Meeting the Challenges of Blood Safety in the 21st Century: Pathogen Inactivation

Transfus Med Hemother 2004;31(suppl 1):I–IV Vol. 31, Supplement 1, January 2004 Meeting the Challenges of Blood Safet...

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Transfus Med Hemother 2004;31(suppl 1):I–IV

Vol. 31, Supplement 1, January 2004

Meeting the Challenges of Blood Safety in the 21st Century: Pathogen Inactivation

Guest Editor J.C. Osselaer, Yvoir (Belgium)

Basel Freiburg Paris London New York Bangalore Bangkok Singapore Tokyo Sydney

Contents Vol. 31, Supplement 1, January 2004

Editorial 1 Meeting the Challenges of Blood Safety in the 21st Century: Pathogen Inactivation Osselaer, J.C. (Yvoir)

Review Articles 2 Why ‘Safer than Ever’ May Not Be Quite Safe Enough Barbara, J. (London)

11 Targeting DNA and RNA in Pathogens: Mode of Action of Amotosalen HCl Wollowitz, S. (Concord)

17 The Science of Safety: Toxicological Review of Amotosalen HCl Dayan, A.D. (London)

24 Protection against Transfusion-Associated Graft-versus-Host Disease in Blood Transfusion: Is Gamma-Irradiation the Only Answer? Schlenke, P. (Lu¨beck)

Quality Management 32 Practical Experience of Implementing the INTERCEPTTM Blood System for Buffy Coat Platelets in a Blood Center Hervig, T.; Aksnes, I. (Bergen)

II Imprint C3 Guidelines for Authors (Inside back cover)

# 2004 S. Karger GmbH, Freiburg Fax +49 761 4 52 07 14 E-mail [email protected] www.karger.com

Accessible online at: www.karger.com/tmh

Editorial Transfus Med Hemother 2004;31(suppl 1):1

Meeting the Challenges of Blood Safety in the 21st Century: Pathogen Inactivation

The past few decades have witnessed enormous improvements in our ability to assure the safety of blood components against viral infections; however, serious threats remain. As pinpointed by J. Barbara [1], bacterial contamination of platelet concentrates in particular is a major ongoing concern. Furthermore, despite the fact that the introduction of nucleic acid testing appears to have brought under control the risk of infection by viruses classically associated with transfusion, newly emerging viruses, mutant strains, protozoa, and known viruses mistakenly regarded as harmless remain a constant threat for both patients and the blood supply. Although major improvements in safety have been achieved through better collection techniques, more stringent donor selection, more and better screening and less donor exposure, and although blood transfusion has become safer than ever before, it does not appear to be possible to completely eradicate the residual risk of infection due to inherent limitations in each of these methods. Pathogen inactivation, in contrast, affords a proactive approach, taking advantage of the fact that all DNA and RNA present in blood products is unwelcome, whether from microorganisms or from leukocytes. Amotosalen’s mechanism of action is described by S. Wollowitz [2]. Amotosalen in combination with UVA presently is indicated for pathogen inactivation of platelet concentrates, and will soon become available for plasma. The efficiency of the method has been extensively proven against a vast array of microorganisms classically or potentially associated with transfusion complications. In order to be acceptable for clinical use, pathogen inactivation must be proven to have no adverse effects on either platelets or the recipient of platelet transfusions. Preclinical studies have shown that platelet integrity and function are maintained following treatment with amotosalen. Furthermore, the INTERCEPTTM Blood System for platelets has been subjected to extensive clinical testing, which has examined the recovery and survival, safety and tolerability, and haemostatic and therapeutic efficacy of treated platelets. Successful phase-I and -II studies led to two phase-III trials, each focussing on the therapeutic efficacy of amotosalen-treated platelets (SPRINT and euroSPRITE), which revealed no clinical safety concerns. Moreover, the proportion of patients who developed persistent immunologic platelet refractoriness was similar between the two groups, with no evidence of antibodies to neoantigens seen in either group. Compounds that target DNA and RNA necessarily have the potential not only for acute and chronic toxicity but also for genotoxicity of both the parent compound and its breakdown products. Results of toxicological studies with amotosalen are reviewed by A. D. Dayan [3], who concludes that there is no genotoxic risk to patients.



The INTERCEPT Blood System for platelets not only inhibits pathogen replication, but also prevents leukocyte proliferation and cytokine generation. As suggested by P. Schlenke [4], the INTERCEPT Blood System for platelets may become an alternative to gamma-irradiation. The full benefits of this innovative technology, however, will only be felt when similar inactivation procedures cover the entire range of blood components. T. Hervig and I. Aksnes [5] report on their experience of validating and implementing the INTERCEPT Blood System for platelets in a Norwegian blood centre, and conclude that it is feasible even for smaller transfusion centres. Similar observations were made in the Blood Transfusion Centre of Mont-Godinne, where INTERCEPT was implemented in October 2003 and led to a very reasonable increase in workload. Although the cost of introducing the INTERCEPT Blood System for platelets for pathogen inactivation remains substantial, it needs to be considered in the light of the wide array of benefits provided by the avoidance of new testing, unnecessary donor exclusion on merely hypothetical grounds, the possibility of an extended platelet shelf life and, even more importantly, the satisfaction of delivering safer products to our patients. Transfusion accidents constitute a serious threat for the blood supply – they are not only disastrous for the recipients, they are also highly frustrating for both donors and blood centre staff. Their real cost is therefore difficult to estimate. When considering the cost-effectiveness of this new invention, the whole spectrum of these advantages should be taken into account. J.C. Osselaer, Yvoir, Belgium

References 1 Barbara J: Why ‘Safer than Ever’ may not be quite safe enough. Transfus Med Hemother 2004;31(suppl 1):2–10 (this issue). 2 Wollowitz S: Targeting DNA and RNA in pathogens: Mode of action of amotosalen HCl. Transfus Med Hemother 2004;31(suppl 1):11–16 (this issue). 3 Dayan AD: The science of safety: Toxicological review of amotosalen HCl. Transfus Med Hemother 2004;31(suppl 1):17–23 (this issue). 4 Schlenke P: Protection against transfusion-associated graft-versus-host disease in blood transfusion: Is gamma-irradiation the only answer? Transfus Med Hemother 2004;31(suppl 1):24–31 (this issue). 5 Hervig T, Aksnes I: Practical experience of implementing the INTERCEPTTM Blood System for buffy coat platelets in a blood center. Transfus Med Hemother 2004;31(suppl 1):32–36 (this issue).

J. C. Osselaer, MD, Master in Law BTC Mont Godinne Mont-Godinne University Hospital B-5530 Yvoir Tel. +32 81 42 32-42, Fax -39

Review Article Transfus Med Hemother 2004;31(suppl 1):2–10

Why ‘Safer than Ever’ May Not Be Quite Safe Enough J. Barbara National Blood Service, London, England

Key Words Blood safety Pathogen inactivation Residual risk Transfusion infections Abstract Although the blood supply in developed countries is now very safe, residual microbial risks can still be identified. These are mainly due to bacteria. A wide range of sophisticated (and resource-consuming) interventions are in place which are generally successful in dealing with the risk from known viral agents. However, newly emergent viral risks can usually only be addressed after they have been shown to transmit and after tests have been developed. Parasitic and, even more significantly, bacterial risks (especially in platelet preparations which have to be stored at 22 C) have often not been managed as effectively as the risk for the ‘known’ viruses such as HIV, HBV and HCV. Pathogen inactivation (PI) offers an approach to remove the vast majority of microbial risks. In the future, if the full inventory of transfusable components can be effectively pathogen-inactivated, PI could form an alternative basis for blood safety in terms of microbial risk, rather than just another incremental safety intervention.

Introduction

The transfusion of safe blood components is a crucial part of modern advances of medicine, including complicated cardiac surgery, multiple organ transplantation and aggressive chemotherapeutic treatments. It is likely that the need for increasingly safe blood will continue in the quest for ‘zero-risk’ transfusion. In addition, there is a growing range of patients who can benefit from the transfusion of blood and its components. Decades of steady input from science and modern technology have resolved many of the early deficiencies in the safety of blood transfusion. These included the provision of enhanced methods to improve donor–recipient compatibility, effective blood storage and preservation methods, and the development of sterile plastic bags for collection of blood to allow safe preparation of individual blood com-



ponents. More recent preoccupations with blood safety have been greatly influenced by the emergence, in the 1980s, of the human immunodeficiency virus (HIV) and its clinical manifestation – acquired immunodeficiency syndrome (AIDS). Following the HIV pandemic, reducing the risk from transfusion-transmitted infections (TTIs) became of paramount clinical and ethical importance. As sophisticated and sensitive laboratory tests to screen for pathogens became available, many were added to blood processing protocols in developed countries. Since the emergence of HIV in the developed world, seven new laboratory interventions (assays and processes including leukodepletion) have been available on a routine basis. The timing, detail and extent of implementation of these interventions vary from country to country (fig. 1). In contrast, in the 40 years prior to the HIV pandemic, only two laboratory assays (for syphilis and hepatitis B surface antigen [HBsAg]) were routinely used to screen blood for infectious agents. As each new microbial threat is identified and a further test added to the routine screening program, costs, complexity and the potential for error inevitably increase. Furthermore, an increasing number of these incremental interventions will not be affordable in many countries, thereby widening the gap in blood safety standards around the world. As will be discussed in more detail later in this review, a ‘pan-effective’ intervention would certainly seem attractive in the face of the ever-growing number of tests. Extensive prospective laboratory testing and other safeguards, including careful selection of donors to try and exclude those at risk of contracting TTIs, have resulted in a blood supply that is protected from many known transfusion-transmitted viral diseases. However, even current donor screening and testing procedures have limitations. If the current trend to achieve ‘zero microbial risk’ continues, improvement of existing tests or the addition of new tests will be required to detect known or new pathogenic risks to the blood supply. To provide prospective protection, and possibly abate the continual addition of new laboratory tests, rou-

Dr. John Barbara National Blood Service Colindale, London NW9 5BG UK

tine pathogen inactivation (PI)/reduction of labile blood components is now under serious consideration. One of the first available approaches for PI of platelet preparations is provided by the INTERCEPT Blood System (Baxter/Cerus), the first to be approved in Europe. The INTERCEPTTM Blood System for routine PI of platelets utilizes amotosalen HCl and UVA light to prevent DNA and RNA replication. Since mature platelets are void of nucleic acids this process can inactivate pathogens and also prevent leukocyte proliferation without blocking platelet function.

Risks of TTIs in Platelets

The key points relating to the risk of transfusion-transmitted disease in platelets are summarized in table 1. Platelet concentrates can be collected from a single donor by automated blood cell processors using apheresis technology or can be prepared from units of whole blood collected from random donors. US-based blood centers typically use centrifugation protocols to derive platelet concentrates, either in the form of platelet-rich plasma or buffy coats. However, in Europe, buffy coat-derived platelets are commonly used. To prepare one adult dose of buffy coat-derived platelet concentrate (24 1010/pool), 4–6 whole-blood donations are commonly required. Platelet concentrates are stored for up to 5 days in the US, and up to 7 days in some parts of Europe (when tested

HBsAG

1958

Syphilis

1970

Anti-HBc

1985

Anti-HIV-1

1986

Anti-HCV

1986

Anti-HTLVI and HTLVII

1991

HIV p24 antigen (subsequently combo HIV Ag/Ab available)

1992

Anti-HIV-2

1996

1998

NAT (HCV/HIV-1)

1999

Leukodepletion

2002

Pathogen inactivation

Fig. 1. Availability of routine tests/processes to prevent transfusiontransmitted diseases in developed countries. Ab ¼ Antibody; Ag ¼ antigen; HBsAG ¼ hepatitis B surface antigen; HTLV ¼ human T-cell lymphocytic virus; Anti-HBc ¼ hepatitis B core antibody; Anti-HCV ¼ HCV antibody; HIV p24 ¼ HIV p24 antigen; NAT ¼ nucleic acid testing; Anti-HIV-1 ¼ HIV antibody; Anti-HTLVI ¼ HTLVI antibody; Anti-HIV-2 ¼ HIV antibodies. Routine use of tests/processes and dates of uptake vary from country to country.

with a bacterial detection assay), with continuous agitation at 20–24 C, rather than the 2–6 C as recommended for red blood cells. Platelets are precluded from refrigeration due to a disc-to-sphere morphological transformation at temperatures 105.

