Pathogen Reduction Methods Research Articles

Transfusion-transmitted hepatitis E: What we know so far?

Hepatitis E virus (HEV) is a major cause of viral hepatitis globally. There is growing concern about transfusion-transmitted HEV (TT-HEV) as an emerging global health problem. HEV can potentially result in chronic infection in immunocompromised patients, leading to a higher risk of liver cirrhosis and even death. Between 0.0013% and 0.281% of asymptomatic blood donors around the world have HEV viremia, and 0.27% to 60.5% have anti-HEV immunoglobulin G. HEV is infectious even at very low blood concentrations of the virus. Immunosuppressed patients who develop persistent hepatitis E infection should have their immunosuppressant regimen reduced; ribavirin may be considered as treatment. Pegylated interferon can be considered in those who are refractory or intolerant to ribavirin. Sofosbuvir, a nucleotide analog, showed modest antiviral activity in some clinical studies but sustained viral response was not achieved. Therefore, rescue treatment remains an unmet need. The need for HEV screening of all blood donations remains controversial. Universal screening has been adopted in some countries after consideration of risk and resource availability. Various pathogen reduction methods have also been proposed to reduce the risk of TT-HEV. Future studies are needed to define the incidence of transmission through transfusion, their clinical features, outcomes and prognosis.

Viral reduction of human blood by ultraviolet A-photosensitized vitamin K5.

Blood product transfusion can transmit viral pathogens. Pathogen reduction methods for blood products have been developed but, so far, are not available for whole blood. We evaluated if vitamin K5 (VK5) and ultraviolet A (UVA) irradiation could be used for virus inactivation in plasma and whole blood. Undiluted human plasma and whole blood diluted to 20% were spiked with high levels of vaccinia or Zika viruses. Infectious titers were measured by standard TCID50 assay before and after VK5/UVA treatments. Up to 3.6 log of vaccinia and 3.2 log of Zika were reduced in plasma by the combination of 500 μM VK5 and 3 J/cm2 UVA, and 3.1 log of vaccinia and 2.9 log of Zika were reduced in diluted human blood (20%) by the combination of 500 μM VK5 and 70 J/cm2 UVA. At end of whole blood treatment, hemolysis increased from 0.18% to 0.41% but remained below 1% hemolysis, which is acceptable to the Food and Drug Administration for red cell transfusion products. No significant alteration of biochemical parameters of red blood cells occurred with treatment. Our results provide proof of theconcept that a viral pathogen reduction method based on VK5/UVA may be developed for whole blood.

Impact of pathogen reduction methods on immunological properties of the COVID-19 convalescent plasma.

Background and objectivesCOVID‐19 convalescent plasma is an experimental treatment against SARS‐CoV‐2. The aim of this study is to assess the impact of different pathogen reduction methods on the levels and virus neutralizing activity of the specific antibodies against SARS‐CoV2 in convalescent plasma.Materials and methodsA total of 140 plasma doses collected by plasmapheresis from COVID‐19 convalescent donors were subjected to pathogen reduction by three methods: methylene blue (M)/visible light, riboflavin (R)/UVB and amotosalen (A)/UVA. To conduct a paired comparison, individual plasma doses were divided into 2 samples that were subjected to one of these methods. The titres of SARS‐CoV2 neutralizing antibodies (NtAbs) and levels of specific immunoglobulins to RBD, S‐ and N‐proteins of SARS‐CoV‐2 were measured before and after pathogen reduction.ResultsThe methods reduced NtAbs titres differently: among units with the initial titre 80 or above, 81% of units remained unchanged and 19% decreased by one step after methylene blue; 60% were unchanged and 40% decreased by one step after amotosalen; after riboflavin 43% were unchanged and 50% (7%, respectively) had a one‐step (two‐step, respectively) decrease. Paired two‐sample comparisons (M vs. A, M vs. R and A vs. R) revealed that the largest statistically significant decrease in quantity and activity of the specific antibodies resulted from the riboflavin treatment.ConclusionPathogen reduction with methylene blue or with amotosalen provides the greater likelihood of preserving the immunological properties of the COVID‐19 convalescent plasma compared to riboflavin.

Inactivation of Zika virus in plasma and derivatives by four different methods.

