Gregor Caspari

Pathogen Inactivation of Cellular Blood Products - More Security for the Patient or Less?

Numerous measures have been introduced to prevent infection of patients during transfusion of blood components and plasma derivatives. These efforts have drastically reduced the residual risk of classical transfusion-related infections by hepatitis B (HBV) and C (HCV) and human immunodeficiency virus (HIV). In addition to excluding donor risk groups, these measures include continuously improved serological diagnosis and, in recent years, molecular biological diagnosis. In the meantime, the residual risks for infection with these agents have become exceedingly small. In Germany, the number of blood product-transmitted HIV infections reported to the Paul Ehrlich Institute (PEI) amounted to less than 1 infection per year (residual risk < 1:5,000,000) [1]. A further reduction can be expected with the obligatory introduction of HIV-PCR [2]. Since the general implementation of HCV-PCR, not a single transfusion-related infection has been reported [1]. Seifried et al. [2] reported a residual risk of 1:13,000,000 [2]. As a result of these successful efforts some pathogens that received less attention in the past are now coming to the fore: In particular thrombocyte concentrates offer, as a result of their storage temperature, excellent conditions for bacterial growth. According to data in the literature, 1 out of 2,000 concentrates is reported to be contaminated with bacteria [3], and according to Montag et al. [4], 1 out of 3,000–5,000 single-donor concentrates, with 1 out of 50,000 thrombocyte transfusions leading to sepsis in the recipient. Incidents resulting from transfusion of erythrocyte concentrates are less frequent but, due to nature of the pathogens, are generally more serious. Data from France showed that 1 out of 600,000 recipients of cellular blood products died as a result of the presence of bacteria in the transfused blood product [4]. In Germany this would correspond to 8 deaths per year. Figures obtained by the PEI, however, are considerably lower [1]. It is unclear so far if some of these infections were not recognized as transfusion related, if they were not reported, if the French figures are an overestimation or if there is a significant regional difference [3] in the frequency of these incidents. The number of incidents are expected to clearly drop as a result of the introduction of predonation sampling. Until the beginning of the 1980s, almost all recipients of coagulation factor concentrates became infected with HBV and/or HCV because the single risks of each plasma sample were multiplied by pooling. Consequently, procedures were developed to achieve inactivation of these pathogens in the preparations, but these only came into general use as a result of the HIV epidemic. However, with the first generation of pathogen-inactivated blood products a number of infection events still occurred [surveyed in 5]. Model experiments led to improved validation of security for the individual treatment steps. Today, virus inactivation of plasma derivatives provides a very high degree of safety. To some extent fresh plasma has also been subjected to pathogen inactivation. The increased level of safety is purchased at the price of a reduction of the activity of some coagulation factors and inhibitors, the clinical relevance of which is controversially discussed. A 4-month quarantine is an alternative to pathogen inactivation of fresh frozen plasma and leads to a similar level of safety for those pathogens that have been tested. Quarantine storage of cellular blood products is hardly possible. In view of the fact that the public senses transfusion-related infections, in spite of their very small number, as a threat, and with respect to the repeated appearance of new pathogenic agents which in the first instance cannot reliably be detected or the transmission of which cannot be ruled out by the exclusion of risk groups, the question arises whether or not the introduction of pathogen inactivation of cellular blood products would be reasonable and increase the safety of blood component transfusion. Pathogen inactivation techniques for cellular blood products have not yet been approved even though some of them are presently undergoing clinical testing. Basically, three aspects of the evaluation of methods for pathogen inactivation are matter of debate:

Validation of the Serological Testing for Anti-HIV-1/2, Anti-HCV, HBsAg, and Anti-HBc from Post-mortem Blood on the Siemens-BEP-III Automatic System