The Photochemical Reaction with Nucleic Acid

The PCT process (see fig. 2) is the reaction of psoralens with the double bonds of the nucleic acid pyrimidine bases. The interaction with the bases is highly specific and allows for the inactivation of very small amounts of nucleic acids in the presence of large amounts of other biological molecules. In blood components, the amotosalen – PCT process stops replication of 99.999% (5 log) of a broad spectrum of pathogens while maintaining viability of platelets and key plasma proteins. At the molecular level, the PCT process occurs in three steps, as depicted in figure 2. These are discussed briefly below.

Monoadduct Formation Upon UVA light activation, the intercalated psoralens undergo a [2 þ 2] photoaddition with a pyrimidine base (thymidine, cytosine, uridine) to form a covalent monoadduct. Either end ring of the three-ring psoralen structure can react. The 5-member ring that is furan-side of the psoralen typically predominates over the 6-member pyrone side in this first reaction, though the monoadduct ratios are dependent on psoralen substituent patterns [20]. See figure 3 for a molecular depiction of the monoadduct and crosslink formation.

Intercalation The psoralen first intercalates into a helical nucleic acid strand. The PCT process requires rapid intercalation into NH2

O

O

O

O

Fig. 1. Amotosalen (S-59).

Crosslink Formation The psoralen can undergo another photoaddition with a pyrimidine base on the opposite strand [21] forming a diadduct or interstrand crosslink. Furan-side monoadducts lead to interstrand crosslinks far more than pyrone monoadducts [22]. Crosslink formation in single-strand genomes of DNA and RNA viruses also occurs. Single-stranded nucleic acids have secondary structures including hairpin turns and loops due to the pairing of short complementary nucleotide sequences in different locations. These doublestranded regions allow for intercalation and intrastrand crosslinks [8, 11]. Sequence Specificity Psoralens have no significant sequence specificity and are therefore not selective for genomic material from any one organism. In DNA, thymine is the primary target for psoralen photocycloaddition, a reaction with cytosine occurs less frequently. For some psoralens that have been studied, monoadducts and crosslinks most frequently occur at 50 -TA sites. Some preference has been shown for sites

Fig. 2. Photochemical treatment (PCT) process using amotosalen.

H N

O

H

O

O

O

O

O

N

NH

dR

O

Furan-side monoadduct

12

H

O CH3

O

H 3C dR

H3C

H

H

N

O

NH O

Pyrone-side monoadduct

Transfus Med Hemother 2004;31(suppl 1):11–16

O

O O

H3C dR

N

O N

NH O

Interstrand crosslink

Wollowitz

dR O

Fig. 3. Structures of the psoralen adducts of thymidine as isolated from reactions with DNA.

with specific adjacent nucleotides [23] but the effect is minimal. The preferred sequences are relatively short and occur with high frequency in any whole genome. The aminated psoralens may have even less sequence specificity based on the high frequency of adducts that can be generated [24]. The total number of monoadducts and crosslinks can occur as frequently as 1 adduct for every 10–15 base pairs (bp). Interaction with RNA Unlike many compounds that interact with nucleic acids, psoralens are relatively indiscriminate in their affinity for DNA and RNA. Relative binding constants typically vary by less than an order of magnitude [3, 25] under favorable conditions. Uridine is the primary nucleotide for adduct formation in RNA [26].

one crosslink per genome will be greater for small genomes. Since the genome size varies among organisms (as shown in table 2), the frequency of adduct formation needed to inactivate the genome also varies considerably. Inhibition of human cell replication should require about 14.6 adducts per 3 billion bp (about 1 per 100,000,000 bp), a statistical event that is relatively easy to achieve. A bacterium will require a higher frequency of interaction (one adduct per 100,000 bp) and a small virus requires a frequency of crosslinks that is about one per several hundred bp. Figure 4 depicts this phenomenon more clearly. Shown in the bottom left corner of the figure, hepatitis B virus (HBV) requires an average crosslink efficiency of 1 : 100 bp to assure 6 logs of inactivation. Human cells require orders of magnitude fewer adducts per bp to achieve inhibition of replication.

Theory of Crosslink Frequency and Inactivation

How Adduct Formation Relates to Genome Inactivation In principle, a single permanent crosslink in a genome should block replication of the organism and result in inactivation. But crosslinks occur with a statistical frequency – for example, the genome of each and every HIV virion does not get one adduct before the first one gets two adducts. Using a Poisson distribution to describe the probability of adduct formation, table 1 shows that one needs an increasing number of crosslinks, on average, to increase the percent of genomes in solution that will have at least one crosslink. To successfully inactivate 6 logs of a virus, 99.9999% of all viral particles need at least one crosslink and the average crosslink frequency required would be 14.6 per strand. The adduct frequency needed to achieve

Experimental Data on Adduct Frequency and Inactivation

Adduct Frequency in the Actual PCT Process In the psoralen–PCT process the parameters have been set to provide a high adduct-per-bp frequency to ensure that even small viruses are inactivated. Using radiolabeled amotosalen, the adduct frequency on leukocyte DNA has been found to be 1 adduct for every 100 bp [27] under conditions used for the pathogen inactivation process. Using polymerase chain reaction (PCR), it has been shown that inactivation of leukocytes, duck HBV, and hepatitis C virus (HCV) results in inhibition of amplification for amplicons of 300–500 bp [28]. In other words, the adduct frequency is such that a large fraction of the genome pool

Table 1. Frequency of crosslinking relative to log reduction Average crosslinks per genome

Percent of genomes having 1 crosslink

Log reduction

2.3 14.6

90 99.9999

1 6

Average adduct:base ratio Genome = 1 Mbp

Genome = 5,000 b

1 : 435,000 1 : 68,000

1 : 2,200 1 : 340

Table 2. Genomic size of various organisms Organism

Genome size

Genome type

Human cell Plasmodium falciparum (malaria) Typical bacterium Cytomegalovirus (CMV) HIV HCV Parvovirus B19 HBV

3,000,000 kbp 23,000 kbp 600–7,000 kbp 250 kbp 9.2 kb (two copies) 9.4 kb 5 kb 3.2 kbp for longer strand

ds DNA ds DNA ds DNA ds DNA ss RNA ss RNA ss DNA DNA, one long, one short strand, about 50% ds

ds ¼ Double-stranded; kbp ¼ kilo base pairs; ss ¼ single-stranded.

Targeting DNA and RNA in Pathogens with Amotosalen HCl


13

in the sample has at least one crosslink in the specific 300–500 base sequence being amplified. The shaded band in figure 4 shows the approximate crosslink frequency found in the amotosalen–PCT process. Those pathogens with genome sizes to the right of the region where the shaded band meets the 6-log inactivation line will be effectively inactivated. In practice, good inactivation is seen even with very small viruses. Table 3 provides inactivation data for the amotosalen–PCT process against a wide variety of viruses, bacteria, and protozoa in platelet products. It is appropriate to also mention the anomaly of latent viruses or integrated viral genetic material (pro-viral DNA) inserted in the nucleic acid of contaminating leukocytes. Under conditions typical for pathogen inactivation in blood products, inactivation of pro-viral HIV has been demonstrated [3] as expected based on the adduct frequency observed. Differences between Self-Replicating Organisms and Viruses Bacterial and parasite genomes may continue to undergo gene transcription even if they are unable to fully replicate

[33]. Cells and bacteria, but not viruses, also contain mechanisms for the repair of DNA lesions, including psoralen adducts. Crosslinks cannot be repaired as easily as monoadducts and the resultant repair process is more error prone [34]. The INTERCEPT Blood System process parameters provide a crosslink frequency that effectively overwhelms the repair system of these organisms. Effect of Inactivation on NAT Nucleic acid testing (NAT) for detection of low levels of viruses in blood products requires the use of very short amplicons (90–120 bp) to achieve the desired level of assay sensitivity. The psoralen adducts that are sufficient to block replication occur, on average, far enough apart on the genome to allow significant amplification of these small amplicons. Additional amplification procedures used for RNA viruses limit further the ability of NAT to distinguish between untreated genomic material and amotosalen–PCT-inactivated viruses. Further complicating the picture is the fact that viruses replicate relatively inefficiently such that only a small fraction (0.001–10%) of the replicated genomic material is packaged in a manner that results in an infectious pro-

1 log (90%) inactivation

1 per 100,000,000

6 logs (99.9999% inactivation) 1 per 1,000,000 1 per 10,000 1 per 100 Crosslink frequency (adducts:bases)

bacteria

HBV

103

human cell

frequency } Crosslink generated by amotosalen - PCT

109

106 Genome size (bases or base pairs)

Fig. 4. Theoretical average crosslink frequency for inactivation. PCT ¼ photochemical treatment.

Table 3. Inactivation of pathogens in platelet concentrates by amotosalen-PCT Organism

Log inactivation

Parasite Spirochetes Gram-positive bacteria Gram-negative bacteria

Enveloped viruses

Non-enveloped viruses

a

[29];

14

b

[30]; c [31];

d

Plasmodium falciparum (malaria) Trypanosome cruzi (Chagas) Borrelia burgdorfia (Lyme) Treponema pallidum (syphilis) Staphylococcus epidermidis Streptococcus pyogenes Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa CMV HIV-1, cell-associated HTLV I/II HCV West Nile Virus HBV Parvovirus B19 Human adenovirus 5 Bluetongue virus

>7.0a >5.3b >7.5a 6.8–7.0d >6.6c >6.8c >6.4c >5.5c 4.5c >5.9c >6.1c 4.7/5.1a >4.5c >6.0a >5.5c 4 to >4.9d >7.1d 6.1–6.4c

[32].


Wollowitz

Table 4. Comparison of HIV inactivation with results from nucleic acid testing [35]

a b

Platelet concentrate spiked with HIV

Log HIV infectivity/mLb

Log HIV genome Eq/mL Amplicor1 (Roche Diagnostics)

NucliSens1 (Organon Technika)

Untreated Treateda

6.3 0

9.3 8.5

9.4 9.3

150 mM amotosalen HCl, 3 J/cm2 UVA, 35% platelet concentrate in 65% InterSolTM solution (Baxter Healthcare). Tissue culture plaque assay.

geny virion (one that is able to replicate again). NAT detects genomic material regardless of whether or not it is infectious. Both these concepts are clearly demonstrated in table 4 comparing observed titers of HIV by infectivity and by NAT when the virus, in platelet concentrate, is treated with amotosalen–PCT [35]. Both NAT methods detect approximately 3 logs more genome than is detected by infectivity, indicating that only about 0.1% of the genomic material present represents infectious virions. In addition, while treatment with amotosalen and UVA resulted in complete inhibition of HIV infectivity, the small size of the amplicon used in the NAT assays resulted in little, if any, impact of treatment on genome amplification. Factors That May Have a Negative Impact on Inactivation Non-enveloped viruses are the most challenging organisms to inactivate mostly due to their largely impermeable capsids with highly structured internal packing. The PCT process does not effectively inactivate the non-enveloped picornaviruses (eg, HAV [32] and poliovirus); however, a variety of other non-enveloped pathogens are successfully inactivated under the PCT conditions, see table 3 for examples.

Psoralens can bind nonspecifically to other biological molecules such as proteins and lipids [36]. For some psoralens, the binding constant to albumin and other proteins can be significant and cause substantial inhibition of the photochemical inactivation process. However, protein concentration has only a minimal effect on the activity of amotosalen and good viral inactivation can be easily obtainable even in whole plasma [37].

