTH levels were found in black as compared to white males (19). Apart from the problem of a reference upper cutoff value, the serum PTH concentration should always be interpreted with regard to concomitant calcemia. The measurement of PTH concentration should be performed in the morning samples of blood serum or plasma (EDTA) and, if not assayed within 6 hours, samples should be frozen because of the peptide stability problem. Four to six freeze-thaw cycles do not affect the peptide concentration (10,11). PTH has been reported to be more stable in EDTA plasma than in serum. The same type of sample (serum or plasma) should be used for PTH measurement in monitoring the patient, because PTH concentration in EDTA plasma is higher than that in serum by 10%–20%, depending on the assay used (20,21). The determination of PTH concentration is not free from interference from heterophilic antibodies. Recently, it was reported that after treatment of patients with murine monoclonal antibody directed against the CD23 of human T-cell immunoglobulin (OKT3), human antimouse antibodies synthesized as a consequence of treatment were the source of interference in Elescys PTH assay (23). It has been stressed that the detection of such an interference is not always easy, particularly in the case of hemodialyzed or transplant patients, in whom high concentrations of PTH can be expected (22). In summary, PTH determination by immunochemistry, although fast and simple if performed automatically, is still challenging due to the existence of many forms of PTH

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in serum or plasma. It is one of the best examples of the heterogeneity problem in immunochemistry measurement, because various PTH forms present in the patient’s sample may differ only by a single amino acid. This translates into practical problems with the interpretation of PTH results, especially in patients with kidney disease. Because of that, laboratory professionals who interpret the PTH results and consult the physician must be familiar with the assay format and reagent antibodies used in the method and always be ready to answer the simple question, what is being measured by my PTH assay? However, the answer is not always straightforward.

References 1. Friedman PA, Goodman WG. PTH (1–84)/PTH (7–84): A balance of power. Am J Physiol Renal Physiol 2006;290: F975–84. 2. D’Amour P, Brossard JH, Rousseau L, Roy L, Gao P, Cantor T. Amino-terminal form of parathyroid hormone (PTH) with immunologic similarities to hPTH (1–84) is overproduced in primary and secondary hyperparathyroidism. Clin Chem 2003;49: 2037–44. 3. Rubin MR, Silverberg SJ, D’Amour P, et al. An N-terminal molecular form of parathyroid hormone (PTH) distinct from hPTH (1–84) is overproduced in parathyroid carcinoma. Clin Chem 2007;53: 1470–6. 4. Arakawa T, D’Amour P, Rousseasu L, et al. Overproduction and secretion of a novel aminoterminal form of parathyroid hormone from a severe type of parathyroid hyperplasia in uremia. Clin J Am Soc Nephrol 2006;1: 525–31. 5. D’Amour P, Brossard JH. Carboxy-terminal parathyroid hormone fragments: role in parathyroid hormone physiopathology. Curr Opin Nephrol Hypertens 2005;14: 330–6. 6. Ratcliffe WA, Heath DA, Ryan M, Jones SR. Performance and diagnostic application of a two-site immunoradiometric assay for parathyrin in serum. Clin Chem 1989;35: 1957–61. 7. Amour R, Brossard JH, Rousseau L, et al. Structure of non-(1–84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism. Kidney Int 2005;68: 998–1007. 8. Souberbielle JC, Friedlander G, Cormier C. Practical considerations in PTH testing. Clin Chim Acta 2006;366: 81–9. 9. Lepage R, Roy L, Brossard JH, et al. Non-(1–84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples. Clin Chem 1998;44: 805–9. 10. Inaba M, Nakatsuka K, Imanishi Y, et al. Technical and clinical characterization of the BioPTH(1–84) immunochemiluminometric assay and comparison with a second-generation assay for parathyroid hormone. Clin Chem 2004;50: 385–90. 11. Gao P, Scheibel S, D’Amour P, et al. Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: Implications for improvement of accurate assessment of parathyroid function. J Bone Miner Res 2001;16: 605–14. 12. John MR, Goodman WG, Gao P, Cantor TL, Salusky IB, Jüppner H. A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: Implications for PTH measurements in renal failure. J Clin Endocrinol Metab 1999;84: 4287–90. 13. Savoca R, Bock A, Kraenzlin ME, Schmid HR, Huber AR. An automated “bio-intact” PTH assay: A step towards standardisation and improved correlation with parathyroid function in renal disease. Clin Chim Acta 2004;343: 167–71.

