Implications of a switch to a 100% apheresis platelet supply for patients and for blood donors: a risk benefit analysis.

BACKGROUND: A 100% apheresis platelet supply is considered to increase transfusion safety by lowering donor exposures for transfusion recipients. We performed a risk benefit analysis to contrast the reduction of donor exposures and the risk of contaminated blood products in the nation-wide inventory with the donor risks associated with a switch to a 100% apheresis platelet supply in Germany. METHODS: Donor exposures and the number of contaminated blood products resulting from HIV-like, HBV-like, HCV-like pathogens and two theoretical agents with infection rates of 10 and 1000 in 100 000, respectively, were calculated for a 100% apheresis platelet supply in Germany based on the 2006-2012 hemovigilance reports. These numbers were compared with the current mixed platelet supply of pooled and apheresis platelets. Moreover, additional donation time and apheresis donor complications resulting from a 100% apheresis platelet supply were estimated. RESULTS: Per million total blood products (red cells, platelets, fresh frozen plasma), a 100% apheresis platelet supply would reduce donor exposures by 87 100 and the number of contaminated blood products ranging from 0·8 to 871·1. On the other hand, this requires additional 29 478 apheresis donations, 3·4 years additional donor time, and would be associated with 58 additional donor complications, respectively. CONCLUSIONS: A 100% apheresis platelet supply would reduce donor exposures and the number of contaminated blood products in the inventory, but would increase apheresis complications in donors. Potential risks for patients must be carefully weighed against the risks for donors, dependent on the specific pathogen scenario.

HNA antibody-mediated neutrophil aggregation is dependent on serine protease activity.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is often caused by antibodies against human neutrophil alloantigen-2 (HNA-2) and HNA-3a. Neutrophil aggregation is considered as a major cause of TRALI, but little is known about how HNA antibodies initiate this process. We explored mechanisms involved in neutrophil aggregation induced by HNA-2 and HNA-3a antibodies. MATERIALS AND METHODS: Isolated neutrophils were pretreated with broad-spectrum or specific inhibitors against different cell functions or proteases. Granulocyte agglutination test (GAT) was performed with serially diluted anti-HNA-2 and anti-HNA-3a plasmas or control plasma, and reactivity was evaluated microscopically. Reactive oxygen species (ROS) production in neutrophils was investigated using a lucigenin-based chemiluminescence assay. RESULTS: HNA-2 and HNA-3a antibody-mediated neutrophil aggregation was inhibited by pretreatment with formaldehyde, iodoacetamide and the serine protease inhibitors Pefabloc-SC, N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Nα-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK). In contrast, inhibition of actin polymerization, respiratory burst, cysteine proteases, metalloproteases or aspartic proteases did not affect neutrophil aggregation. Furthermore, HNA-3a antibodies did not directly cause ROS production in neutrophils. CONCLUSION: Aggregation of neutrophils induced by HNA-2 and HNA-3a antibodies is an active process and depends on trypsin- or chymotrypsin-like serine proteases but is not dependent on the production of ROS. These findings may open new prospects for the pharmacologic prevention of neutrophil-associated acute lung injury.

Routine bacterial screening of platelet concentrates by flow cytometry and its impact on product safety and supply.

BACKGROUND AND OBJECTIVES: Bacterial contamination represents the major infectious hazard associated with transfusion of platelet concentrates (PCs). As bacterial screening of PCs is not mandatory in Germany, the BactiFlow flow cytometry test has been introduced as a rapid detection method to increase product safety. MATERIALS AND METHODS: During a period of 25 months, a total of 34 631 PCs (26 411 pooled and 8220 apheresis-derived PCs) were tested at the end of day 3 of their shelf life using the BactiFlow system. PCs initially reactive in BactiFlow testing and expired PCs not reactive in BactiFlow on day 3 were also investigated by the BacT/ALERT system and by microbiological cultivation in order to identify the contaminating bacterial species and to confirm reactive BactiFlow results. RESULTS: Two hundred and twenty-eight PCs (0.7%) had an initially reactive result, 24 of them remained reactive in a second test run. Out of these reproducible reactive BactiFlow results, 12 could not be verified by parallel BacT/ALERT culturing, resulting in a confirmed false-positive rate of 0.03%. The bacterial species were identified as S. aureus, S. epidermidis, S. dysgalactiae ssp. equisimilis and B. cereus. In 10 out of 9017 expired PCs (0.11%), a confirmed-positive result was obtained in the BacT/ALERT system which had a negative result in the BactiFlow system. CONCLUSION: Testing of PCs by BactiFlow was successfully implemented in our blood donation service and proved sufficient as a rapid and reliable screening method. False reactive results are in an acceptable range since the transfusion of 12 bacterially contaminated PCs was prevented.