Conclusions

A wide variety of DNA- and RNA-based pathogens, as well as leukocytes, may be present in blood products for transfusion thus compromising the safety of the products [31]. The photochemical reaction of amotosalen and UVA light with nucleic acids is ideal for the inactivation of these pathogens and leukocytes in sensitive blood components. It has the dual nature of being broad spectrum in its activity yet extremely specific to nucleic acids in the presence of other biological molecules. Its application in the blood center setting offers the opportunity to significantly enhance the safety of transfusion medicine.

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Targeting DNA and RNA in Pathogens with Amotosalen HCl

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24 Oroskar A, Olack G, Peak MJ, Gasparro FP: 40 -Aminomethyl-4,5’,8-trimethylpsoralen photochemistry: The effect of concentration and UVA fluence on photoadduct formation in poly(dA-dT) and calf thymus DNA. Photochem Photobiol 1994;60:567–573. 25 Rodighiero G, Musajo L, Dall’Acqua F, Marciani S, Caporale G, Ciavetta I: Mechanism of skin photosensitization by furocoumarins: Photoreactivity of various furocoumarins with native DNA and ribosomal RNA. Biochim Biophys Acta 1980;607:215–220. 26 Bachelleri J-P, Thompson JF, Wegnez MR, Hearst JE: Identification of the modified nucleotides produced by covalent photoaddition of hydroxymethyltrimethylpsoralen to RNA. Nucleic Acids Res 1981;9:2207–2222. 27 Grass JA, Hei DJ, Metchette K, Cimino GD, Wiesehahn GP, Corash L, Lin L: Inactivation of leukocytes in platelet concentrates by photochemical treatment with psoralen plus UVA. Blood 1998;91:2180–2188. 28 Lin L, Cook D, Londe H, et al: Use of variable length amplicons to determine the efficiency of viral inactivation by psoralen mediated photochemistry. Blood 1993; 82(suppl 1):10(abstr 401a). 29 Sawyer LS, Dupuis K, Bernard K, Lane R, Van Voorhis W, Grellier P, Guillemain B: Helinx technology inactivates high titers of a variety of blood-borne pathogens in platelets. 43rd Interscience Conf Antimicrobial Agents and Chemotherapy, Chicago, Sept 2003.


30 Van Voorhis WC, Barrett LK, Eastman RT, Alfonso R, Dupuis K: Trypanosoma cruzi inactivation in human platelet concentrates and plasma by psoralen (amotosalen HCl) and long-wavelength UV. Antimicrob Agents Chemother 2003;47:475–479. 31 Summary of Intercept Platelet Product Characteristics. Irish Medicines Board, 2003. 32 Dupuis K, Cerus Corporation: Personal communication, 2003. 33 Truitt RL, Johnson BD, Hanke C, Talib S, Hearst JE: Photochemical treatment with S59 psoralen and ultraviolet A light to control the fate of naı¨ve or primed T lymphocytes in vivo after allogenieic bone marrow transplantation. J Immunol 1999;163:5145–5156. 34 Barre FX, Asseline U, Harel-Bellan A: Asymmetric recognition of psoralen interstrand crosslinks by nucleotide excision repair and the error-prone repair pathways. J Mol Biol 1999;286:1379–1387. 35 Hanson CJ: Personal communication, 2003. 36 Veronese FM, Bevilacqua F, Schiavon O, Rodighiero G: Drug-protein interaction: Plasma protein binding by furocoumarins. Il Farmaco, [Sci] 1979;34:716–725. 37 Alfonso R, Lin C, Dupuis K, et al: Inactivation of viruses with preservation of coagulation function in fresh frozen plasma. Blood 1996;88(suppl):526a(abstr).

Wollowitz

Review Article Transfus Med Hermother 2004;31(suppl 1):17–23

The Science of Safety: Toxicological Review of Amotosalen HCl A.D. Dayan London, UK

Key words Amotosalen Neoantigenicity Platelets Toxicity testing UVA illumination Summary The strategy used to plan toxicity testing of the INTERCEPT Blood System platelets is discussed and the results of the comprehensive non-clinical studies as provided to regulatory agencies are presented. The studies covered the toxicity of the psoralen amotosalen HCl added to platelets, the effect of residual photoproducts after controlled UVA illumination and any induced change in platelet antigens. Independent studies had demonstrated the normality of the physiology, survival and functions of treated platelets. The formal toxicity tests covered the pharmacokinetics of amotosalen and its photoproducts, their actions in safety pharmacology, acute and chronic toxicity, genotoxicity, full reproduction toxicity and p53 carcinogenicity tests. Local irritancy, phototoxicity and sensitisation potential were also examined, as well as platelet neoantigenicity and any risk from the physical components of the system. The ‘safety margin’ between high dose effects in laboratory models and the exposure of patients in the clinic is 45- to >1,000-fold, factors much higher than for many medicines and substances in the diet.

Introduction

The toxicological investigation of amotosalen HCl provides an excellent example of how a conventional, general approach to the detection of chemical hazards has been adapted, on scientific grounds, to the special circumstances of pathogen reduction treatment of platelet transfusions exposed to amotosalen and controlled UVA illumination. Comprehensive investigations specifically devised to study the unique conditions of the INTERCEPTTM Blood System for platelets have explored the risks of toxicity, reproduction and genotoxicity, carcinogenicity, pharmacokinetics and metabolism, local tolerance, phototoxicity and sensitisation.



The purpose of toxicity testing is to provide information about the occurrence or absence of chemical hazards and the relationship between exposure to the test substance and the dose used. In this way, the demonstration of hazard can be extrapolated to determine the likelihood of any potential risk to patients as a consequence of their particular treatment. An understanding of the hazards can then be used to make a judgment on safety, which is based on balancing the benefits against the risks. The toxicity of amotosalen and its metabolites in the body has been investigated in the same way as any other chemical to which the body may deliberately be exposed. The potential toxicity of free or bound photoproducts of amotosalen produced by UVA illumination and any consequences of UVA exposure of platelets have also been investigated. In the benefit–risk assessment, the toxicological findings have been correlated with the equally important studies demonstrating the preservation of platelets and retention of their normal physiological state and functions after treatment, ultimately providing clinical proof of effectiveness and safety in humans. Amotosalen treatment of platelets prior to transfusion occurs in a sophisticated medical device, the INTERCEPT Blood System for platelets, comprising various collection and holding bags, connectors and tubes, additive solution (InterSolTM), a controlled UVA illumination system and a compound adsorption device (CAD) to remove photoillumination products and any unused amotosalen. In addition to the comprehensive toxicity studies mentioned above, a further complete set of device toxicity evaluations was designed to explore the possible biological and chemical effects of any substances that might be leached from the components of the device. Consideration has also been given to the safety-in-use of the INTERCEPT Blood System for platelets, ie, would there be any risk to staff if part of the system were to leak, releasing reagents and storage solutions, and how are INTERCEPT Blood System units

Prof. Anthony D. Dayan c/o Remedica Publishing 32–38 Osnaburgh Street UK-London NW1 3ND

safely disposed of after use? A simplified outline of the components of the INTERCEPT Blood System is shown in figure 1 and the principal aspects of toxicological concern in planning comprehensive studies are listed in table 1. Toxicological studies for the INTERCEPT Blood System for platelets have been broader than those conventionally performed on new drugs because it was necessary to take into account several features unique to a system of combined photochemical treatment of platelets for transfusion. The investigative methods used have been based on procedures common to any comprehensive suite of toxicity tests but adapted to reflect the special circumstances associated with the processing of platelets for transfusion. In the European Union the overall strategy of testing and the procedures employed were discussed beforehand with the German TV, which regarded the INTERCEPT Blood System for platelets as a hybrid between a ‘pharmaceutical’ and a medical device, requiring assessment by specialised official agencies responsible both for ‘Medicines’ and ‘Devices’. The results obtained were reviewed and accepted by those official bodies as part of the official procedures awarding the ‘CE’ mark to the INTERCEPT Blood System for platelets. A1 DONOR

A2

A3 G

C Blood Cells Protein B

Add chemical

Store

D Irradiate

H

E

PATIENT

As the objectives of the toxicity-testing programme have been to study the complete INTERCEPT Blood System for platelets, the following potential hazards have been analyzed: amotosalen levels of amotosalen post-treatment with the CAD bound photoproducts attached to platelets the pharmacokinetics and metabolism of amotosalen and its photoproducts as part of the evaluation of exposure and toxicity, and the efficacy of the CAD possible changes in the antigenicity of platelets after treatment with amotosalen and UVA illumination effects of amotosalen treatment and UVA illumination on the structure and function of platelets and on their viability after storage any effects of leachates from the containers, tubing, and other components of the INTERCEPT Blood System. All of these aspects were examined in a wide-ranging set of experimental studies, based on adaptations of standard methods, to test acute, subchronic, and reproduction toxicity, safety pharmacology, genotoxicity, carcinogenic potential, local tolerance, phototoxicity and photosensitisation, and neoantigenicity of treated platelets. The pharmacokinetics and metabolism of amotosalen and its photoproducts have also been examined. In parallel, there have been extensive investigations of the morphology, functional state, survival, viability and efficacy of treated platelets. Much of the work has been done in vitro, some of necessity has been done in animals, and findings regarding the safety and efficacy of treated platelets have subsequently been confirmed by extensive clinical investigations in patients.

Clean up F

What Has Been Tested and Why?

± γ-rad

Fig. 1. Outline of the INTERCEPT Blood System for pathogen reduction treatment of platelets. Areas of toxicological importance are denoted by the letters A–H and are explained in table 1.

In conventional toxicity tests, in vitro systems are exposed to the drug that is to be tested and it is directly administered to animals. Patients given INTERCEPT platelets receive only 1 mg/kg of residual free amotosalen. In these

Table 1. Areas of toxicological importance shown in figure 1 Area

Stage

Potential complications

A1, 2, and 3

plastic connectors, bags

biocompatibility, leachates, toxicity

B

cell/protein separation system

as above

C

chemical

toxicity, phototoxicity, neoantigenicity?

D

irradiation

photoproducts, toxicity, cells/protein? neoantigenicity?

E

clean up

efficacy, leachates, toxicity, neoantigenicity?

F

g-irradiation?

cells/protein? container? neoantigenicity?

G

storage

container leachates? toxicity, neoantigenicity?

H

patient

product survival and efficacy, susceptibilities, toxicity, phototoxicity, neoantigenicity?