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14. Terry AH, Orrock J, Meikle AW. Comparison of two third-generation parathyroid hormone assays. Clin Chem 2003;49: 336–7. 15. Boudou P, Ibrahim F, Cormier C, Chabas A, Sarfati E, Souberbielle JC. Third- or secondgeneration parathyroid hormone assays: A remaining debate in the diagnosis of primary hyperparathyroidism. J Clin Endocrinol Metab 2005;90: 6370–2. 16. Koller H, Zitt E, Staudacher G, Neyer U, Mayer G, Rosenkranz AR. Variable parathyroid hormone (1–84)/carboxyl-terminal PTH ratios detected by 4 novel parathyroid hormone assays. Clin Nephrol 2004;61: 337–43. 17. Souberbielle JC, Boutten A, Carlier MC, et al. Inter-method variability in PTH measurement: implication for the care of CKD patients. Kidney Int 2006;70: 345–50. 18. Marcus R, Madvig P, Young G. Age-related changes in parathyroid hormone and parathyroid hormone action in normal humans. J Clin Endocrinol Metab 1984;58: 223–30. 19. M’Buamba-Kabangu JR, Fagard R, Lijnen P, Bouillon R, Lissens W, Amery A. Calcium, vitamin D-endocrine system, and parathyroid hormone in black and white males. Calcif Tissue Int 1987;41: 70–4. 20. Omar H, Chamberlin A, Walker V, Wood PJ. Immulite 2000 parathyroid hormone assay: Stability of parathyroid hormone in EDTA blood kept at room temperature for 48 hours. Ann Clin Biochem 2001;38: 561–3. 21. Holmes DT, Levin A, Forer B, Rosenberg F. Preanalytical influences on DPC Immulite 2000 intact PTH assays of plasma and serum from dialysis patients. Clin Chem 2005;51: 915–17. 22. Cavalier E, Carlisi A, Chapelle JP, et al. Human anti-mouse antibodies interferences in Elecsys PTH assay after OKT3 treatment. Transplantation 2009;87: 451–2.

Example 2 Human chorionic gonadotropin (hCG) – Problems of heterogeneity and lack of standardization

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein composed of two nonidentical, noncovalently bound glycoprotein subunits, A-subunit and B-subunit, both existing in multiple forms. hCG is synthesized in the syncytiotrophoblast cells of the placenta in all normal and abnormal pregnancies, gestational trophoblastic disease, and certain other pathological conditions. It is also secreted by the pituitary gland in menopausal women as well as in men with hypogonadism (1,2). The measurement of hCG in plasma is used for the confirmation and monitoring of pregnancy, pregnancyrelated disorders, prenatal screening and diagnosis, and monitoring of treatment of trophoblastic tumor of placenta and tumors of germ cell origin. Among many problems of hCG measurement in blood plasma and urine, the protein heterogeneity and confusion of nomenclature of various forms of hCG are the main issues. At least six different forms of hCG have been identified in blood serum: nonnicked and nicked intact hCG (hCG and hCGn), nonnicked and nicked free B-subunit (hCGB and hCGnB), and regular and hyperglycosylated free hCGA (3). The nicking process causes inactivation of the hormone and is the reason for reduced ability of assay reagent antibodies to recognize such hCG forms. Regular hCG and hyperglycosylated hCG have the same polypeptide structures, but hyperglycosylated hCG contains much larger N- and O-linked oligosaccharides. Although both regular hCG and hyperglycosylated hCG present in plasma have similar structures, they differ in biological functions, and thus their measurement in patient samples serves different clinical purposes. In urine, hCGB core fragment of regular and nicked hCG can be found. Because of hepatic and renal clearance, the early morning urine specimen contains hCG concentration comparable with that in serum (4). Depending on antibodies used in immunoassays, different forms of hCG are detected. From a clinical point of view, the most appropriate hCG form for the particular pathological state should be measured. Intact hCG predominates in normal and abnormal pregnancies, but the carbohydrate content and composition of hCG depends on the pregnancy progress. In early pregnancy (3–5 weeks), hyperglycosylated hCG predominates (5). In neoplasia, regular hCG predominates in quiescent trophoblastic disease. The hyperglycosylated free hCGB is present in trophoblastic disease, testicular tumors, or other nongestational malignancies. The hyperglycosylated hCG is characteristic for choriocarcinoma or testicular germ cell tumor (5). Most current immunoassays measure intact hCG (nicked and nonnicked hCG) or hCG plus hCGB. All assays vary in specificity toward different forms of hCG. For calibration of hCG assays, the third (IS 75/537) or the fourth (IS 75/589) international standards are widely used for all immunoassay platforms including point-of-care-testing devices and OTC testing. Both standards are calibrated in biological units (international units per liter, IU/L). Based on sequence analysis, it was shown that these hCG preparations contain 9% nicked hCG, varying levels of hCG free B, hyperglycosylated hCG free B, and significant proportions (15%–18%) of hyperglycosylated hCG and B-subunit