Evaluation of a new German blood donor questionnaire.

BACKGROUND AND OBJECTIVES: In Germany, blood donors must complete a donor history questionnaire (DHQ). In 2011, a new standardized DHQ for Germany was proposed which included questions about sexual risk behaviour that raised concerns about acceptance by donors. MATERIALS AND METHODS: We assessed the acceptability and ease of use of the new DHQ in two German donor populations, by asking the donors to complete a paper survey evaluating the design and clarity of the questions of the new DHQ with a focus on the questions addressing sexual risk behaviour. RESULTS: In Greifswald, 2000 donors (mean age 32 ± 12 years) participated, and in Hagen 2088 donors (mean age 48 ± 14 years). Ratings between the two populations showed only minor differences. Donors reported that the questions in the new DHQ were understandable and clearly structured. Perception of comfort level with questions addressing sexual risk behaviour differed significantly depending on donor characteristics such as age, gender and educational level. Overall, the new DHQ seems to deter approximately 5% of potential donors from blood donation. CONCLUSION: The new DHQ was acceptable to the vast majority of donors. Potential donors who were older, male gender and less educated were most at risk of refusing to donate and may benefit from educational interventions.

HLA class II haplotypes differentiate between the adult autoimmune polyglandular syndrome types II and III.

BACKGROUND: Genetics of the adult autoimmune polyglandular syndrome (APS) is poorly understood. AIM: The aim of this study was to gain further insight into the genetics of the adult APS types. SITE: The study was conducted at a university referral center. METHODS: The human leukocyte antigen (HLA) class II alleles, haplotypes, and genotypes were determined in a large cohort of patients with APS, autoimmune thyroid disease (AITD), and type 1 diabetes and in healthy controls by the consistent application of high-resolution typing at a four-digit level. RESULTS: Comparison of the allele and haplotype frequencies significantly discriminated patients with APS vs AITD and controls. The HLA class II alleles DRB1*03:01 *04:01, DQA1*03:01, *05:01, DQB1*02:01, and *03:02 were observed more frequently (P<.001) in APS than in AITD and controls, whereas the alleles DRB1*15:01, DQB1*03:01, and *06:02 were underrepresented in APS vs AITD (Pc<.001) and controls (Pc<.01), respectively. The DRB1*03:01-DQA1*05:01-DQB1*02:01 (DR3-DQ2) and DRB1*04:01-DQA1*03:01:DQB1*03:02 (DRB1*04:01-DQ8) haplotypes were overrepresented in APS (Pc<.001). Combination of both haplotypes to a genotype was highly prevalent in APS vs AITD and controls (Pc<.001). Dividing the APS collective into those with Addison's disease (APS type II) and those without Addison's disease but including type 1 diabetes and AITD (APS type III) demonstrated DR3-DQ2/DRB1*04:01-DQ8 as a susceptibility genotype in APS III (Pc<.001), whereas the DR3-DQ2/DRB1*04:04-DQ8 genotype correlated with APS II (Pc<.001). The haplotypes DRB1*11:01-DQA1*05:05-DQB1*03:01 and DRB1*15:01-DQA1*01:02-DQB1*06:02 are protective in APS III but not in type II (Pc<.01). CONCLUSIONS: HLA class II haplotypes differentiate between the adult APS types II and III. Susceptible haplotypes favor the development of polyglandular autoimmunity in patients with AITD.

External quality assessment of human neutrophil antigen (HNA)-specific antibody detection and HNA genotyping from 2000 to 2012.