Zika virus (ZIKV) is an emerging arbovirus with increasing prevalence in recent years. To reduce the risk of ZIKV transmission by transfusion, some mitigation strategies were recommended based on pathogen reduction technologies for blood products. In this study, we aimed to study the efficacy of several common pathogen reduction methods in the inactivation of ZIKV. The fresh frozen plasma and derivatives were spiked with a high titer of ZIKV or Sindbis virus (SINV). Viral titers and ZIKV RNA were measured before and after the inactivation treatment by methylene blue (MB), solvent/detergent (S/D), pasteurization, and low pH. The mean ZIKV infectivity titers in plasma and derivatives were 7.08 ± 0.14, 5.17 ± 0.14, 7.08 ± 0.14, and 5.80 ± 0.14 log10 TCID50 /mL, respectively before MB, S/D, pasteurization, and low pH inactivation. We found no detectable ZIKV RNA after five successive passages of inoculation on host cells, indicating there is no infectivity after inactivation. Similar inactivation results were observed for SINV. In conclusion, we achieved robust ZIKV inactivation through the four inactivation procedures in several blood products. These findings suggest that the pathogen reduction technologies commonly applied in plasma and derivatives have the capacity to mitigate the risk of ZIKV transmission by transfusion.

Study of CD69 antigen expression and integrity of leukocyte cellular membrane in stored platelet concentrates following irradiation and treatment with Mirasol® PRT System.

Leukocytes in transfused blood components, particularly residual lymphocytes, have been shown to contribute to the occurrence of various adverse reactions. One of the most severe is transfusionassociated graft versus host disease (TA-GvHD) following transfusion of blood components contaminated with immunocompetent T lymphocytes. Irradiation is a routine method for protection against TA-GvHD. According to the literature, some pathogen reduction methods have also been proven effective for the inactivation of T lymphocytes, and so they may be considered as an alternative to irradiation. Comparison of CD69 antigen expression and the integrity of the leukocyte cellular membrane in stored platelet concentrates (PCs) following irradiation with the Gammacell 3000 Elan (Nordion Inc., Ottawa, Canada) and treatment with the Mirasol® Pathogen Reduction Technology (PRT) System (Terumo BCT, Lakewood, USA). The study included seven experiments. For each experiment we used 3 PCs, for Mirasol® PRT System treatment (M), for Gammacell 3000 Elan irradiation (R), and for the control (C). 7-amino-actinomycin D (7-AAD, Becton Dickinson, Franklin Lakes, USA) permeability was used to determine lymphocyte viability while CD69 antigen expression was the marker of lymphocyte activation. Analyses of 7-AAD and CD69 antigen expression were performed in a FACS Canto I flow cytometer (Becton Dickinson, USA). During 6 storage days, viable lymphocyte count decreased to 28% (p = 0.001) in the Mirasol® PRT System treated PCs and to 65% (p = 0.004) in the irradiated PCs. A statistically significant increase in CD69 expression in the irradiated PCs was observed; 1.3-fold on day 3 and 1.5-fold on day 6. In the Mirasol ® PRT System treated PCs, no statistically significant increase was observed. The in vitro results suggest that the Mirasol® PRT System is as effective as irradiation due to donor leukocyte inactivation capacity.

Preventing bacterial infection in blood components

BackgroundThe prevention of bacterial infections by blood components can be classified into general procedures and blood component‐specific procedures.General proceduresDonor selection programmes with a temporary deferral of blood donors are sufficient measures to prevent bacterial contaminations of blood components. Secondly, an efficient donor arm disinfection is important to reduce the resident skin flora to a minimum. Since the beginning of this century, all countries have implemented the predonation sampling with the diversion of the first 30–40 ml.Blood components specific proceduresMany countries have already implemented methods of detecting bacteria in platelets using culture methods. A residual risk for culture methods, however, is the occurrence of sample errors by collecting the test volume within the first 24 h. Some countries have implemented rapid bacterial detection systems like Bactiflow or NAT systems. Different pathogen reduction methods were developed for platelets, plasma and packed red cells. The advantage of these methods is the reduction of all bacteria and potential new pathogens. A disadvantage might be their high cost and limited efficiency in detecting bacteria spores.ConclusionGeneral procedures are not sufficient to prevent all bacterial transmissions. Culture methods and pathogen reduction methods that have focused on the prevention of bacterial contamination in platelets are currently in development for erythrocytes and whole‐blood components and might not be feasible for stem cell components. Therefore, the world is still waiting for a new general procedure that will be an addendum to existing systems – such a system is eagerly awaited.

Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products.

Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion reactions, alloimmunization to HLA antigens, and transfusion-associated graft-versus-host disease (TA-GVHD). Pathogen reduction (PR) methods such as the ultraviolet C (UVC) light-based THERAFLEX UV-Platelets system were developed to reduce the risk of transfusion-transmitted infection. As UVC light targets nucleic acids, it interferes with the replication of both pathogens and WBCs. This preclinical study aimed to evaluate the ability of UVC light to inactivate contaminating WBCs in platelet concentrates (PCs). The in vitro and in vivo function of WBCs from UVC-treated PCs was compared to that of WBCs from gamma-irradiated and untreated PCs by measuring cell viability, proliferation, cytokine secretion, antigen presentation in vitro, and xenogeneic GVHD responses in a humanized mouse model. UVC light was at least as effective as gamma irradiation in preventing GVHD in the mouse model. It was more effective in suppressing T-cell proliferation (>5-log reduction in the limiting dilution assay), cytokine secretion, and antigen presentation than gamma irradiation. The THERAFLEX UV-Platelets (MacoPharma) PR system can substitute gamma irradiation for TA-GVHD prophylaxis in platelet (PLT) transfusion. Moreover, UVC treatment achieves suppression of antigen presentation and inhibition of cytokine accumulation during storage of PCs, which has potential benefits for transfusion recipients.

Paired analysis of plasma proteins and coagulant capacity after treatment with three methods of pathogen reduction

The effect of photochemical pathogen reduction (PR) methods on plasma quality has been the subject of several reports but solid comparative data for the different technologies are lacking. Plasma (n = 24) photoinactivated with methylene blue (MB), riboflavin (RF), or amotosalen (AS) was compared using a pool-and-split design. Samples were taken before and after treatment with each method and tested for coagulation factors (fibrinogen, Factor [F] II, FV, FVIII, F IX, FXI), natural coagulation inhibitors (Protein C [PC], protein S [PS], antithrombin III [AT]), prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin generation (TG). The three methods were mutually compared by repeated-measures analysis of variance. All three PR methods cause significant reduction (p < 0.01) of activity of the procoagulant proteins fibrinogen, FII, FV, FVIII, F IX, and FXI. Coagulation is also affected, with significant changes in PT, APTT, and TG. RF treatment causes a significantly higher decrease in concentration of coagulation factors, PS, and AT than the other methods (p < 0.01). PT, APTT, and TG are also affected most by RF treatment. FII, FVIII, F IX, PC, AT, and PT are best preserved with the MB method and FV, FXI, and TG after AS treatment (p < 0.01). Coagulation factor loss due to the volume loss during PR treatment is more important for MB and AS than for RF. PR treatment of plasma affects coagulation proteins and coagulant capacity. For the RF method this effect is most pronounced, although to some extent compensated by a smaller volume loss.

Bacterial contamination of blood products

Background and ObjectivesThe residual risk for transfusion‐transmitted viral infections has been reduced to less than 1:1 000 000 via improvements in various techniques (e.g. donor selection, leukocell depletion, introduction of third‐ or fourth‐generation ELISAs, mini‐pool nucleic acid testings and pre‐donation sampling) over the last two decades. In contrast, the risk for bacterial infections in general has remained stable and is estimated to range between 1:2000 and 1:3000, and for fatal septic reactions, the incidence ranges between 1:100 000 and 1:500 000. All blood components can be contaminated. Platelets are the main focus of bacterial contamination due to their storage at room temperature. However, transfusion‐transmitted septic reactions have also been reported for red cell concentrates and stem cell preparations.Materials and MethodsIn this review, an overview of residual bacterial contamination risks is presented for different blood components. Different strategies have been developed and implemented in different countries to improve blood safety. In principle, improving blood safety by reducing bacterial contamination can be accomplished by (1) general procedures, such as the diversion of 10–40 ml after blood puncture and reduction of the platelet shelf life; (2) implementation of pathogen reduction methods (e.g. Intercept, Mirasol or Theraflex); and (3) the implementation of bacterial screening methods, which can be subdivided into culture methods and rapid detection systems. A combination of all these procedures is possible and it reflects the current blood safety strategy of different countries.ResultsThe introduction of pre‐donation sampling with the diversion of the first few millilitres has been accepted and implemented worldwide. The maximum platelet shelf life varies between 3 days (Japan) and 7 days (the Netherlands); however, in the majority of countries, the platelet shelf life is 5 days. Many countries have already implemented bacterial screening methods with culture systems in combination with the ‘negative‐to‐date’ release concept. Pathogen reduction systems and rapid bacterial detection methods are only used in a few countries.ConclusionsThe bacterial contamination of blood components is an on‐going challenge in transfusion medicine. Currently, there is no doubt that blood safety must be improved due to bacterially transmitted septic reactions in all countries. Therefore, all countries have implemented the diversion of the first few millilitres after donation. Additional safety procedures are still a point of debate to determine the most effective and economic solution.