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Example 2 Human chorionic gonadotropin (hCG)

core fragments (3,6). WHO standards for free B-subunit and free A-subunit are also calibrated in arbitrary international units where 1 IU/L equals 1 μg/L. These standards are somewhat incompatible with hCG standards, since 1 IU of free B represents 0.045 nmol of free B, and 1 IU of hCG represents 0.0029 nmol of hCG. As such, 1 IU of free B contains 15.5-fold more B-subunit than 1 IU of hCG (5). Because of heterogeneity of hCG, proper assay standardization is very important in order to know what is really measured in the patient’s sample. Recently, the first WHO International Reference Reagents (IRRs, WHO code 99/688) was prepared for six hCG-related molecule variants along with WHO codes and International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) nomenclature. The first IRRs contains intact hCG (hCG, IRR 99/688, molar units), nicked hCG (hCGn, IRR 99/642, molar units), hCG B-subunit (hCGB, IRR 99/650, molar units), nicked hCG B-subunit (hCGBn, IRR 99/692, molar units), hCG B core fragment (hCGBcf, IRR 99/708, molar units), and hCG A-subunit (hCGA, IRR 99/720, molar units) (7). These IRRs are the first standards for glycoprotein hormone to be calibrated in substance concentration, which allow comparison of the relative reactivity of each molecule and serve for the cross-reactivity experiments in different immunoassays. On behalf of the IFCC Working Group for Standardization of hCG, Sturgeon (7) based on first IRR 99/688 investigated the analytical specificity of commercially available hCG immunoassays. It was shown that currently used hCG immunoassays differ considerably in their recognition of various forms of hCG in serum. This variability is the most important cause of method-related differences in hCG results in serum and an even more important cause of method-related differences of hCG measurements in urine (7). Currently, no standards are available for hyperglycosylated intact hCG (an important marker for very early pregnancy), hyperglycosylated free B-subunit (important as a marker for cancer patients), and hyperglycosylated free A-subunit (important for pregnancy). However, much research is performed at present to produce the assay for hyperglycosylated hCG (8). Also, there is a need for the quantitative urine hCG test (9). Immunoassays for hCG suffer not only from lack of proper standardization because of heterogeneity of hCG, but also from interference, which affects the assay result. Two typical forms of interference cause the problems in routine hCG concentration measurement, namely, interference from heterophilic antibodies and the hook effect. Interference from heterophilic antibodies posed a big problem between 1999 and 2002. It was because the assays most frequently used at that time contained assay buffer without animal serum as an additive to protect from the presence of heterophilic antibodies in patient samples, but animal serum was added to assay diluent; thus all diluted samples were protected from interference, while undiluted samples were prone to interference (10). In those assays, many falsely positive results were seen when the patient’s samples were assayed undiluted causing many unnecessary clinical interventions (11,12). Ten-year USA hCG reference service report analyzed in detail the false-positive serum hCG results (frequently called “phantom hCG”) (13). Although since that time many assays for hCG have been improved, still laboratories should be aware of this problem with devastating consequences to not only female but also male patients (14). If an unexpected serum hCG concentration is obtained, frequently urine hCG is measured. Normally, interfering antibodies are not present in urine, whereas hCG appears in urine following increased hCG secretion. However, the measurement of hCG in urine might not be helpful if the serum hCG concentration is relatively low (30 mIU/mL) is justified by the presence of