18


Dayan

patients, most of the exposure will be to residual photoproducts: a small quantity in the platelet-suspending medium (the platelet storage solution comprises 65% InterSol solution and 35% plasma) and some bound to platelets and plasma macromolecules. Another factor considered in designing the non-clinical testing programme was the need to employ autologous plasma and platelets whenever possible to avoid the complication of heterospecific immune responses after repeated doses. As there had been little experience of the production of rodent and canine plasma and platelets in ways suitable for toxicity testing prior to the amotosalen programme, means had to be devised to provide suitable materials for treatment and testing. Many of the in vivo toxicity tests have used a 35% solution of plasma in the appropriate platelet additive solution, InterSol as this represents the matrix and vehicle used in medical treatment. In some experiments, autologous canine INTERCEPT platelets were given up to 3 times weekly for 3 months, a treatment regimen that reflects multiple and long-term human clinical use of platelet transfusions. Toxicity studies were also conducted with amotosalen alone, as much higher doses could be used and the corresponding ‘safety margin’ could be established. For functional testing, human platelets were prepared, as in real-life treatment, and the physiological, biochemical, and functional state of the platelets were tested in vitro and in vivo. All the studies have been performed in compliance with Good Laboratory Practice requirements. Ciaravino et al. have published a full account of the toxicity testing programme, the results obtained and a critical evaluation of these results [1]. The detailed findings discussed in this paper have been taken from this report. Wagner et al. provide a helpful review from a broader perspective [2]. Pharmacokinetics and Metabolism A conventional approach was used to investigate pharmacokinetics and metabolism. Studies included: (i) cold as-

says for amotosalen; and (ii) experiments with 14C-amotosalen, which permitted analysis of the overall disposition and metabolism of the compound and its photoproducts after UVA illumination in the presence and absence of platelets, as well as the effects of using the CAD. The principal pharmacokinetic variables are summarised in table 2. Experiments have shown that amotosalen and its principal photoproducts are rapidly cleared (half-life 6 h) from plasma and accumulation did not occur, even after 3 months of repeated transfusions of amotosalen alone in plasma. The CAD led to a 4- to 5-fold reduction of exposure to photoproducts and the small amount of residual amotosalen after incubation for 6–24 h as in clinical practice. Experiments with radiolabelled-amotosalen demonstrated that, during photoillumination, 80% of amotosalen was converted to photoproducts: two-thirds of these photoproducts were free in the supernatant and one-third were bound to plasma macromolecules (lipids with a molecular mass >5 kDa) and platelets. The clearance of labelled photoproducts was slower than the clearance of amotosalen itself, but the unbound fraction was eliminated quite rapidly. The bound fraction in plasma was removed more slowly, reflecting the physiological turnover of normal plasma constituents. Elimination occurred by excretion in faeces and urine. There was considerable similarity between the kinetics and metabolism in man and animals, which strengthens the value of the animal toxicity tests. Safety Pharmacology and Acute Toxicity Studies Several experiments, using instrumental and clinical techniques, were carried out to determine the acute effects of high i.v. doses of amotosalen and its photoproducts on the function of the principal physiological systems of the body. The systems examined were the cardiovascular (cardiac rate, rhythm, electrocardiogram [ECG] and blood pressure), respiratory, nervous and gastrointestinal systems. Single doses up to 25–80 mL/kg i.v. treated 35% autologous plasma in InterSol solution, an appropriate surrogate

Table 2. Pharmacokinetics and toxicokinetics of amotosalen and its photoillumination products after i.v. administration. AUC ¼ Area under the curve; CAD ¼ compound absorption device. In patients given a conventional transfusion of INTERCEPT platelets, the AUC of the photoproducts was 0.6 ng h/mL, Cmax was 0.9 ng/mL, and t1/2 was 6.5 h [1] Rat

Dose amotosalen or equivalents

Dog

Amotosalen

Photoproducts no CAD

Photoproducts with CAD

Amotosalen

Photoproducts no CAD

Photoproducts with CAD

18 mg/kg

260 mg eq/h

50 mg eq/h

25 mg/kg

232 mg eq/h

70 mg eq/h

Cmax, ng/mL

1,681

1,110

770

3,458

1,510

1,290

tmax, min

3

2

2

2

3

3

AUC, ng h/mL

1,952

11,400

8,900

–

56,600

51,800

t1/2l1,2,3, h

0.7

1, 11, and 52

1, 7, and 44

–

4, 36, and 180

7, 41, and 177

Toxicological Review of Amotosalen HCl


19

for the preparation given to humans, had no effect on any of these systems. The cardiac rhythm and ECG were not affected. In acute toxicity tests, treated 35% plasma was only toxic when a large volume was administered i.v., causing temporary volume overload. The minimum toxic dose of free amotosalen i.v. was about 220 mg/kg in the rat; it resulted in piloerection, weakness and development of a hunched posture. Administering amotosalen itself at 40 mg/kg i.v. in the dog caused convulsions. To put these values into perspective, in a patient given INTERCEPT platelets, the maximum exposure to amotosalen is about 1 mg/kg. Subchronic Toxicity Testing These experiments are a major part of the backbone of toxicity testing and risk evaluation because they involve a wide range of clinical, laboratory, and pathological assessments of the animal. To gain an understanding of its properties, certain full toxicity studies were performed on amotosalen itself; however, most work has been carried out on treated 35% plasma (35% autologous plasma in InterSol platelet additive solution after admixture with amotosalen, photoillumination and either testing as such or after exposure to the CAD). These studies most closely represent the preparations to which patients will be exposed. Toxicity tests in the rat and dog involved i.v. treatment for up to 28 days, once and 3 times weekly in the dog for 13 weeks, and once daily in the rat for 13 weeks. Doses of amotosalen up to 75 mg/kg/day i.v. for 28 days were used. When treated 35% plasma was tested, with and without CAD treatment, there was exposure to UVA illumination products up to 350 mg/kg/day for 28 days in the initial study and subsequently for 13 weeks in the dog and rat. Two other special studies were also carried out. First, dogs were given canine INTERCEPT platelets i.v. once weekly for 13 weeks; this resulted in exposure to UVA illumination products up to a level of 350 mg/kg/week. Second, cynomolgus monkeys were infused i.v. with human INTERCEPT platelets, with and without use of the CAD, every 2 days for 14 days. Without CAD treatment, exposure to amotosalen and free photoproducts was 350 mg/kg; after CAD treatment exposure was reduced to about 8 mg/kg. The limiting factor in dosing animals was the risk of overloading the circulation with a large volume of a proteinrich solution. In no study was there any evidence of toxicity in the repeated dose tests of treated plasma when animals were comprehensively examined at intervals during dosing and terminally. Enlargement of the liver was not observed and there was no sign of enzyme induction. The veins through which the repeated i.v. infusions were given did not show any particular signs of local irritation. Considering the pattern of clinical usage of platelet transfusions, it was con-

20


cluded that there would be no value in undertaking further toxicity tests. Genetic Toxicity Testing These experiments were of critical importance because amotosalen, as a psoralen, was itself expected to be genotoxic under certain experimental conditions, and the key question was whether this property would be manifested under circumstances that could pose a risk to patients. Amotosalen, as expected, was positive in the Ames test, but only in strain TA1537 at a concentration of 103 mg/mL with S9 metabolic activation and at a concentration of 44 mg/mL in the absence of S9. Amotosalen was also positive in the mouse lymphoma test (strain L5178Y TK+/– ) at a concentration >7.5 mg/mL with S9 and at >65 mg/mL without metabolic activation. In addition, it gave a positive result, in the Chinese hamster ovary (CHO) cell chromosome aberration test at a concentration of 40 mg/mL with S9 and at a concentration 5 mg/mL without S9. It is important to note that amotosalen gave a negative result in the mouse bone marrow micronucleus test at doses up to 60 mg/kg i.v. and in the rat liver unscheduled DNA synthesis (UDS) test at doses up to 34 mg/kg i.v., the maximum tolerable doses under the test conditions. These doses gave plasma concentrations of 40 mg/mL. Human platelets treated with amotosalen and UVA illuminated in 35% plasma, as they would be prepared for clinical use, but without CAD treatment, gave clear negative results in the Ames test, chromosome aberration and mouse lymphoma tests in vitro. Negative results were also given in the mouse bone marrow micronucleus and rat liver UDS tests in vivo at up to a maximum tolerable dose of 200 mg/kg of residual amotosalen. A further intensive test was performed in which a human platelet preparation in 35% plasma was treated 25 times with amotosalen and UVA illumination according to the standard protocol, but without CAD exposure. Despite the presence of much higher levels than normal of bound and free products, the Ames (TA1537) and CHO cell chromosome aberration tests were negative except at levels of amotosalen predicted to be positive in studies with amotosalen alone. It was concluded that treatment of human platelets in 35% plasma plus the appropriate electrolyte solution (InterSol) using the INTERCEPT Blood System for platelets does not present any genotoxic risk to humans. Carcinogenicity Test The lack of carcinogenic activity in the p53 mouse test complemented the results of in vivo genotoxic testing. It was considered that performing conventional carcinogenicity tests in rodents would be impossible because it would not be feasible to inject the test solutions i.v. for 2 years. Instead,

Dayan

the new procedure of a shorter-term test in heterozygous transgenic p53+/ – mice was used to overcome this problem. Animals were dosed i.v. 3 times weekly for 6 months, with treated 35% plasma 20 mL/kg (this corresponded to 200 mg eq/kg of residual amotosalen and free and bound photoproducts). Another group of mice was given 20 mL/kg of 150 mM amotosalen (approximately 1 mg/kg) in plasma; this corresponded to approximately 1,000 times the normal clinical exposure. There was no evidence of carcinogenicity in any animal group studied. The positive control in the study, p-cresidine, a known bladder carcinogen, was found to induce tumours. Reproduction Toxicity Testing Clinical consideration of the circumstances under which INTERCEPT platelet transfusions may be given suggests that the underlying diseases themselves will often be a considerable threat to reproduction by the patient. However, for regulatory reasons and to permit a comprehensive assessment of the toxicity of the INTERCEPT Blood System for platelets, complete conventional fertility (Segment I) and maternal and fetal toxicity (Segment II) tests were performed on treated plasma with and without CAD treatment. This ensured maximum exposure to free and residual photoproducts. The maximum tolerable dose of the 35% treated plasma was 25 mL/kg/h i.v., corresponding to 350 mg eq/kg residual amotosalen. No harmful effect was found on fertility, maternal health or fetal growth and development. Furthermore, there was no evidence of teratogenicity. A peri- and postnatal test in the rat did not indicate any harmful effects on pups [V. Ciaravino, personal communication, 2003].

Neoantigenicity In the INTERCEPT Blood System, platelets are exposed to amotosalen and UVA illumination under circumstances specifically designed to damage DNA. It is, therefore, reasonable to query whether there could also be an effect on the antigenicity of platelets and plasma macromolecules due to the demonstration of covalent binding products. Is there a possibility that the resultant neoantigens could cause harmful immune responses in patients? This has been indirectly examined in the repeated dose toxicity tests by demonstration of the absence of anaphylactic effects and changes in the immune system, or any other system, in the animal. In an experiment in which dogs were given repeated allogeneic platelet transfusions over a 3-month period, there was no variation in the response to the platelet transfusions. This suggests an absence of harmful neoantigens in treated platelets. Direct studies have also been carried out in which untreated human platelets, INTERCEPT platelets and photochemically treated plasma (both the latter carrying covalently bound photoproducts) were used to hyperimmunise rabbits. When sera from these animals were tested against human platelets and plasma by FACScan, crossed-immunoelectrophoresis, and Ouchterlony methods, no neoantigens were found.

Risk Assessment of Platelets Treated with the INTERCEPT Blood System

These experiments are important because amotosalen was deliberately chosen for development because of its special phototoxic activity under controlled circumstances. Theoretically, this activity could pose a risk to patients after infusion of INTERCEPT platelets. When amotosalen was tested in preliminary experiments, it was shown to cause phototoxicity after 1 and 10 mg/kg i.v. An intensive multidose study was then performed. To mimic a ‘worst case’ treatment schedule, treated 35% plasma was infused i.v. up to the maximum tolerable dose of 25 mL/kg 3 times weekly for 4 weeks, with exposure to UV illumination at the end of each week. In this experiment CAD treatment of the plasma was reduced to 1 h, resulting in the presence of a higher than normal concentration of amotosalen (5 mM instead of the normal 0.5 mM) and photoproducts. There was no evidence of phototoxicity or photosensitisation even after such prolonged and repetitive exposures. It was concluded that there was no indication of any risk of phototoxicity or photosensitisation in patients receiving INTERCEPT platelets.