Inactivation of Plasmodium falciparum in whole blood by riboflavin plus irradiation

Malaria parasites are frequently transmitted by unscreened blood transfusions in Africa. Pathogen reduction methods in whole blood would thus greatly improve blood safety. We aimed to determine the efficacy of riboflavin plus irradiation for treatment of whole blood infected with Plasmodium falciparum. Blood was inoculated with 10(4) or 10(5) parasites/mL and riboflavin treated with or without ultraviolet (UV) irradiation (40-160 J/mL red blood cells [mL(RBCs)]). Parasite genome integrity was assessed by quantitative amplification inhibition assays, and P. falciparum viability was monitored in vitro. Riboflavin alone did not affect parasite genome integrity or parasite viability. Application of UV after riboflavin treatment disrupted parasite genome integrity, reducing polymerase-dependent amplification by up to 2 logs (99%). At 80 J/mL(RBCs), riboflavin plus irradiation prevented recovery of viable parasites in vitro for 2 weeks, whereas untreated controls typically recovered to approximately 2% parasitemia after 4 days of in vitro culture. Exposure of blood to 160 J/mL(RBCs) was not associated with significant hemolysis. Riboflavin plus irradiation treatment of whole blood damages parasite genomes and drastically reduces P. falciparum viability in vitro. In the absence of suitable malaria screening assays, parasite inactivation should be investigated for prevention of transfusion-transmitted malaria in highly endemic areas.

Two pathogen reduction technologies—methylene blue plus light and shortwave ultraviolet light—effectively inactivate hepatitis C virus in blood products

Contamination of blood products with hepatitis C virus (HCV) can cause infections resulting in acute and chronic liver diseases. Pathogen reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light as well as irradiation with shortwave ultraviolet (UVC) light were developed to inactivate viruses and other pathogens in plasma and platelet concentrates (PCs), respectively. So far, their inactivation capacities for HCV have only been tested in inactivation studies using model viruses for HCV. Recently, a HCV infection system for the propagation of infectious HCV in cell culture was developed. Inactivation studies were performed with cell culture-derived HCV and bovine viral diarrhea virus (BVDV), a model for HCV. Plasma units or PCs were spiked with high titers of cell culture-grown viruses. After treatment of the blood units with MB plus light (Theraflex MB-Plasma system, MacoPharma) or UVC (Theraflex UV-Platelets system, MacoPharma), residual viral infectivity was assessed using sensitive cell culture systems. HCV was sensitive to inactivation by both pathogen reduction procedures. HCV in plasma was efficiently inactivated by MB plus light below the detection limit already by 1/12 of the full light dose. HCV in PCs was inactivated by UVC irradiation with a reduction factor of more than 5 log. BVDV was less sensitive to the two pathogen reduction methods. Functional assays with human HCV offer an efficient tool to directly assess the inactivation capacity of pathogen reduction procedures. Pathogen reduction technologies such as MB plus light treatment and UVC irradiation have the potential to significantly reduce transfusion-transmitted HCV infections.

Developments in the prevention of transfusion‐associated graft‐versus‐host disease

The transfusion of blood products can result in a variety of consequences, including transfer of pathogens and the induction of immune responses, such as the almost invariably fatal transfusion-associated graft-versus-host disease (TA-GVHD). Exposure of blood products to γ-irradiation is currently the standard of care for the prevention of TA-GVHD. Regulatory, technical and clinical challenges associated with the use of γ-irradiators are driving efforts to develop alternatives. Initially, pathogen reduction methods were developed to reduce the risk of microbial transmission by blood components. Through modifications of nucleic acids, these technologies interfere with the replication of both pathogens and leucocytes. These methods have been found to be as effective as γ-irradiation in preventing T lymphocyte proliferation and TA-GVHD responses. Additional benefits of pathogen reduction protocols potentially include inhibition of antigen presentation, cytokine production and activation of lymphocytes.

Treatment of whole blood with riboflavin plus ultraviolet light, an alternative to gamma irradiation in the prevention of transfusion‐associated graft‐versus‐host disease?