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Example 2 Human chorionic gonadotropin (hCG)

peri- or postmenopause and the presence of pituitary hCG. The reflex FSH testing in all female patients between 41 and 55 years of age with serum hCG concentration in the range 5.0–14 IU/L was proposed to rule out pregnancy with the cutoff of FSH concentration equal to 45 mIU/mL or higher (25). If there is a concern about the source of the hCG, hormone replacement therapy is advocated for confirming or rejecting a pituitary source of hCG (25). Attention should also be paid if hCG concentration is measured in men with hypogonadism because slightly higher values are seen in such patients (2). Heterogeneity of hCG, confusion about the nomenclature of hCG forms, and lack of standardization across all the assays used for hCG measurement calls for laboratories’ attention. First, laboratories should know the extent to which different isoforms of hCG are recognized by assay reagent antibodies. There are multiple antibody-binding sites on each of the hCG molecules, and different antibodies are used by assays manufacturers. Specificity of the reagent antibody is not always known to laboratory professionals. It is important to realize that current assays for hCG measurement are approved to detect pregnancy and are not approved for diagnosis and management of throphoblastic disease or other cancers. It is the responsibility of laboratory professionals to inform clinicians which hCG isoform is being measured by the assay and what the assay limitations are, including the possibilities of false-positive results due to the presence of heterophilic antibodies in the patient’s sample or of falsely low results due to the hook effect. Many excellent reviews concerning hCG biology and laboratory measurement of this protein have been published recently (5,10,26,27,28). However, taking into account the needs for improvement of hCG assay standardization and for better markers for cancer patient tracing, laboratory professionals should get acquainted with any accessible information in this regard, because the patient’s safety might be jeopardized if the wrong hCG results are reported.

References 1. Cole LA, Sasaki Y, Muller CY. Normal production of human chorionic gonadotropin in menopause. N Eng J Med 2007;356: 1184–6. 2. Lempiälinen A, Hotakainen K, Blomqvist C, Alfhan H, Stenman UH. Increased human chorionic gonadotropin due to hypogonadism after treatment of a testicular seminoma [letter]. Clin Chem 2007;53: 1560–1. 3. Cole LA. Immunoassay of human chorionic gonadotropin, its free subunits and metabolites. Clin Chem 1997;43: 2233–43. 4. Burtis CA, Ashwood ER, Bruns DE, ed. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics.: Elsevier Saunders; St. Louis, Missouri, USA 2006. 5. Cole LA. New discoveries on the biology and detection of human chorionic gonadotropin. Reprod Biol Endocrinol 2009;26: 7–8. 6. Elliot MM, Kardana A, Lustbader JW, Cole LA. Carbohydrate and peptide structure of the A- and B-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine 1997;7: 15–32. 7. Sturgeon CM, Berger P, Bidart JM, et al., on behalf of the IFCC Working Group in hCG. Differences in recognition of the 1st WHO international reference reagents for hCGrelated isoforms by diagnostic immunoassays for human chorionic gonadotropin. Clin Chem 2009;55: 1484–91. 8. Sasaki Y, Ladner DG, Cole LA. Hyperglycosylated human chorionic gonadotropin and the source of pregnancy failures. Fertil Steril 2008;89: 1781–6.