As mentioned initially, nonclinical studies can only be used to explore hazards, predict risks or to demonstrate the absence of detectable dangers; the actual decision about safety is a judgment based on weighing risks against the benefits. This brief review of several years work on amotosalen and UVA treatment of human platelets for transfusion, the INTERCEPT Blood System, has described the pharmacokinetics and disposition of amotosalen and its photoproducts, rigorous systemic and phototoxicity testing of amotosalen and the properties of allogeneic INTERCEPT platelets in a model system in the dog. As repeated treatments will require continual use of the complete INTERCEPT Blood System, there has also been testing of any materials leached from the plastics of the device, e.g., tubing, connectors and bags; all plastics used are of medical grade. No evidence of any harmful effect of leachates was found. Based on the complete toxicological information, the potential for occupational and environmental risk has also been assessed and has been shown to be very low. Disposal of used units is acceptable by the means normally employed for medical devices contaminated by body fluids. In broad terms, there has been little or no evidence of toxicity from autologous INTERCEPT platelets, or asso-



Phototoxicity and Photosensitisation

21

ciated with free or bound photoproducts, except at very high doses. Human INTERCEPT platelets retain their physiological properties and functions and survive subsequent storage as well as untreated platelets. Whenever possible, it is helpful to try to express evidence about the risk of toxicity of any substance in a quantitative manner, as the ‘safety margin’ relative to clinical use and patient exposure, in order to give some understanding of the strength of statements about safety. The ‘safety margin’ is calculated as the ratio between the lowest toxic dose in laboratory tests and the highest human dose. As a very simple calculation this ignores differences in pharmacokinetics between species and begs the question of what is the most important toxic risk? In the case of a conventional medicine, it can be replaced by direct consideration of the therapeutic plasma level and the level associated with toxicity. An alternative is to calculate the so-called ‘safety factor’, which is taken as a small fraction of the highest non-toxic dose in appropriate laboratory tests. An acceptable ‘safety factor’ calculated in this way, for example of a food component to which there may be life-long exposure, is often taken as 1/100, representing the combination of 1/10 for extrapolation between species and 1/10 to represent variation in susceptibility between individuals. This approach is commonly used when considering the toxicity of foodstuffs and contaminants; in this case it is termed the Acceptable Daily Intake. The fraction can be adjusted to take limited data into account and to allow for particular risks [3]. Unlike the ‘safety margin’, it does reflect possible species differences in kinetics, and it is based on the most critical toxic response. In the case of relatively brief or intermittent treatments the value of the calculated factor must be considered in relation to conventional factors, such as 1/100, which are based on lifetime exposure. Blindly applying conventional values for ‘safety factors’ to substances

administered for only a short period of time may be misleading. For these reasons and to remind us of the extrapolations involved in the calculations, many instead prefer the term ‘uncertainty factor’. As neither factor directly reflects the consequences of exposure of humans, they must be seen as no more than guides that require re-evaluation in the light of findings in clinical use. They should be most helpful in early risk–benefit assessments but become of more limited importance later on when both benefit and risk can be assessed from findings in the real target of treatment – patients – taking into account any consequences of the underlying diseases, therapeutic efficacy and adverse events. Although there is now extensive clinical experience of INTERCEPT platelet transfusions, it may still be helpful to at least consider the calculated ‘safety margins’ (see table 3). These cover a very wide range of possible toxic hazards. Many possible harmful effects on bodily systems have been excluded experimentally, e.g., effects on cardiac activity and other physiological systems, or effects on platelet antigenicity. The calculations show that there is a considerable margin between the human exposure and the highest non-toxic or lowest toxic dose across the entire range of completed studies, which represent a comprehensive range of potential toxic actions. Taking into account the extent of the toxicological coverage, the overall similarity between species in their known responsiveness to psoralens, and the similarity between the kinetics of amotosalen in man and animals, it is apparent that the INTERCEPT Blood System for platelets has been extensively studied and it does not carry any particular risk to patients. Acknowledgement I am grateful for the advice of Dr. V. Ciaravino in the preparation of this report.

Table 3. ‘Safety margins’ for the INTERCEPT Blood System for platelets. The toxicity data for laboratory experiments come from the studies summarized in this paper. The corresponding human exposure of 1 mg/kg is taken from Ciaravino et al. [1] Residual amotosalen

Free photoproducts

Bound photoproducts

Acute toxicity test: rat and dog

>1,120

>49

>29

1- and 3-month toxicity tests: rat and dog

>350

>16

>9

Genotoxicity of amotosalen

>7 106 to 2,000 depending on test method >40,000 for in vivo tests

inactive

inactive

Phototoxicity

100–1,000

no effect of maximum technically feasible dose


Reproduction toxicity

>350



Carcinogenicity

>1,000



22


Dayan

References 1 Ciaravino V, McCullough T, Dayan AD: Pharmacokinetic and toxicology assessment of INTERCEPT (amotosalen and UVA treated) platelets. Hum Exp Toxicol 2001;20:533–550.


2 Wagner SJ: Pathogen Inactivation in Blood Components. In: Simon TL, Dzik WH, Snyder EI, Stowell CP, Strauss RG (eds): Rossi’s Principles of Transfusion Medicine, ed 3. Philadelphia, Lippincott Williams Wilkins, 2002, pp 806–814. 3 Klaassen CD (ed): Casarett and Doull’s Toxicology, ed 6. New York, McGraw-Hill, 2001, pp 91–93.


23

Review Article Transfus Med Hemother 2004;31(suppl 1):24–31

Protection against Transfusion-Associated Graftversus-Host Disease in Blood Transfusion: Is Gamma-Irradiation the Only Answer? P. Schlenke Institute of Immunology and Transfusion Medicine, University of Schleswig-Holstein, Campus Lu¨beck, Germany

Key words Graft-versus-host disease Transfusion Irradiation Pathogen inactivation Summary Transfusion-associated graft-versus-host disease (TA-GvHD) is an infrequent, but fatal, complication associated with transfusion of any cellular blood component. At present, gamma-irradiation of cellular blood components is the only acceptable method for preventing TA-GvHD. All blood components can be subjected to gamma-irradiation, which irreversibly inactivates leukocytes, especially T lymphocytes, while preserving the functional integrity of the pharmaceutically effective cellular blood components. Pathogen inactivation technologies have been developed to eliminate the minimal transfusion-associated risks caused by viral or bacterial contaminants. The INTERCEPTTM Blood System for platelets is based on the use of amotosalen HCl and UVA-irradiation. It sufficiently inhibits the replication of parasitic, bacterial, and viral genomes and inactivates T cell replication and cytokine generation on at least an equivalent level to gamma-irradiation. The INTERCEPT Blood System for platelets shows great robustness in inactivating viable T lymphocytes with more than a 5 log10 reduction in platelet concentrates, and it may have the potential to replace gamma-irradiation of platelet concentrates.

Introduction

Modern transfusion medicine is a clinically oriented subject, ideally combining the traditional fields of immunohaematology, blood component supply, and the novel challenges emphasising patient care and treatment [1, 2]. In the last decade, specialists in transfusion medicine have become more involved in designing appropriate transfusion strategies or participating in blood-derived cellular therapies with their clinical colleagues. Therefore, transfusion



medicine, accompanied by emerging biotechnologies in the field of blood safety and blood supply, is now an ideal professional platform where research and development can be translated into clinical practice. One promising approach uses pathogen inactivation procedures to reduce some of the side effects currently associated with blood transfusions, such as the risk of transfusion-associated infections or the transfusion-associated graft-versus-host disease (TAGvHD) caused by blood products contaminated with viable allogeneic leukocytes [3]. Furthermore, recent biotechnological advances, such as the development of perfluorocarbons, may partly replace conventional cellular blood supply by artificial oxygen carriers [4]. Both technologies are currently being evaluated in clinical trials and have the potential to improve the standard of the blood that is supplied to patients [5, 6].

Transfusion-Associated Graft-versus-Host Disease

Incidence Graft-versus-host disease (GvHD) is a well-known complication of allogeneic bone marrow or haematopoietic stem cell transplantation [7]. Until now, post-transplant morbidity and mortality have been predominantly affected by severe GvHD and its treatment. However, TA-GvHD is a very infrequent, but nonetheless fatal, complication associated with transfusion of any cellular blood component [8]. Most TA-GvHD cases reported are caused by a failure in the prescription of gamma-irradiated blood or are a result of laboratory errors such as omitting the procedure or because of its ineffectiveness. Primarily, TA-GvHD occurs in immunologically disparate recipients who are incapable of mounting a sufficient immune reaction to reject donor lymphocytes. Immunocompromised patients who are at risk are summarised in table 1. Foetuses and premature/

PD Dr. med. Peter Schlenke University of Schleswig-Holstein Campus Lu¨beck Institute of Immunology and Transfusion Medicine Ratzeburger Allee 160, D-23538 Lu¨beck Tel. +49 451 50028-41, Fax -57 E-mail [email protected]

mature neonates with an indication for intrauterine or exchange transfusion are included in this at-risk group [9]. Taking recently published data into account, even organ transplant recipients, patients suffering from non-Hodgkin’s lymphoma, or patients receiving fludarabine treatment are at risk for TA-GvHD [10–16]. In this context, it is amazing that there have been no reports of TA-GvHD in HIV-patients, with the exception of 1 case of a child with mild cutaneous TA-GvHD [17]. However, on the other hand, there are some case reports of TA-GvHD in patients with an apparently intact immune function or supposed immunocompetence [18]. Because of its rarity and the delayed onset of clinical features, the incidence of TA-GvHD is likely to be underestimated. In 1990, a survey by the American Association of Blood Banks (AABB) published 12 cases of TA-GvHD reported on the basis that almost 14 million blood units per annum were transfused without irradiation [19]. In the UK Serious Hazards of Transfusion (SHOT) Steering Group report, 13 cases of TA-GvHD were reported over a 5-year period (1996–2000) [14]. Interestingly, only 1 case was reported in the last 2 years of this study. This case documented a 14-year-old girl with acute lymphoblastic leukaemia, which suggests that currently practiced leukoTable 1. Patients at risk for TA-GvHD Immunosuppressed patients: Congenital immunodeficiency syndromes Foetuses (intrauterine transfusion) Neonates (exchange transfusion) Mature neonates ð*Þ Haematological malignancies (acute and chronic leukaemia, Hodgkin’s disease, non-Hodgkin’s lymphoma) Solid tumors ð*Þ Solid organ transplantation ð*Þ All bone marrow and stem cell recipients Fludarabine therapy (purine analog) Monoclonal antibody therapy ð*Þ Nonimmunosuppressed patients: Recipients of blood of HLA homozygous donors Populations with restricted HLA polymorphism (Japan) Directed blood donations from relatives HLA ¼ Human leukocyte antigen. ð*Þ ¼ Indication for gamma-irradiation not generally accepted.

cyte depletion processes cannot always prevent TA-GvHD in susceptible patients. In a Japanese study, the risk of developing TA-GvHD in immunocompetent hosts was estimated to be about 1 : 500 without preventive gamma-irradiation; this comparatively high risk is explained by the relative homogeneity of human leukocyte antigen (HLA) haplotypes and the common practice of blood donations by relatives in the Japanese population [20]. The inability to accurately determine the frequency of TAGvHD in different at-risk patient groups is further compounded by two factors: (i) gamma-irradiation for prevention of TA-GvHD is subject to patient selection; and (ii) the availability of novel modifications in blood component production, such as universal leukocyte depletion, which have a high degree of efficacy. To date, there are no data available to indicate that TA-GvHD strongly correlates with exceeding a specific threshold of viable and proliferative T lymphocytes. Clonable T lymphocytes at a concentration of 104 to 106 per unit may result in TA-GvHD; nevertheless, this range is virtually identical to the degree of leukocyte reduction achieved by filtration or physical separation [21]. In the near future, the impact of reduced lymphocyte contamination in blood components and the adequate irradiation dosage required to destroy T lymphocytes irreversibly have to be evaluated in order to answer these questions. Since TA-GvHD prevention has already been well established using gamma-irradiation, the FDA require that any new T-cell inactivation method must be tested in vitro and be as effective as irradiation with 2,500 cGy, as per the clonal T-cell expansion assay devised by Pelzynski [22], before it can be tested in a clinical trial. Clinical Manifestation For unknown reasons, the clinical features of TA-GvHD differ significantly from GvHD related to allogeneic bone marrow transplantation (see table 2). The early signs of TA-GvHD are fever, skin rash, diarrhea, and hepatitis. None of these symptoms are specific for TA-GvHD and each can often be attributed to other complications associated with severely immunosuppressed patients, such as bacterial or viral infections, or pharmaceutical side effects.