Background: Some properties of blood are modified post mortem. These modifications might give false-negative or false-positive results in infectious disease testing. Most CE-marked test equipment for infectious serology testing is not validated for testing post-mortal blood. Validation, however, is obligatory, if the results are used for the release of tissues for transplantation. Methods: Samples of pre- and post-mortem sera were obtained from 20 cornea donors, and the results were compared for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc on the Siemens-BEP-III Automatic System. Negative post-mortem sera were spiked with standard sera (PEI anti-HCV IgG, PEI HBsAg ad 1000 standard, anti-HBc IgG (WHO) NIBSC 95/522, PEI anti-HIV-IV) in concentrations which give low- and high-positive results for the respective marker. Results: All pre-mortem sera were negative for all markers. None of the post-mortem samples was false-positive. None of the spiked post-mortem samples was false-negative. Technical errors occurred during the validation process but could be detected and eliminated. Serum samples should be centrifuged immediately after collection, and it must be taken into account that post-mortem serum could rarely lead to blockage of pipetting systems due to clotting phenomena. Conclusion: There is no indication that post-mortem samples give false-negative or false-positve results with the test system and test kits used. The procedure described might serve as a model for validating other test kits on post-mortem samples.

Pathogen Inactivation of Cellular Blood Products – Still Plenty of Reason to Be Careful

In a recent editorial [1] we discussed reasons for and against the introduction of pathogen-inactivated cellular blood products. Our main argument was and is that serious morbidity and mortality through transfusion-transmitted infections are exceedingly rare in Germany. Pathogen-inactivated cellular blood products only have an advantage over non-treated products if the net damage done to patients if they receive inactivated products is lower than the damage caused by infections transmitted through non-treated products. Despite extensive studies of the manufacturer of the inactivated products we had serious doubts that a lower damage through the inactivated products had indeed been proven, and we have recommended, in accordance with European recommendations [2], caution when considering the introduction of the inactivated products. Schlenke et al. [3] have then written an extensive response concluding that ‘[with pathogen inactivated platelets] there is an opportunity to improve patient security beyond that currently available by testing alone’. Thus, it is necessary to look again at the epidemiology, i.e. the frequency and possible damage done by transfusion-associated infectious diseases on the one hand and the efficacy and the possible damage done by the pathogen-inactivation procedure on the other hand. Schlenke et al. [3] claim that, according to Barbara et al. [4], the residual risk per blood donation ranges from 1:86,505 to 1:249,750. However, at least in Germany, and probably also in other Northern European countries, the risk of HCV and HIV transmission is distinctly lower. The risk of HIV transmission has been recently further diminished by additional NAT testing, thus older risk estimates are not applicable. Only 1 HCV infection through cellular blood products has been reported to the Paul-Ehrlich-Institut (PEI) after the official introduction of HCV-NAT in 1999 [5, updated by personal communication by Keller-Stanislawski for 2004 and 2005]. Risk estimates vary between 1 in 4.4 million and 1 in 13 million transfusions [6, 7]. About 3 clinically apparent HBV transmissions are reported to the PEI per year (on more than 5 million blood products transfused) [5]. Risk estimates based on the window phase method give somewhat higher risk estimates [6], but have to rely on several unproven assumptions and include asymptomatic infections. Even if 1 death occurred through a transfusion-transmitted HBV infection in 2003 (M. Chudy, personal communication), the overall disease burden is lower than for HCV and HIV. The number of HBV transmissions will further be reduced by the additional testing for antibodies to the hepatitis B core antigen which has recently been recommended by the Arbeitskreis Blut [20] and will be subject of a phased plan of the PEI which is likely to prepare its obligatory introduction. Furthermore, HBV vaccination should be given to all patients who need frequent transfusion of cellular blood products. West Nile virus infections through blood might have been prevented by pathogen-inactivated blood products [8] but the US authorities choose to introduce laboratory testing for the pathogen instead. In Germany, North-America-acquired West Nile virus infections through cellular blood products are reliably excluded by a 4-week donor deferral. Infections with other pathogens acquired in tropical regions are likewise dealt with appropriate donor deferrals. A problem might theoretically arise from persons who come from tropical regions and stay permanently in Germany. They would not loose a Plasmodium vivax, Plasmodium ovale or Plasmodium malariae malaria infection or a Trypanosoma cruzi infection just because they acquire a German passport. However, transmissions of parasitic infections have been extremely rare. According to published studies the most important risk comes from bacteria [9, 10]. But these figures have not been generated in Germany, and they were obtained before general predonation sampling [11] reduced bacterial pathogen load by up to Letter to the Editors · Brief an die Herausgeber

Prevalence of antibodies to recombinant hepatitis C virus protein C100-3 and of elevated transaminase levels in blood donors from Northern Germany.