Exposure of blood products to gamma irradiation is currently the standard of care in the prevention of transfusion-associated graft-versus-host disease (TA-GVHD). Regulatory, technical, and clinical challenges associated with the use of gamma irradiators are driving efforts to develop alternatives. Pathogen reduction methods were initially developed to reduce the risk of microbial transmission by blood components. Through modifications of nucleic acids, these technologies interfere with the replication of both pathogens and white blood cells (WBCs). To date, systems for pathogen and WBC inactivation of products containing red blood cells are less well established than those for platelets and plasma. In this study, the in vitro and in vivo function of WBCs present in whole blood after exposure to riboflavin plus ultraviolet light (Rb-UV) was examined and compared to responses of WBCs obtained from untreated or gamma-irradiated blood by measuring proliferation, cytokine production, activation, and antigen presentation and xenogeneic (X-)GVHD responses in an in vivo mouse model. In vitro studies demonstrated that treatment of whole blood with Rb-UV was as effective as gamma irradiation in preventing WBC proliferation, but was more effective in preventing antigen presentation, cytokine production, and T-cell activation. Consistent with in vitro findings, treatment with Rb-UV was as effective as gamma irradiation in preventing X-GVHD, a mouse model for TA-GVHD. The ability to effectively inactivate WBCs in fresh whole blood using Rb-UV, prior to separation into components, provides the transfusion medicine community with a potential alternative to gamma irradiation.

Adverse effects of plasma transfusion

Plasma utilization has increased over the past two decades, and there is a growing concern that many plasma transfusions are inappropriate. Plasma transfusion is not without risk, and certain complications are more likely with plasma than other blood components. Clinical and laboratory investigations of the patients suffering reactions after infusion of fresh-frozen plasma (FFP) define the etiology and pathogenesis of the panoply of adverse effects. We review here the pathogenesis, diagnosis, and management of the risks associated with plasma transfusion. Risks commonly associated with FFP include: 1) transfusion-related acute lung injury, 2) transfusion-associated circulatory overload, and 3) allergic and/or anaphylactic reactions. Other less common risks include 1) transmission of infections, 2) febrile nonhemolytic transfusion reactions, 3) red blood cell alloimmunization, and 4) hemolytic transfusion reactions. The effects of pathogen inactivation or reduction methods on these risks are also discussed. Fortunately, a majority of the adverse effects are not lethal and are adequately treated in clinical practice.

Establishment of the first International Repository for Transfusion‐Relevant Bacteria Reference Strains: ISBT Working Party Transfusion‐Transmitted Infectious Diseases (WP‐TTID), Subgroup on Bacteria

Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

The development of high hydrostatic pressure processes as an alternative to other pathogen reduction methods

In biology, scientist's interest for high hydrostatic pressure (HHP) has increased over the last 20 years, for both research and industrial developments, mainly because of the low energy associated with its application in liquid phase and its capacity to inactivate pathogens. It is now considered as an interesting alternative to heat treatments for the inactivation of contaminants in many products, from foods to pharmaceutical preparations. This last statement implies different objectives according to the type of product. The therapeutic properties of pharmaceutical preparations or other biological media of physiological importance are in general associated with specific and well-defined molecules such as proteins. Their activity mainly depends on their spatial conformation, maintained by weak chemical bonds that are often pressure sensitive. In this case, the optimization of a HHP process can be more complex than for foods, for which the organoleptic molecules are less pressure sensitive, and the evaluation of their preservation is more subjective and highly dependent on the consumers acceptance. The objective of this review is therefore to underline how, even if the basic concept for the optimization of a pathogen reduction process using HHP is the same whatever the product, major differences arise from the product itself and its final use.

Alternative strategies in assuring blood safety: An Overview

Assuring transfusion safety is an essential element of health care in all countries, requiring government commitment, national policy and a legal framework. Fundamental safety strategies include selection of low risk donors, Good Manufacturing Practices in preparation of blood components, and appropriate clinical use including avoidance of unnecessary transfusions. Hemovigilance, including surveillance for known adverse events and sentinel reporting of unexpected adverse events, enhances safety through benchmarking to promote best practices and by enabling rapid responses to new threats. Preventing transmission of infectious diseases is a principal safety concern. Selection of low risk donors includes use of screening questions to elicit risk factors known to be associated with transmissible infections. Laboratory testing for specific infectious disease markers is an established strategy for interdicting contaminated donations. The sensitivity, specificity, and operational convenience of laboratory testing have improved over time and newer technologies are imminent. Donor screening and laboratory testing, while highly effective in reducing risk, cannot eliminate all risk from known agents and must be developed de novo to address emerging infections. In contrast, pathogen reduction technologies offer the possibility for robust inactivation of a broad spectrum of blood transmissible agents and provide an added safeguard against newly emerging infectious threats of most types. Current pathogen reduction methods also inactivate leukocytes, adding safety benefits similar to leukocyte removal and product irradiation. However, to date, concerns about the safety and efficacy of cellular blood components treated by pathogen reduction have prevented approval of these technologies in the U.S. and Canada. FDA is promoting clinical and basic scientific studies to clarify these issues and would consider alternative approaches to assuring blood safety if pathogen reduction technologies are proven to be safe and effective.