Table 2. Differences in the clinical manifestation of GvHD versus TA-GvHD Symptoms

Classical GvHD

TA-GvHD

Onset after BMT/transfusion Fever Skin rash Diarrhoea Hepatitis Bone marrow aplasia Pancytopenia Improvement during treatment Mortality

1–3 months yes yes yes yes (obstructive) no no yes (response rate up to 90%) 10–20%

within days or weeks yes yes yes yes (cellular) yes yes no (response rate 10%) near 100%

BMT ¼ Bone marrow transplant.

Prevention of TA-GvHD


25

The clinical outcome for patients with TA-GvHD is usually fatal, with death occurring in more than 90% of cases due to life-threatening infections as a consequence of profound bone marrow hypoplasia and peripheral pancytopenia. The diagnosis of TA-GvHD is made by skin (dermal infiltrate of lymphocytes) and/or bone marrow biopsy (‘empty marrow’) [3]. The detection of donor–recipient microchimerism by DNA or human HLA testing may also contribute to the diagnosis; however, donor T lymphocytes persist for weeks to years in a patient’s circulation [23, 24]. This is also reported for conventional blood component transfusions, exchange transfusions in neonates, and intrauterine transfusions without the development of TA-GvHD [25–27].

tive immune response may fail to occur because of nonrecognition of foreign antigens [28–30]. The probability of HLA-homozygous transfusions in unrelated individuals who are HLA heterozygous but share the same HLA haplotype has been calculated for different populations [31]. Subsequently, the clonal expansion of T lymphocytes, the cellular infiltration of tissues, and the cytokine release due to the inflammatory response are responsible for the systemic nature of TA-GvHD and its clinical manifestations. Although the complex interactions between cytokines and different cellular compartments (the ‘Th1/Th2 paradigm’) involved in the pathogenesis of acute GvHD have been reviewed, it remains unclear if these observations can be directly compared to TA-GvHD [32].

Pathogenesis Several prerequisites are necessary for the development of GvHD as a transfusion-associated complication [3]. 1. The individual blood component involved must be contaminated with viable donor T lymphocytes. 2. The transfusion recipient must express tissue antigens, such as major histocompatibility complex (MHC) antigens, that are not present in the blood donor. 3. The recipient’s immune system must be unable to recognise and destroy the allogeneic T lymphocytes that are transfused. In general, donor lymphocytes are destroyed by lymphocytolysis in an immunocompetent host (see table 3). This process is initiated by the recognition of foreign antigens followed by an effective immune response mediated by T lymphocytes and natural killer cells. In the case of TAGvHD, there are several reasons for the immune surveillance failure that leads to donor-derived lymphocyte engraftment with a high proliferative capacity in the host (see table 3). Especially in the case of HLA-homozygous blood donors who share one HLA haplotype with an HLA-heterozygous blood transfusion recipient, an effec-

TA-GvHD Prevention by Gamma-Irradiation

Gamma-Irradiation of Blood Components Gamma-irradiation is currently the only recommended approach to prevent TA-GvHD [33–35]. This prophylactic strategy aims to irreversibly inactivate leukocytes, especially T lymphocytes, while preserving the functional integrity of the pharmaceutically effective cellular blood component. Irradiation of cellular blood components damages nuclear DNA, either directly or via free radicals (DNA single-stranded or double-stranded breaks are formed), leading to the inactivation of T lymphocytes and antigen presenting cells [36]. Most of the blood components transfused contain viable lymphocytes with proliferative capacity. In practice, experience has revealed that TA-GvHD can develop following the transfusion of: whole blood red blood cell (RBC) units buffy coat platelets or platelets collected by apheresis unfrozen plasma fresh frozen plasma.

Table 3. Immunological response of transfused leukocytes depending upon the immune status and the human leukocyte antigen (HLA) system

Case 1 Immune status HLA Response Case 2 Immune status HLA Response Case 3 Immune status HLA Response

26

Recipient

Donor

normal heterozygous (A/B) rejection by the recipient’s immune system

normal heterozygous (C/D)

immunodeficient normal heterozygous (A/B) heterozygous (C/D) no rejection by the recipient’s immune system TA-GvHD caused by the donor’s immunocompetent cells normal normal heterozygous (A/B) homozygous (B/B) no rejection by the recipient’s immune system TA-GvHD caused by the donor’s immunocompetent cells


Schlenke

Cesium-137 source

In addition, repetitive granulocyte transfusions have had a renaissance in treating severely immunocompromised septic patients. These transfusions are accompanied by a particular risk of inducing TA-GvHD, since components are contaminated with high concentrations of fresh T lymphocytes. A more controversial issue is the actual risk of developing TA-GvHD by transfusing fresh frozen plasma containing up to 107 leukocytes per unit; leukocytes are rarely resistant to freezing procedures without the use of a cryoprotectant [37]. The likelihood of T lymphocytes surviving with proliferative potential cannot be ruled out; therefore, from a more sophisticated point of view, gamma-irradiation is strongly recommended to prevent rare TA-GvHD per se. In general, there are no data available to indicate that leukocyte depletion technologies will replace gamma-irradiation for one of the above-mentioned blood components in the near future.

Chamber 1

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= spacer (center and edge)

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95 mm

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Dose of Gamma-Irradiation, Equipment, and Dosimetry

46.8

37.0

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31.8

33.0

37.0

46.8

Historically, an effective dose of gamma-irradiation was presumed to be 15 Gy, which was thought to cause complete suppression of the mixed lymphocyte culture. Two observations brought this dose into question: (i) TAGvHD still occurred after gamma-irradiation with a dose of 15 Gy; and (ii) experimental data revealed that clonable T cells, although very infrequent, could be detected from T lymphocyte expansion in limiting dilution assays. Consequently, a dose of 25 Gy is now generally accepted for gamma-irradiation. However, there is no agreement expressis verbis to recommend 25 Gy as the appropriate mean or minimum dose. It is important to distinguish between the average value and the minimum dose at any position in the container because the dose of gamma-irradiation varies significantly, by almost 30–50%. Using commercially available irradiators, this inhomogeneous irradiation arises in both directions, horizontally (from the center to the periphery) and vertically (from the bottom to the top in the central axis). The limited irradiation field of a freestanding caesium-137 irradiator with a metal canister on a rotating turntable and the annually performed dosimetry in Plexiglas equivalents (qualified substitutes) are given in figure 1. In the US, the Food and Drug Administration demands a central dose of 25 Gy (minimum 15 Gy), whereas the recommendations in the UK, Germany, and the guidelines of the European Council (EC) require that a 25 Gy minimum dose is achieved [38–42]. With respect to the legislation and requirements of regulatory authorities, it is a well-established opinion that gamma-irradiation of blood components is a significant part of the manufacturing process and the EC guidelines for good manufacturing practice have to be applied [41].

49.1

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65 mm 35 mm 5 mm

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Fig. 1. Isodose distribution in a metal canister within and outside the irradiation field measured in Plexiglas as the material equivalent to blood. Freestanding gamma-irradiator (OB29/4-BA, STS-Buchler, Germany) with a caesium-137 source (half-life: 30 years) of 48 Tbq radioactivity in 1990. Irradiation time: 22 min; mean dose: 33.8 2.4 Gy (29.4–37.0 Gy).

Usually, special freestanding blood center gamma-irradiators equipped with a long half-life, gamma-emitting source (e.g., caesium-137 or cobalt-60) are used. The activity must be specified by the manufacturer and will fix the irradiation time (which must be a reasonable time for routine use) necessary to achieve a minimum dose of 25 Gy. In addition, the manufacturer provides the initial dosimetry at specified points in the irradiation canister when it is filled with material equivalent to blood according to the national standards. The dosimeters or dose mapping systems have to be calibrated prior to use. To achieve a higher level of irradiation uniformity, the irradiation field can be limited by using spacers at the bottom or top of the canister. On the basis of the dosimetry performed, the irradiation time can be calculated for a given time period (e.g., 1 year) and can be adapted by annually performed recalibrations. During design, installation, and maintenance of gamma-irradiators, it is strongly recommended that all other national requirements concerning the qualification of rooms, staff, and additional equipment be considered (e.g., radiation protection and fire protection).


27

Irradiated blood units have to be fully labelled to include the date of irradiation, the mean dose of irradiation, and, if necessary, the reduction of shelf life. In addition, to guarantee actual exposure to irradiation, gamma ray labels, which change from ‘not irradiated’ to ‘irradiated’, are used. These radiation-sensitive labels, however, are not accepted as a substitute for regular and precise dosimetry. Furthermore, gamma ray labels may not be necessary if qualified irradiators and validated procedures are able to clearly document the exposure for each given blood component (position) without any doubt. Manufacture of Gamma-Irradiated Blood Components Validation studies have suggested that gamma irradiation affects the membrane stability of RBCs, resulting in a decreased post-transfusion recovery in vivo [43]. Furthermore, this phenomenon is more pronounced when RBC units with a prolonged storage after gamma-irradiation are used. To avoid the unacceptably high extracellular potassium levels that result from storage and irradiation, it has become general practice to limit the irradiation procedures for RBC units collected within the previous 14 days to an additional 14-day storage period. However, a physician orders the supplement gamma irradiation for each patient, individually, as soon as the clinical decision has been made that transfusion of blood components is indicated. At our university hospital, the general storage of already gamma-irradiated blood components is not necessary, thanks to the use of an in-house radiation source that allows blood components to be irradiated within a reasonable time immediately prior to transfusion. The extracellular potassium peaks within the first 24 h after gamma-irradiation (the concentration may be twice as high as in nonirradiated controls). With this knowledge, it is recommended that intrauterine or exchange transfusions are performed with almost fresh and immediately gamma-irradiated units. In the context of platelet transfusion, there is accumulating evidence that gamma-irradiation with a dose of up to 50 Gy does not affect the platelet recovery in a patient’s circulation or the platelet shelf life in vivo [44–45]. Sweeney and co-workers published a mean in vivo recovery of 51 versus 52% and a 147 versus 146 h in vivo survival time when comparing apheresis platelet concentrates irradiated with 25 Gy on day 1 and transfused on day 5 with matched controls [46]. Results from in vitro studies suggesting a slightly higher surface expression of CD62 (P-selectin) in gamma-irradiated platelet concentrates did not correlate with a clinically significant functional impairment. More recently, Zimmermann et al. studied the effect of gamma-irradiation on white blood cell-reduced single-donor apheresis platelet concentrates. No adverse effect on platelet quality was observed on the basis of CD62 surface

28


expression, platelet aggregability, and supernatant b-thromboglobulin [47].

Pathogen Inactivation as an Alternative to Gamma-Irradiation

At present, gamma-irradiation of cellular blood components is the only acceptable method for preventing TAGvHD. However, a novel, innovative methodology for pathogen inactivation has now become commercially available in Europe [48]. The INTERCEPTTM Blood System is based on amotosalen HCl, a psoralen derivate (S-59) that intercalates into nucleic acids to form monoadducts and irreversibly crosslinks with DNA and RNA upon illumination with long-wavelength UVA light. This interaction with nucleic acids is highly specific and occurs at a high frequency (once per 83 base pairs) in comparison to gammairradiation, which causes strand breaks only once per 37,000 base pairs. In blood components, amotosalen stops the replication of a broad spectrum of pathogens – including bacteria, viruses, and protozoa – and leukocytes that contaminate blood products, while, at the same time, maintaining the functional integrity of the pharmaceutically active cellular compound, such as platelets. Examination of a variety of cell types has shown a unique response of amotosalen with respect to control of replication and shut down of gene expression [49]. The effectiveness of overall inactivation depends upon the concentration of amotosalen and the UVA light dose, and has been optimized to apply this technology to platelet transfusions in routine manufacturing procedures (150 mM amotosalen, 3 J/cm2 UVA). Using these specifications, the INTERCEPT Blood System is able to inactivate >5.4 log10 T lymphocytes, which is greater than the degree of inactivation produced by gamma-irradiation (see fig. 2) [50]. However, the pathogen inactivation technology is the more robust of the two procedures because complete T-cell inactivation is achieved, even when the amotosalen concentration is reduced by more than 1,000-fold [50]. In contrast, only a small reduction in the dose of gammairradiation significantly reduces the efficacy to suppress Tcell replication (5 Gy: 1.0 log10 reduction) [22, 50]. These in vitro results are in line with the clinical observation that TAGvHD can occur when transfusing blood components irradiated with a dose of less than 25 Gy as the minimum [51]. In addition, the loss of T-cell replication competence using PCR amplification of genomic DNA sequences, the inhibition of cytokine synthesis by contaminating leukocytes, and the surface expression of proliferation-associated T-cell activation markers, such as CD25 and CD69, has been studied in more detail [50, 52]. Grass et al. impressively demonstrated the complete shut down of interleukin-8 synthesis during platelet storage after photochemical treatment [53].