Antibody to recombinant hepatitits C virus protein C100-3 (anti-C100-3) was assayed by a first generation enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostics) in 116,700 blood donors who had not been tested before. Total prevalence of repeatably positive donors was 0.72% (n = 842). Prevalence increased significantly from 0.42% at 18–27 years of age to 1.26% at ≥58 years. Donors with elevated serum transaminase levels were significantly more often anti-C100-3 positive, but in 98.7% of donors with current or 99.1% with previous transaminase elevations, anti-C100-3 was not found. Elevated transaminases were more often associated with positive anti-C100-3 in females than in males. However, in the total donor population no significant differences of anti-C100-3 prevalence were found between the sexes. During follow up at three subsequent blood donations, 1.08 % of donors were positive at least once, but only 0.48 % were consistently positive. The cutoff of the Ortho ELISA was not in the minimum of the frequency distribution between positive and negative samples, but far within the range of the negative signals, i.e. the test is likely to produce a significant number of false-positive results. In retesting positive samples with two ELISAs from other producers only a 22% to 65% agreement was found. In a low prevalence group such as German blood donors, the first generation ELISAs for anti-C100-3 produced more false than specific positive results. Most donors with elevated alanine aminotransferase (ALT) are anti-C100-3 negative.

Nukleinsäure-Amplifikationstests für HIV, HBV und HCV bei Gewebespendern: Sinnvoll oder überflüssig?

Mit der Umsetzung der EU-Richtlinien 2004/23/EG und 2006/17/EG im Gewebegesetz sowie den begleitenden Verordnungen (Arzneimittel- und Wirkstoffherstellungsverordnung, Transplantationsgesetz-Gewebeverordnung) wurden die grundlegenden Anforderungen an die Virussicherheit der Gewebespenden allgemein definiert. Während infektionsserologische Testungen (Anti-HIV 1/2, Anti-HCV, Hepatitis-B-Oberflächenantigen, Anti-Hepatitis-B-Core-Antigen, Treponema-pallidum-Hämagglutinationsassay) vorgeschrieben sind, wird der Nukleinsäure-Nachweis für HIV, HBV und HCV nicht explizit gefordert. Anhand in der Literatur berichteter Virusübertragungen, gewebespezifischer Besonderheiten sowie Herstellungs- und gegebenenfalls Inaktivierungsverfahren wird eine Bewertung des Stellenwertes des HIV/HCV/HBV-Nachweises mittels Nukleinsäure-Amplifikationstechniken (NAT) bei Spendern unterschiedlicher Gewebe vorgenommen und mit den Erfahrungen des Blutspendewesens verglichen. Muskuloskelettale Gewebe besitzen infolge des zumeist hohen Blutgehalts der Gewebe, des umfangreichen Entnahmespektrums, der bisher beschriebenen Übertragungen, der unterschiedlichen Herstellungsverfahren sowie der hohen Spender-Empfänger-Ratio ein signifikantes Risiko, HIV/HCV/HBV zu übertragen. Daher sollte bei Spendern muskuloskelettaler Gewebe, die keinem effektiven Virusinaktivierungsverfahren unterzogen werden, eine HIV-, HBV- und HCV-NAT-Testung erfolgen. Kardiovaskuläres Gewebe hat bei Durchführung der serologischen Testung ein sehr geringes Restrisiko der HIV/HCV/HBV-Übertragung. Aufgrund der fehlenden Möglichkeit einer effektiven Virusinaktivierung (Erhalt der Gewebemorphologie) und der Spender-Empfänger-Ratio von bis zu 1:10 sollte die HIV-, HCV- und HBV-NAT jedoch als zusätzliche Sicherheitsmaßnahme erfolgen. Augenhornhäute besitzen aus physiologisch-morphologischer sowie epidemiologischer Sicht das geringste HIV/HCV/HBV-Übertragungsrisiko, jedoch sollte die HCV-NAT durchgeführt werden. Eine Quarantänelagerung der Gewebe eines Spenders kann bei negativem Ergebnis der HIV/HCV/HBV-NAT grundsätzlich entfallen. Aufgrund der vielfältigen Synergieeffekte mit der Transfusionsmedizin bietet es sich für Gewebebanken an, die Testung der infektionsserologischen bzw. molekularbiologischen Laborparameter in Kooperation mit Blutspendediensten durchzuführen.