Since 2000, Quality Assurance (QA) exercises for the detection and identification of granulocyte antibodies and DNA typing for human neutrophil antigens (HNA) have been distributed within the International Granulocyte Immunobiology Workshops, which are linked to International Society of Blood Transfusion. The exercises were standardised at the outset to enable laboratory performance to be monitored. Between 2000 and 2012, nine exercises were distributed to 20 laboratories. Overall, 45 examples of 42 unique samples containing defined granulocyte reactive antibodies were distributed for serological analysis together with 20 samples for HNA genotyping. The level of satisfactory serological performance was initially set at 50% and later increased to 70%, while the 'cut-off' for HNA genotyping was set at 100% after 2008. Failure to achieve the minimum score in the QA exercises in consecutive years resulted in temporary exclusion. In 2000, the 15 participating laboratories had a mean score of 56.1% for serological analysis and 13 laboratories attempted HNA-1a and -1b genotyping, while 11 attempted HNA-1c typing. Steady improvements in proficiency for serological testing and HNA typing occurred in subsequent exercises. In 2012, the mean score for serology was 88.5% and 12/13 laboratories scored 100% for HNA-1a, -1b, -1c, -3a, -3b, -4a, -4bw, -5a and -5bw genotyping. These QA exercises have provided an invaluable tool to monitor and improve the standard of granulocyte immunology investigations for participating laboratories, thereby enhancing performance for both clinical investigations and donor screening programmes to reduce the incidence of TRALI.

Detection of bacterial contamination in platelet concentrates by a sensitive flow cytometric assay (BactiFlow): a multicentre validation study.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk. As a result of septic complications, particularly observed with older PCs, the shelf life of PCs has been reduced in Germany to 4 days. In this study, bacterial screening of PCs by BactiFlow (BF) flow cytometry was introduced in three German blood services to evaluate the robustness and applicability of the assay. Results were used to discuss the potential for the extension of PC shelf life to 5 days. STUDY DESIGN AND METHODS: A total of 1956 PCs were tested on days 4 or 5+ after PC production using the BF, whereas the BacT/Alert culture system served as reference method. RESULTS: Two PCs were confirmed positive by culture only and were identified as Propionibacterium acnes and Staphylococcus species. Two PCs were confirmed positive for Streptococcus mitis by BF and culture. Additionally, two PCs were culture-positive only in one culture bottle (aerobic: S. mitis and anaerobic: S. hominis). Retrospective analysis of bacterial growth kinetics provide the indication that corresponding bacterial titres were most likely below the BF analytical detection limit (<150 CFU mL(-1) ) and had probably no transfusion relevance. All remaining specimens were tested negative. CONCLUSIONS: Testing of PCs by BF was successfully implemented. The BF proved sufficient as a rapid screening method to improve PC safety. This study further provides data supporting the extension of PC shelf life to 5 days after negative BF testing on day 4.

Inter-laboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates.

BACKGROUND: Bacterial contamination of platelet concentrates still represents a major risk in transfusion medicine, and a variety of screening methods have been available to improve the safety of PCs. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry, Pan Genera Detection Assay, 23S rRNA RT-PCR) was evaluated in an inter-laboratory comparison in three different German blood services. METHODS: Samples were inoculated with different bacteria [Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli (two strains), Klebsiella pneumoniae (two strains), Enterobacter aerogenes (one strain), Serratia marcescens (one strain)] at different counts (4·5 × 10(3) -4·5 × 10(8) CFU/ml) alternating with negative samples in one transfusion facility. Samples were blinded with a random order for each screening method, shipped to partners and analysed immediately after receipt with different rapid screening methods. RESULTS: The inter-laboratory comparison revealed that the BactiFlow assay and 23S rRNA RT-PCR-screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan Genera Detection Assay test detected only four of the positive samples. Four of the non-detected positive samples were below the assay's detection limit. Another four inoculated samples with comparatively high bacteria counts were detected false negative (E. coli (two strains): 9·87 × 10(5) and 2·10 × 10(7) CFU/ml, respectively, K. pneumoniae: 4·79 × 10(6) CFU/ml, S. aureus: 6·03 × 10(5) CFU/ml). All rapid screening methods revealed no false-positive results. CONCLUSIONS: Both BactiFlow and 23S rRNA RT-PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs. The Pan Genera Detection Assay had some shortcomings regarding sensitivity, especially for the detection of Gram-negative strains.