Schlenke

INTERCEPT Blood System >5.4 logs No loss of inactivation even when amotosalen concentration is reduced by >1,500-fold

1

Gamma-irradiation (2,500 cGy) >5.0 logs Small reduction in dose results in significant loss of efficacy

Experiment 1

6

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0 -1 -2 -3 150 µM amotosalen

-4 -5 -000.1 0.001

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10

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100

1,000

0

3

10

30

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300 1,000 3,000 10,000

Radiation dose (cGy)

Fig. 2. T-cell inactivation using the INTERCEPT Blood System or gamma-irradiation. With respect to irreversible T-cell inactivation, the INTERCEPT Blood System has a great robustness whereas a slight under-irradiation leads to a significant loss of efficacy. Reprinted with permission from Grass et al. [50].

Table 4. Principal advantages of pathogen inactivation via the INTERCEPT Blood System for TA-GvHD prevention compared with gammairradiation Advantages High frequency of DNA modifications by covalent monoadducts and crosslinks (loss of DNA replication competence) Great robustness in T-cell inactivation even when amotosalen is reduced (safety margin) Photochemical treatment during manufacturing process of platelet concentrates ‘ready for use’ application, no failure in delivering non-pathogen inactivated units) No necessity to apply radioactive material (No need to fulfill additional national requirements for radiation protection, e.g., room and staff qualification [24 h on-call service], radioactivity leakage) No necessity to install and uninstall a radioactive source (investment of 150.000 $) No need for annual dosimetry and to lengthen the time of irradiation to correct for decay of the radioactive source used (failure to treat outpatients in reasonable time) No need to use radiation indicators and label modified blood components (‘irradiated’ and new expiry date) Ability to inactivate pathogens as well as leukocytes simultaneously

Taken together, the results of these studies suggest that the INTERCEPT Blood System may well be an alternative to gamma-irradiation. The advantages of the INTERCEPT Blood System over gamma-irradiation are listed in table 4. In the first instance, and from a more practical point of view, this technology may have the potential to replace gamma-irradiation of platelet concentrates in particular; however, at the moment, this approach is not applicable to RBC units due to the inability of UVA light to penetrate these blood components. This limitation may be overcome by using frangible anchor-linker-effector molecule (FRALE) (S-303) in connection with a pH shift, which targets nucleic acids without the need for UVA illumination (but a pH shift is required).

At present, in contrast to GvHD, no effective treatment for TA-GvHD is available. Due to severe bone marrow aplasia, resistance to immunosuppressive therapies, and infectious complications, TA-GvHD leads to death, often within 1 month of transfusion. Therefore, the prevention

of TA-GvHD is of paramount importance. Worldwide, gamma-irradiation is currently the only recommended strategy for TA-GvHD prevention. All blood components can be subjected to gamma-irradiation (minimum dose 25 Gy), which inactivates T lymphocytes while preserving the functional capacity of the blood cells transfused. Over the past few years, pathogen inactivation technologies have been developed to eliminate the minimal transfusion-associated risks caused by viral or bacterial contaminants. It has been shown that the INTERCEPT Blood System, which is based on the use of amotosalen and UVA-irradiation, sufficiently inhibits the replication of parasitic, bacterial, and viral genomes and also inactivates T-cell replication and cytokine generation on at least an equivalent level as gamma-irradiation. These studies suggest that the INTERCEPT Blood System shows great robustness in inactivating viable T lymphocytes with more than a 5 log10 reduction in platelet concentrates. Therefore, the INTERCEPT Blood System will become an alternative to gammairradiation, particularly when clinical trials confirm adequate in vivo survival and equivalency to the already established gold standard.



Conclusion

29

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17 Klein C, Fraitag S, Foulon E: Moderate and transient transfusion-associated cutaneous graft versus host disease in a child infected by human immunodeficiency virus. Am J Med 1996;101:445–446. 18 Juji T, Takahashi K, Shibata Y, et al: Posttransfusion graft-versus-host disease in immunocompetent patients after cardiac surgery in Japan. N Engl J Med 1989;321:56. 19 Anderson KC, Goodnough LT, Sayers M, et al: Variation in blood component irradiation practice: Implications for prevention of transfusion-associated graft-vs.-host disease. Blood 1991;77:2096–2102. 20 Ohto H, Anderson KC: Survey of transfusion-associated graft-versus-host disease in immunocompetent recipients. Transfus Med Rev 1996;10:31–43. 21 Mu¨ller-Steinhardt M, Hennig H, Kirchner H, et al: Prestorage WBC filtration of RBC units with soft-shell filters: Filtration performance and impact on RBCs during storage for 42 days. Transfusion 2002;42:153–158. 22 Pelszynski MM, Moroff G, Luban NL, et al: Effect of gamma irradiation on T-cell inactivation as assessed by limiting dilution analysis: Implications for preventing transfusion associated graft vs. host disease. Blood 1994;83:1683–1689. 23 Kunstmann E, Bocker T, Roewer L, et al: Diagnosis of transfusion-associated graft-versushost disease by genetic fingerprinting and polymerase chain reaction. Transfusion 1992;32:766–770. 24 Wang L, Juji T, Tokunaga K, et al: Polymorphic microsatellite markers for the diagnosis of graft-versus-host disease. N Engl J Med 1994;330:398–401. 25 Lee TH, Donegan E, Slichter S, et al: Transient increase in circulating donor leukocytes after allogeneic transfusions in immunocompetent transfusion recipients compatible with donor cell proliferation. Blood 1996;85:1207– 1214. 26 Wang-Rodriguez J, Fry E, Fiebig E, et al: Immune response to blood transfusion in very low birthweight infants. Transfusion 2000;40: 25–34. 27 Vietor HE, Hallensleben E, van Bree SP, et al.: Survival of donor cells 25 years after intrauterine transfusion. Blood 2000;95:2709–2714. 28 McMilan KD, Johnson RL: HLA-homozygosity and the risk of related-donor transfusionassociated graft-versus-host disease. Transfus Med Rev 1993;7:37–41. 29 Thaler M, Shamiss A, Orgad S: The role of blood from HLA-homozygous donors in fatal transfusion-associated graft-versus-host disease after open-heart surgery. N Engl J Med 1989;321:25–28. 30 Kruskall MS: The major histocompatibility complex: The values of extended haplotypes in the analysis of associated immune diseases and disorders. Yale J Biol Med 1990;63:477– 486. 31 Wagner FF, Flegel WA: Transfusion-associated graft-versus-host disease: Risk due to homozygous HLA haplotypes. Transfusion 1995;35:284–291.


32 Ferrera JM, Krenger W: Graft-versus-host disease: The influence of type 1 and type 2 Tcell cytokines. Transfus Med Rev 1998;12:1– 17. 33 Leitman SF: Use of blood cell irradiation in the prevention of post-transfusion graft-vshost disease. Transfus Sci 1989;10:219–232. 34 Leitman SF: Dose, dosimetry and quality improvement of irradiated blood components. Transfusion 1993;33:447–449. 35 Linden JV, Pisciotto PT: Transfusion-associated graft-versus-host disease and blood irradiation. Transfus Med Rev 1992;6:116–123. 36 Shlomchik WD, Couzens MS, Tang CB, et al: Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 1999;285:412–415. 37 Gresens CJ, Paglieroni T, Moss CB, et al: Tcells in fresh frozen plasma are viable and can respond to mitogen, superantigen and allogeneic monocytes (abstract). Transfusion 1999;39:99S. 38 FDA: Recommendations regarding license amendments and procedures for gamma irradiation of blood products. Food and Drug Administration, 1993. 39 BCSH Blood Transfusion Task Force: Guidelines on gamma irradiation of blood components for the prevention of transfusion associated graft-versus-host disease. Transfus Med 1996;6:261–271. 40 Richtlinien zur Gewinnung von Blut und Blutbestandteilen und zur Anwendung von Blutprodukten (Ha¨motherapie). Aufgestellt vom Wissenschaftlichen Beirat der Bundesa¨rztekammer und vom Paul-Ehrlich-Institut. Deutscher rzteverlag 2000/2001. 41 EC Guide to Good Manufacturing Practice for Medicinal Products including Annex 12: Use of Ionizing Radiation in the Manufacture of Medicinal Products. Commission of the European Communities, 1992. 42 Guide to the preparation, use and quality assurance of blood components, ed 8. Council of Europe Strasbourg: Council of Europe Publishing, 2002. 43 Davey RJ, McCoy NC, Yu M, et al: The effect of prestorage irradiation on post-transfusion red cell survival. Transfusion 1992;32: 525–528. 44 Moroff G, George VM, Siegl M, et al: The influence of irradiation on stored platelets. Transfusion 1986;26:453–456. 45 Read EJ, Kodis C, Carter CS, et al: Viability of platelets following storage in the irradiated state. A pair-controlled study. Transfusion 1988;28:446–450. 46 Sweeney JD, Holme S, Moroff G: Storage of apheresis platelets after gamma irradiation. Transfusion 1994;34:779–783. 47 Zimmermann R, Schmidt S, Zingsem J, et al: Effect of gamma radiation on the in vitro aggregability of WBC-reduced apheresis platelets. Transfusion 2001;41:236–242. 48 Lin L, Cook DN, Wiesehahn GP, et al: Photochemical inactivation of viruses and bacteria in human platelet concentrates using a novel psoralen and long wavelength UV light. Transfusion 1997;37:423–435.

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49 Wollowitz S: Fundamentals of the psoralenbased Helinx Technology for inactivation of infectious pathogens and leukocytes in platelets and plasma. Sem Hematol 2001;38: 4–11. 50 Grass JA, Hei DJ, Metchette K, et al: Inactivation of leukocytes in platelet concentrates by psoralen plus UVA. Blood 1998;91:2180– 2188.


51 Lowenthal RM, Challis DR, Griffiths AE: Transfusion-associated graft-versus-host disease: Report of an occurrence following the administration of irradiated blood. Transfusion 1993;33:524–529. 52 Hei DJ, Grass J, Lin L: Elimination of cytokine production in stored platelet concentrate aliquots by photochemical treatment with psoralen plus ultraviolet A light. Transfusion 1999;39:239–248.

53 Grass JA, Wafa T, Reames A, et al: Prevention of transfusion-associated graft-versushost disease by photochemical treatment. Blood 1999;93:3140–3147.