Evaluation of potential immune response and in vivo survival of riboflavin‐ultraviolet light–treated red blood cells in baboons

Pathogen reduction methods have the potential to modify blood components, resulting in immunologic reactions or compromised blood components. This study evaluated the hypothesis that there is no immune response to riboflavin-and-ultraviolet [UV]-light-treated red blood cells (RBCs), as observed by serology and by survival of RBCs in circulation. Three baboons were in each treatment group: 1) untreated (negative control), 2) quinacrine mustard (QM)-treated (positive control), and 3) riboflavin-and-UV light-treated (test group) RBCs. In the immunization phase, autologous test or control RBCs were injected subcutaneously on Days 0, 21, 42, and 49. Plasma samples from these days were tested against test or control RBCs by flow cytometry and standard serology. On Day 56, autologous (51)Cr-labeled test or control RBCs were injected. Blood samples were taken over 21 days after injection to determine RBC survival (t(1/2)). Untreated and riboflavin-and-UV-light-treated RBCs showed no evidence of significant immunoglobulin G (IgG) binding after incubation with autologous plasma. RBC-bound IgG was detected on QM-treated RBCs after incubation with autologous plasma. This antibody was inhibited by QM, as demonstrated by a hapten inhibition study. t(1/2) values for the untreated and riboflavin-and-UV-light-treated RBCs were 7.3 +/- 0.8 and 7.5 +/- 1.7 days, respectively; the t(1/2) value for QM-treated RBCs was 2.3 +/- 2.9 days. Treatment with riboflavin and UV light did not render RBCs immunogenic. Positive controls indicated that immunization promoted an immune response. In the (51)Cr-labeled RBC survival phase of the study, riboflavin-and-UV-light-treated RBCs exhibited behavior similar to negative control RBCs. Detrimental immunologic or functional side effects were not observed.

Strategies of bacteria screening in cellular blood components

Since the impressive reduction of transfusion-transmitted virus infections, bacterial infections by blood transfusion represent the most important infection risk. Platelet concentrates are the current focus of attention, as they are stored under temperature conditions which allow growth of contaminating bacteria up to 10(10) and more microbes per platelet bag. This paper does not consider the pathogen reduction methods but will assess suitable screening methods. Beside conventional microbiological approaches or surrogate markers, several efficient methods able to detect bacterial contamination in platelets are available on the market. They need to be divided into two different methodological principles: the cultivation methods and rapid methods. Cultivation or incubation methods require some time for signal production as they depend on growth of microbes. Thus, they have to be combined with early sampling, i.e., the sample to be examined has to be drawn from the blood component 1 day after donation. Their advantage is the relatively uncomplicated implementation into the logistics of blood banks. Because of the initially very low count of bacteria after donation, a certain small sampling error in application of that strategy remains. Rapid methods are able to produce the diagnosis within a short time. Therefore, they allow postponing of sample drawing, ideally up to the time immediately before transfusion. However, this procedure causes logistic complications. On the other hand, late sampling combined with a rapid method will prevent the transfusion of highly contaminated platelet concentrates leading to acute septic shock up to the death of the patient. Considering the sum of different aspects including the supply of patients, the potential improvement of microbial safety of platelet concentrates is comparable in both strategies.

Transfusion‐transmitted bacterial infection: risks, sources and interventions

Records of the transmission of bacterial infections by transfusion date back to the beginning of organized blood banking. Despite tremendous strides in preventing viral infection through careful donor screening and viral testing, there has been little improvement in reducing the risk of bacterial sepsis since the introduction of closed collection systems. Based on the French Haemovigilance study, the British Serious Hazards of Transmission (SHOT) study and fatality reports to the United States Food and Drug Administration, the risk of clinically apparent sepsis exceeds the risk of HIV, HBV, and HCV transmission. Sources of contamination include the skin, blood, disposables, and the environment. Potential interventions to reduce transfusion-associated bacterial sepsis include improvements to donor arm preparation, diversion of the first aliquot of whole blood, introduction of bacterial testing and/or implementation of pathogen reduction methods.