Fresh frozen plasma quality: relation to age and gender of blood donors.

BACKGROUND: In view of demographic changes, it is becoming increasingly necessary to make allowance for the fact that older individuals in good health are generally more willing to donate blood and hence they should be allowed to do so irrespective of an upper age limit. In this respect, evidence has to be produced that there is neither an age-related increase in risks to the donor nor any influence on the safety and efficacy of the blood components. It was therefore of interest to examine the quality of therapeutic plasma in relation to a donor's age and also a donor's gender in view of the preferred use of male donor plasma for reducing the risk of transfusion related acute lung injury. STUDY DESIGN AND METHODS: Citrate-phosphate-dextrose (CPD) units of whole blood taken in each case from 50 blood donors (30 men and 20 women) of three age groups (cohort 1 = 69-71 years, cohort 2 = 66-68 years and cohort 3 = 50-52 years) were filtered, centrifuged and then separated into the relevant blood components. The plasma samples were assessed for international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, factor V (FV), factor VIII (FVIII), antithrombin (AT), protein S and plasminogen. RESULTS: While aPTT showed only slight age-related differences of no significance, the INR levels of cohort 1 were significantly higher than those of cohorts 2 and 3. However, in terms of gender, this applied to male donors only. The differences in INR are demonstrated by lower FV levels of male cohort 1 donors, although this is not statistically significant in comparison with the other two age groups and with female donors. Female donors of all cohorts, however, exhibited noticeably lower aPTT levels than male donors. Lower INR and aPTT values in women could be a sign of a lower dilution affected by CPD anticoagulant as compared to male plasma. An increase in FVIII levels was also apparent with increasing age (P < 0·05), particularly in male donor plasma. The fibrinogen levels suggest a slight, though insignificant, age-related increase and no significant gender-dependent effect. The plasma levels of AT and plasminogen were unremarkable. The plasma of female cohort 3 donors exhibited a protein S concentration that was slightly lower by comparison. Compared with the other two cohorts, cohort 1 plasma levels were similarly above or below the normal range derived from cohort 3 (central 95% range) for all parameters tested. CONCLUSION: International Normalized Ratio, aPTT, FVIII, FV, fibrinogen and protein S, as quality indicators for the efficacy of therapeutic plasma, revealed a moderate correlation with age and gender. Compared with the usual reference ranges, the differences were not significant enough to identify any relevant imbalance between procoagulating, anticoagulating and fibrinolytic factors that might influence product quality where the increasing age of the donors or the preference of male donor plasma was concerned.

TRALI-new challenges for histocompatibility and immunogenetics in transfusion medicine.

Antibodies against human leukocyte antigens (HLAs) have long been associated with transfusion-related acute lung injury (TRALI). In contrast to febrile transfusion reactions and refractoriness to platelet transfusions in immunized patients, the causative antibodies in TRALI are present in the transfused blood component, i.e. they are formed by the blood donor and not by the recipient. Consequently, blood components with high plasma volume are particularly associated with TRALI. In addition to antibodies against HLAs, antibodies directed against human neutrophil antigens (HNAs) present in the plasma of predominantly multiparous female blood donors can induce severe TRALI reactions. Especially, antibodies to HLA class II and HNA-3a antigens can induce severe or even fatal ALI in critically ill patients. Over the last decade, the clinical importance of TRALI as major cause for severe transfusion-related morbidities has led to the establishment of new guidelines aimed at preventing this condition, including routine testing for HLA and -HNA antibodies for plasma donors with a history of allogeneic sensitization. This, in turn, poses new challenges for close collaboration between blood transfusion centers and histocompatibility and immunogenetics laboratories, for sensitive and specific detection of the relevant antibodies.

The safety of blood donation by elderly blood donors.