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Quality Management Transfus Med Hemother 2004;31(suppl 1):32–36

Practical Experience of Implementing the INTERCEPT TM Blood System for Buffy Coat Platelets in a Blood Center T. Hervig

I. Aksnes

Blood Bank, Haukeland University Hospital, Bergen, Norway

Key words Pathogen inactivation Platelet concentrates Product quality Transfusion Summary To ensure the quality and efficiency of pathogen inactivation of buffy coat platelet concentrates by the INTERCEPTTM Blood System it is necessary to standardise the products, to streamline the logistics and to consider data management options thoroughly. During the optimisation period, the number of buffy coat platelets pooled was increased from 4 to 5, and the secondary centrifugation (soft spin) time was increased to 5 min. The additional workload compared with standard procedures was approximately 10 min when few concentrates were prepared simultaneously. The data management system ensured the quality of the process. After optimisation, the average platelet yield was 3.17 1011 with a standard deviation (SD) of 0.39. The average pH on storage day 7 was 6.87 (SD: 0.06), and the swirling was excellent. Due to the documented effects of the inactivation procedure and the documented quality of the products, we have omitted bacterial surveillance and irradiation of the INTERCEPT platelets, and we store the platelets for up to 7 days before transfusion, in accordance with national guidelines.

Background

Minimising the risks related to blood transfusion is a key issue for all blood centres. Although many precautions have been implemented, transfusion-transmitted infection is a major concern, not least in public opinion. Therefore, we welcome the development of systems for pathogen inactivation of cellular blood products. The response generated by the various approaches to pathogen inactivation in blood products has differed significantly in the transfusion medicine community [1–3]. Concerns have been raised regarding costs, loss of function



and the possibility of toxic side effects [1]. We have chosen to introduce INTERCEPT platelets (see fig. 1) in our hospital as we believe in the principle of pathogen inactivation and we want to gain experience with the technology. We also believe that new technologies should be thoroughly monitored. To evaluate the transfusions during the introductory period, we aimed for 50% of all transfusions to be treated with the INTERCEPT blood system. The active substance in the INTERCEPT Blood System is the synthetic psoralen amotosalen HCl (S-59). Photochemical treatment with amotosalen is a nucleic acid specific inactivation process with a broad spectrum of activity against bacteria, viruses and leukocytes. Amotosalen reversibly intercalates into helical regions of DNA and RNA. Upon illumination with UVA, amotosalen reacts with pyrimidine bases to form monoadducts or covalent crosslinks with nucleic acids. Bacteria, viruses and cells that have been modified by amotosalen are unable to replicate [4]. As this paper covers the practical aspects of implementing the INTERCEPT Blood System in a blood centre, it is necessary to include some information on the blood centre being discussed. The blood centre is a department of the Haukeland University Hospital. We collect blood, produce blood components, and issue the components to hospitals in the Bergen area. The total number of blood donations is 20,000. We produce 1,700 buffy coat (BC) platelet concentrates from 6,800 BC platelets and 400 platelet concentrates from apheresis. Before the implementation of the INTERCEPT Blood System, we standardized our platelet concentrates according to the requirements listed below. During implementation, Baxter provided support. 35 concentrates were tested after the standardization process.

Tor Hervig Blood Bank Haukeland University Hospital N-5021 Bergen

Implementation Period

The BC Platelet Concentrate The INTERCEPT Blood System requires standardized BC platelet concentrates (PCs) in order to secure the inactivation procedure. Standardization requires: Percentage of plasma in PCs after addition of amotosalen 30–45% Volume of PC after addition of amotosalen 317.5– 407.5 mL (300–390 mL before addition of amotosalen) Concentration of amotosalen in PCs: 120–180 mM

Fig. 1. INTERCEPT Blood System for platelets: Process flow. The original platelet concentrate is transferred through the amotosalen container. After illumination, the surplus of amotosalen is adsorbed before the product is transferred to the storage container.

Thrombocytes x109

500 400 300 200 100

A

B

Platelet dose at time of treatment 2.5–5 1011 Maximum level of red blood cells before treatment: 4 106/L (or maximum hemoglobin level before process 0.1 g/L) Platelet Dose Our standard platelet concentrates were produced from 4 blood cells (BCs) and aimed to fulfill the requirements listed in the guidelines from the Council of Europe [5]. However, the average platelet dose was 2.28 1011/L, and 40% of the concentrates had platelet dosage below 2.4 1011/L. In order to comply with the requirements, we performed an optimization protocol. However, as indicated in figure 2, there was still a substantial percentage of the concentrates that had platelet content below 2.5 1011/L. We therefore increased the number of BC platelets from 4 to 5. After this adjustment, all except one of the 35 platelet concentrates evaluated during the implementation period fulfilled the platelet dose requirement. The average platelet yield was 3.17 1011/L (standard deviation [SD] ¼ 0.39) (see table 1). A concern when new methods are introduced is the loss of active substances during preparation. Pooling 5 BC platelets added costs to the product; however, it also led to a more standardized product with a higher platelet content, which should, theoretically, reduce the number of platelet transfusions needed. A further advantage was that it became unnecessary to demand that each BC platelet should be produced from a donor with a minimum peripheral blood platelet concentration of 190 109/L (earlier local requirement). The average platelet dose in the concentrates before pooling was 3.54 1011/L. The platelet loss was therefore 10.7% (to 3.17 1011/L). We regarded this as acceptable, as the platelets were transferred successfully in three different containers, agitated with the adsorption device, and finally filtered through an in-line filter (PL 1813/1).

C

Residual Plasma in Platelet Concentrates

0 Total platelet count x 109/unit

Minimum platelet content

Fig. 2. Platelet content in BC concentrates. Region A represents data before standardization started, region B is the standardization period and region C is the validation period.

As shown in figure 3, we had difficulties achieving the residual plasma requirements whilst simultaneously ensuring sufficient platelet content. Our primary separation centrifu-

Table 1. Validation data. The table shows the product quality data from the validation period (n ¼ 35) Post-treatment

Day 1

Day 5

Day 7

Pre-treatment volume (mL) Post-treatment volume (mL) Platelet count (103/mL) Platelet yield (1011) pH at 22 C Swirling*

361 9 337 10 945 119 3.17 0.39 7,300 0.062 30

907 111 3.00 0.36 7,210 0.044 2.94 0.25

879 120 2.86 0.39 7,089 0.060 2.91 0.30

* Optimum swirling is 3 (graded 0–3).

Implementing INTERCEPT for Buffy Coat Platelets in a Blood Center


33

3 on the day of production. On storage day 7, the average swirling score was 2.9 (range 2–3).

% Plasma

50 40 30 20 10

Logistics

A

B

C

0 Plasma ratio

Plasma ratio — minimum

Plasma ratio — maximum

Fig. 3. Plasma content in BC concentrates. Region A represents data before standardization started, region B is the standardization period and region C is the validation period.

gation cycle was 4,260 g (maximum) for 10 min, and the secondary centrifugation cycle was 800 g (maximum) for 4 min. Before implementation began, we increased the secondary centrifugation time to 5 min, and after this adjustment, the residual plasma content was 31.8% (SD: 0.87; range: 30.6–34.7%). PC Volume and Residual Amotosalen and Hemoglobin The average volume of the PCs was 361 mL (SD: 9; range 345–393 mL). Although one of the 35 concentrates had a volume slightly above the upper limit, this was deemed satisfactory, which was confirmed by measurements of residual amotosalen. The average residual hemoglobin content was 0.05 g/L (SD: 0.01; range 0.05–0.09 g/L). Before the implementation period, we experienced some concentrates with excessive hemoglobin concentrations. A repetition of the secondary centrifugation cycle (800 g for 5 min) removed a sufficient amount of red cells without interfering substantially with other parameters. As hemoglobin interferes with the illumination process, it is critical to discard units with high hemoglobin content. A trained eye may pick out the unsuitable concentrates, but a color scale indicating different hemoglobin levels would certainly be helpful. Product Quality During the implementation study, we evaluated the product quality parameters pH, swirling, and platelet count. As shown in table 1, the platelet count was stable during storage. The slight reduction in platelet count during storage is to be expected from evaluation of the counting method [6]. It is important to recognize that different counting methods affect platelet yield reported at the time of production and during the storage period [6, 7]. The average pH on the day of production was 7.30, which was reduced to 6.87 after 7 days’ storage. The SD on day 7 was 0.06, and the variation in pH was 6.75–6.98. These results indicate good and stable product quality. The swirling phenomenon was graded 0–3 (no swirling to excellent). In all concentrates tested, the swirling score was

34


To facilitate the introduction of the INTERCEPT Blood System, it is necessary to organize a production line to reduce working time and avoid transportation of the product. During the implementation period, we were not able to change the layout of the production laboratory, which led to some practical difficulties. It is important to note that the concentrates must be shielded from direct sunlight, as sunlight may interfere with the critical illumination process. Workload It is important to reduce workload. During the implementation process, only a small number of concentrates were produced simultaneously. The addition of amotosalen took 2–3 min per concentrate and the average illumination period was 4.30 min (range 4.08–4.40 min). The working time including transfer to the component adsorption device was 2–3 min and the transfer time to the final container was slightly over 1 min.

Preparation of Concentrates for Routine Transfusions

Staff Training Our standard operating procedures for the INTERCEPT Blood System were based on the evaluation process. The BC concentrate production was only slightly modified, with the use of a platelet additive solution specially developed for pathogen inactivation (InterSol Solution). Training staff to use the inactivation process proved to be straightforward, with experienced technicians having no difficulties following the procedure. We allowed a working period of 1 week to familiarize staff with the process. Data Management and Quality Assurance During the implementation process, the data management system was not available; however, for routine usage, a comprising data management unit is available. In addition, each production site has the opportunity to design its own data configuration. We started our data management process by defining which products can be treated with the INTERCEPT Blood System. This is important, as there are different procedures for treating BC platelet concentrates and apheresis-derived platelet concentrates. Initially, we only treated BC platelet concentrates. Secondly, treatment was re-

Hervig/Aksnes

stricted to freshly prepared concentrates. We also generated a local ISBT 128 code for the product. The INTERCEPT Blood System will refuse to treat non-conforming units. During the process, each step was documented in the data management system. The connection of amotosalen has to be accepted in the IT-system. Illumination data are printed after each procedure. The component adsorption bag location in the platelet agitator is registered, and an ‘agitation list’ is produced. The transfer steps are also registered. Our usage of the data management system has been streamlined while gaining practical experience of the system, and we believe that we have obtained a safe and efficient usage of the various options available. Logistics The production laboratory has been slightly enlarged and rebuilt to improve the working conditions and the production line as compared with the earlier configuration. We have based our platelet production time on working hours from 7.30 am to 8 pm. The accepted agitation time is 6–16 h, making it easy to modify the procedure to the local conditions. For example, larger facilities may find it optimal to produce the components during the night. The BC platelets are produced on the donation day and then kept overnight at ambient temperature. The next morning the BC platelet concentrate production starts at 7.30 am and the inactivation procedure follows immediately after. We aim for 6 h of agitation, which enables us to have the products ready by 7.30 pm. The concentrates are then released for clinical usage.

Consequences for Routine Transfusions

Bacteriological Surveillance For BC platelet concentrates and apheresis platelet concentrates, we collected 10-mL samples for the BacT/ Alert system on the day after donation. Based on the available documentation data for bacterial inactivation [8, 9] and our experience of parallel usage of BacT/ Alert surveillance and the INTERCEPT Blood System, we have decided to rely fully on the inactivation procedure. Irradiation To avoid transfusion-related graft-versus-host reaction, donor lymphocyte inactivation is essential. We use a quality controlled irradiation procedure with a dose of

Implementing INTERCEPT for Buffy Coat Platelets in a Blood Center

2,500 rad to inactivate lymphocytes in our blood products. Again, based on the documentation of the efficiency of the INTERCEPT Blood System, there was no additional irradiation of the platelet concentrates treated with the INTERCEPT Blood System. We consider the INTERCEPT Blood System to be at least equally safe because the inactivation process is documented for each concentrate, and the safety margin itself is broader than for irradiation [10]. Storage Time In Norway, the acceptable storage time for platelet concentrates is 5 days, although this is increased to 7 days when bacterial surveillance or pathogen inactivation is used (when not using bacterial surveillance or pathogen inactivation, platelet concentrates older than 5 days should only be used in emergencies). However, for patients with malignancies, the preferred storage time is

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