HIV-specific antibody among voluntary blood donors in lower saxony (FRG)

SummaryAnti-HIV test results of the Red Cross Blood Transfusion Service of Lower Saxony from 1 June 1985 to 31 July 1986 inclusive were analysed retrospectively. Nine out of 70,936 donors who had not donated blood before 1 June 1985 (first-time donors) and 9 out of 261,231 donors who had donated blood before this date (repeating donors) were found anti-HIV confirmed positive at the time of the first blood donation during the study period. The prevalence of HIV antibody in first-time donors was significantly higher than in repeating donors (p<0.01). It was concluded that some members of risk groups used blood donation to obtain an anti-HIV test result. One out of 30,300 blood donations was confirmed anti-HIV positive. The results of this study justify the transfusion of blood donations that are reactive only in the initial ELISA test.

Assay of antibodies to hepatitis C virus protein C100-3 in blood donors from Northern Germany

The prevalence of anti-C100-3 increases with age from 0.41% to 1.26%. It is more frequent in donors with elevated ALT (4.5%). Most ALT elevations, however, are not related to anti-C100-3. Low EIA signals (< 3 x cutoff) are often non-specific. The cutoff value should be 2.5 times higher. High EIA signals correlate with ALT elevations.

Is screening of blood donors for anti-HBc useful?

Blood donor screening for antibodies to hepatitis B core antigen (anti-HBc) was introduced in the USA because the retrospective evaluation of two studies performed between 1973 and 1980 on posttransfusion hepatitis (PTH) showed a weak correlation between anti-HBc in the donor blood and non-A, non-B P T H in the blood recipient [6, 11]. In a German study performed in 1980/81, a similar correlation was suggested [12]. Since that time the epidemiological association may have been fundamentally changed by measures to prevent persons at risk for HIVinfection from donating blood, and in recent studies the correlation could not be confirmed [1,4, 8, 9]. A crucial prerequisite for such a correlation is a reliable assay for anti-HBc. It is, however, a common experience that current enzyme immunoassays (EIAs) generate up to 30°7o more positive results than radioimmunoassays for anti-HBc [3, 10, 14, 16] and that there are important discrepancies between the currently available EIAs [16]. The latter could recently be confirmed in a large cooperative study of the German Red Cross [2], in which six test kits were applied in parallel to 4,080 blood donor sera. Of 1,838 sera evaluable in all six assays, only 62 produced consistently positive results while 228 gave divergent results. Reference testing by two additional tests and a recently developed direct binding assay identified up to 50°70 false positives in the initial screening and a significant number of false negatives [2]. The estimated efficacy of excluding anti-HBc positive blood units was already low in the first two studies considering that some non-A, non-B PTH infections also occured after transfusions of only anti-HBc negative blood. Only 1 out of 6 1 6 donations discarded for anti-HBc could be assumed infectious. Yet 57°70 to 78°7o of non-A, non-B P T H cases could not be prevented [6, 11].

Validation of Serological Testing for Anti-Treponema pallidum from Postmortem Blood on the Siemens-BEP®-III Automatic System

SummaryBackground: Infectious disease marker testing is obligatory for the release of human tissue for transplantation. Most CE-marked tests are not validated for postmortem blood. In a previous study we have validated the testing for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc. Here, we present the validation of testing for antibodies against T. pallidum, which is the last marker obligatory for tissue release for transplantation. Methods: 17 samples of postmortem sera and 10 samples of both pre- und postmortem sera were obtained from cornea donors and tested for anti-T. pallidum on the Siemens-BEP-III-System. These sera were spiked with anti-T. pallidum-positive standard sera in concentrations which give low- and high-positive results at the respective dilution. Results: Two of the unspiked postmortem sera were false-positive most likely due to intense hemolysis (free hemoglobin > 50 mg/dl). Of the 25 negative postmortem sera, none of the spiked samples was false-negative after 0, 24 and 60 h. Conclusion: There is no indication that postmortem samples give false-negative or false-positive results with the test system and test kits used in cases of low hemolysis. The procedure described might serve as a model for validating other test kits on postmortem samples.