BACKGROUND Due to the ageing population, blood donation by the elderly is necessary to maintain blood supply. We initiated a prospective study, to assess whether there is an increased risk of donor reactions in elderly donors. STUDY DESIGN AND METHODS In this prospective study, regular donors aged from 66 to 68 and 69 to 71 years were invited to continue blood donation on mobile collection sites of the German Red Cross Blood Service West. A control group (50-52 years) was established. Admission of donors in all groups followed the German national guidelines for blood donation. Donor deferrals and all kinds of donor reactions during donation (on-site) and in the 48 h following donation (off-site) were monitored. RESULTS A total of 64 260 valid cases were entered in the study. Donor deferrals increased with age from 1·12% in the control group up to 8·74 in female donors aged 69-71 years. Adverse reactions to blood donation were rare with an overall reaction rate of 0·63% (0·05% on-site; 0·58% off-site). Off-site reactions significantly decreased with increasing age. The relative risk (RR) for adverse reactions in elderly donors compared to the control group (50-52 years) was slightly increased for on-site reactions in the 69- to 71-year-old donors (RR 1·0309; 95% CI 1·0292-1·0325). In all other comparisons, the RR for adverse reactions was distinctively lower in elderly donors (RR 0·3785 - 0·7778). CONCLUSIONS Our data confirm that elderly regular blood donors may safely continue blood donation at least to the age of 71. Based on these data, we increased the upper age limit.

Autoimmune polyglandular syndrome type 2 shows the same HLA class II pattern as type 1 diabetes.

Autoimmune polyglandular syndrome (APS) type 2 is defined by the manifestation of at least two autoimmune endocrine diseases. Only few data exist on genetic associations of APS type 2. In this controlled study, 98 patients with APS type 2, 96 patients with type 1 diabetes (T1D), and 92 patients with autoimmune thyroid disease, both as a single autoimmune endocrinopathy, were tested for association with alleles of the human leukocyte antigen (HLA) class II loci DRB1, DQA1, and DQB1. Patients with APS type 2 had significantly more often the alleles DRB1*03 (P(c) < 0.0001), DRB1*04 (P(c) < 0.000005), DQA1*03 (P(c) < 0.0001), and DQB1*02 (P(c) < 0.05), when compared with controls. Less frequent in APS were DRB1*15 (P(c) < 0.05), DQA1*01 (P(c) < 0.0005), and DQB1*05 (P(c) < 0.005). With regard to frequency and linkage of these alleles, the susceptible haplotypes DRB1*0301-DQA1*0501-DQB1*0201 and DRB1*0401/04-DQA1*0301-DQB1*0302 were deduced. Protective haplotypes in this study were DRB1*1501-DQA1*0102-DQB1*0602 and DRB1*0101-DQA1*0101-DQB1*0501. Comparing APS patients with vs without AD, no significant differences regarding HLA class II alleles were noted in our collective. Patients with T1D as a singular disease had the same susceptible and protective HLA alleles and haplotypes. The prevalence of DRB1*03 and DRB1*04 in APS patients was not because of the presence of diabetes, as the APS type 2 patients without diabetes had the same allele distribution. In conclusion, these data suggest a common immunogenetic pathomechanism for T1D and APS type 2, which might be different from the immunogenetic pathomechanism of other autoimmune endocrine disease.

Antibody-mediated (immune) transfusion-related acute lung injury.

Antibody-mediated transfusion-related acute lung injury (immune TRALI) is now recognized as the most common cause of transfusion-associated major morbidity and death in the Western world. Among the implicated leucocyte antibodies, these ones directed against the human leucocyte antigens (HLA) class II, human neutrophil alloantigens (HNA)-3a and HLA-A2 antigens are frequently associated with severe (artificial ventilation required) and fatal cases. There is accumulating evidence that preventive measures such as transfusion of plasma-rich blood components from male donors or from donors tested negative for leucocyte antibodies are effective in the reduction of severe and fatal immune TRALI.

Blood donor screening with cobas s 201/cobas TaqScreen MPX under routine conditions at German Red Cross institutes.