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9. Cole LA, Khanlian SA. The need for a quantitative urine hCG assay. Clin Biochem 2009;42: 676–83. 10. Cole LA. Hyperglycosylated hCG, a review. Placenta 2010;31: 653–64. 11. Rotmensch S, Cole LA. False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations. Lancet 2000;355: 712–15 12. Cole LA. Phantom hCG and phantom choriocarcinoma. Gynecol Oncol 1998;71: 325–9. 13. Cole LA, Laidler LL, Muller CY. USA hCG reference service, 10-year report. Clin Biochem 2010;43: 1013–22. 14. Ballieux BEPB, Weijl NI, Gelderblom H, van Pelt J, Osanto S. False-positive serum human chorionic gonadotropin (hCG) in a male patient with a malignant germ cell tumor of the testis: a case report and review of the literature. The Oncologist 2008;13: 1149–54. 15. Price A, Gillett G. Needless treatment for presumed malignancy [letter]. Lancet 2000;355: 1724–5. 16. Knight AK, Bingemann T, Cole L, Cunningham-Rundles C. Frequent false positive beta human chorionic gonadotropin tests in immunoglobulin A deficiency. Clin Exp Immunol 2005;141: 333–7. 17. Gronowski AM, Cervinski M, Stenman UH, Woodworth A, Ashby L, Scott MG. Falsenegative results in point-of care qualitative human chorionic gonadotropin (hCG) devices due to excess hCGB core fragment. Clin Chem 2009;55: 1389–94. 18. Snyder JA, Haymond S, Parvin CA, Gronowski AM, Grenache DG. Diagnostic considerations in the measurement of human chorionic gonadotropin in aging women. Clin Chem 2005;51: 1830–5. 19. Cole LA. Background hCG in healthy, non-pregnant women. Clin Chem 2005;51: 1765–6. 20. Cole LA, Khanlian SA, Muller CY. Detection of hCG peri- or post-menopause an unnecessary source of alarm. Am J Obstet Gynecol 2008;198: 275–9. 21. Cole LA, Khanlian SA, Muller C. Normal production of human chorionic gonadotropin perimenopausal and menopausal women and after oophorectomy. Int J Gyn Cancer 2009;19: 1556–9. 22. Beck SDW, Patel M, Sheinfeld J. Tumor marker levels in post-chemotherapy cystic masses: Clinical implications for patients with germ cell tumors. J Urol 2004;171: 168–71. 23. Cole LA, Khanlian SA. Inappropriate management of women with persistent low hCG results. J Reprod Med 2004;49: 423–32. 24. Cole LA, Khanlian SA, Giddings A, et al. Gestational trophoblastic diseases: Presentation with persistent low positive human chorionic gonadotropin test results. Gynecol Oncol 2006;102: 165–72. 25. Gronowski AM, Fantz CR, Parvin CA, et al. Use of serum FSH to identify perimenopausal women with pituitary hCG. Clin Chem 2008;54: 652–6. 26. Gronowski AM. Clinical assays for human chorionic gonadotropin: What should we measure and how? Clin Chem 2009;55: 1900–4. 27. Gronowski AM, Grenache DG. Characterization of the hCG variants recognized by different hCG immunoassays: An important step toward standardization of hCG measurement. Clin Chem 2009;55: 1447–9. 28. Sturgeon CM, Ellis AR. Standardization of FSH, LH and hCG – current position and future prospects. Mol Cell Endocrinol 2007;260–262: 301–9.

Example 3 Troponin measurement by immunoassay – Problem of low assay sensitivity and interference from heterophilic antibodies

Cardiac troponins (cTn’s) play a central role in diagnosis and risk stratification in acute coronary syndrome (1). Although thousands of troponin determinations by immunoassay are performed everyday all over the world, there are many unsolved problems that may affect the patient’s safety by misclassification and misdiagnosis based on erroneous troponin results. According to the Universal Definition of Myocardial Infarction (2) and the guidelines of the National Academy of Clinical Biochemistry (NACB)(3), cardiac troponins have been identified as the preferred biomarkers, while creatine kinase isoenzyme mass is the second marker of choice when troponin measurement is not available. Proper interpretation of troponin results requires basic knowledge of biochemistry of troponin and deeper knowledge concerning immunoassay performance, assay sensitivity and specificity, as well as detection limit. The troponin complex, as well as myosin, tropomyosin, actin, and calcium play a crucial role in the regulation of muscle contraction. Depending on the tissue, two types of troponin exist: cTn and skeletal troponin (sTn). The cTn complex exists in three subunits: troponin T (TnT), which binds the troponin complex to tropomyosin and sensitizes actin for contraction; troponin I (TnI), which modulates the interaction of actin and myosin by actin as an inhibitor of actomyosin ATP activity; and troponin C (TnC), which via Ca2-binding transmits a contraction signal to thin filament. Each of these three subunits of the troponin complex has distinctive properties and function within the thin filaments. Most of the cTnI and cTnT exist as a structural myofilament-bound protein pool. Only a small percentage of total cTn (about 6%–8% of cTnT and 3% of cTnI) exists as free cytosolic pool within myocytes. The troponin subunits cTnI, cTnT, and TnC are released by necrotic myocytes in a biphasic manner and in several forms: after initial release of cytosolic cTn (first phase), an extended release pattern can be observed for days (second phase), originating from continued breakdown of myofibrils from the damaged myofilament structures of myocytes. cTnT is found primarily as free fraction (37 kDa), in the form of the cTnT:cTnI:TnC trimeric complex (114.5 kDa), and smaller immunoreactive fragments (

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