BACKGROUND: In 1997 the German Red Cross (GRC) blood donor services introduced mini-pool nucleic acid testing (NAT) for human immunodeficiency virus (HIV)-1, hepatitis C virus (HCV) and hepatitis B virus (HBV) to increase blood safety. With the new cobas s 201/cobas TaqScreen MPX, a fully automated extraction method and a multiplex amplification system specifically adapted to the needs of blood donation services is available. METHODS: The cobas s 201 system was evaluated at the GRC BTS locations Hagen, Springe and Frankfurt. In phase A, the analytical sensitivity for the detection of HBV, HCV and HIV-1 was investigated and in phase B, at least 60,000 samples at each test site were screened in parallel with the MPX test on s 201 system and the existing routine mini-pool NAT system to compare the diagnostic specificity and the diagnostic sensitivity. RESULTS: Comparable analytical sensitivities in a range of 1.6-3.6 IU/ml, 4.9-10.9 IU/ml and 14.7-26.6 IU/ml for HBV, HCV HIV, respectively, for the MPX test on s 201 system (95% probability based on probit analysis) were determined at all test sites. The diagnostic sensitivity was 99.8% and the diagnostic specificity was 99.85%. CONCLUSIONS: The MPX test on s 201 system is a fully automated NAT system suitable for routine blood donor screening. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements of the Paul Ehrlich Institute for blood donor screening in mini-pools up to 96 donations per pool. A major benefit of the automated NAT system is the reduced personnel time and the extensive complete barcode-controlled process documentation.

[Autoimmune thrombocytopenia, neutropenia and hemolysis]. / Autoimmune Thrombozytopenie, Neutropenie und Hämolyse.

Autoantibodies reduce the life span of platelets, granulocytes, and red blood cells. This may result in thrombocytopenia with bleeding, in neutropenia with infection, and in anemia, respectively. Immune-hemocytopenias can manifest as primary disease without another cause, or they are associated with other underlying morbidities such as autoimmune diseases, lymphoproliferative diseases, immune defects, or viral infections. Diagnosis is confirmed by laboratory tests showing autoantibodies against the respective blood cells. Indication for treatment is the clinical manifestation of symptoms: bleeding in autoimmune thrombocytopenia, infections in neutropenia, and symptomatic anemia, respectively. Especially in case of thrombocytopenia patients should not be treated because of abnormal laboratory values, only. To date steroids are the basic treatment in autoimmune thrombocytopenia and hemolytic anemia, while prophylactic antibiotics are the main treatment in autoimmune neutropenia. Growth factors like the new thrombopoietin receptor agnonists in autoimmune thrombocytopenia or G-CSF in autoimmune neutropenia, and anti-CD20 antibodies are new options for treatment.

Recommendations of the ISBT Working Party on Granulocyte Immunobiology for leucocyte antibody screening in the investigation and prevention of antibody-mediated transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is currently one of the most common causes of transfusion-related major morbidity and death. Among the many TRALI mediators, leucocyte antibodies have been identified as important triggers of severe TRALI. STUDY DESIGN AND METHODS: These recommendations were compiled by experts of the ISBT Working Party on Granulocyte Immunobiology, based on the results obtained in eight international granulocyte immunology workshops, their personal experiences and on published study results. RESULTS: Leucocyte antibody screening has to include the detection of human leucocyte antigen (HLA) class I, class II and human neutrophil alloantigen antibodies using established and validated techniques. HLA class I antibody detection should be restricted to antibodies clinically relevant for TRALI. To avoid unnecessary workload, TRALI diagnosis should be assessed by consultation with the reporting clinician and thorough exclusion of transfusion-associated circulatory overload/cardiac insufficiency. In patients diagnosed with TRALI having donors with detectable leucocyte antibodies, evidence of leucocyte incompatibility should be provided by either cross-matching or typing of patient for cognate antigen. CONCLUSION: Leucocyte antibody screening for the immunological clarification of TRALI cases as well as for identification of potentially alloimmunized blood donors is feasible and can be performed in a reasonable and quality assured manner. This practice can contribute to the prevention of antibody-mediated TRALI.