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BACKGROUNDSerologic determination of the Vel- phenotype is challenging due to variable Vel expression levels. In this study we investigated the genetic basis for weak Vel expression levels and developed a high-throughput genotyping assay to detect Vel- donors. STUDY DESIGN AND METHODSIn 548 random Caucasian and 107 Vel+(w) donors genetic variation in the SMIM1 gene was studied and correlated to Vel expression levels. A total of 3366 Caucasian, 621 black, and 333 Chinese donors were screened with a high-throughput genotyping assay targeting the SMIM1*64_80del allele. RESULTSThe Vel+(w) phenotype is in most cases caused by the presence of one SMIM1 allele carrying the major allele of the rs1175550 SNP in combination with a SMIM1*64_80del allele or in few cases caused by the presence of the SMIM1*152T>A or SMIM1*152T>G allele. In approximately 6% of Vel+(w) donors genetic factors in SMIM1 could not explain the weak expression. We excluded the possibility that lack of expression of another blood group system was correlated with weak Vel expression levels. Furthermore, using a high-throughput Vel genotyping assay we detected two Caucasian Vel- donors. CONCLUSIONWeak Vel expression levels are caused by multiple genetic factors in SMIM1 and probably also by other genetic or environmental factors. Due to the variation in Vel expression levels, serologic determination of the Vel- phenotype is difficult and a genotyping assay targeting the c.64_80del deletion in SMIM1 should be used to screen donors for the Vel- phenotype.

Background Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. In the past decade blood banks have implemented low-risk TRALI donor strategies, including a male-only donor policy for plasma-containing blood products to prevent onset of TRALI. We performed a meta-analysis to determine whether use of low-risk TRALI donor strategies for plasma indeed reduces onset of TRALI. Study Design and Methods We searched MEDLINE and Cochrane Central Register of Controlled Trials from January 1995 up to January 2013. Two reviewers independently extracted data on study characteristics, methods, and outcomes. Primary endpoint was onset of TRALI. Subgroup analyses were performed for patient populations prone to develop TRALI and general patient populations. Results Ten articles were included. Meta-analysis using a random-effects model taking into account all transfused products showed a significant reduction for the risk of TRALI after implementation of low-risk TRALI donor strategies (odds ratio [OR], 0.61; 95% confidence interval [CI], 0.42-0.88). Data from patient populations prone to develop TRALI showed a significant reduction of TRALI risk (OR, 0.51; 95% CI, 0.29-0.90), while data from general patient populations showed a similar nonsignificant trend (OR, 0.66; 95% CI, 0.40-1.09). Results were similar when taking only plasma products into account (OR, 0.62; 95% CI, 0.42-0.92). Conclusion The introduction of low-risk TRALI donor strategies for plasma-containing products results in a reduction of TRALI.

BACKGROUND: A photochemical treatment (PCT) process using the psoralen compound amotosalen HCL (S59) and long wavelength UVA light was developed for inactivation of infectious pathogens and WBCs. In this study the effect of PCT on functional characteristics of the platelets was evaluated in vitro. STUDY DESIGN AND METHODS: Platelet concentrates were treated photochemically using the experimental clinical processing system T-bag S59 Reduction Device (SRD) (n = 4) or the commercially available integral processing system Wafer SRD (n = 4) and compared with control platelet concentrates in plasma/PAS III alone (n = 4). The evaluation included variables with respect to the overall quality of the product (e.g., HSR, pH), the function (aggregation and activation tests), apoptosis (annexin V and caspase 3), and lysis. RESULTS: No differences were found in the product quality variables, in P-selectin expression, and the apoptosis variables. PCT using the T-bag SRD led to a significant decrease in aggregation capacity with collagen and thrombin and a significant increase in plasma LDH, whereas no differences for the Wafer SRD were found. CONCLUSION: PCT using the experimental T-bag SRD led to a significant decrease in platelet function. However, the commercially available Wafer SRD had only minor in vitro effects on the quality of the platelets. latelet concentrates (PCs) are predominantly used for the treatment of thrombocytopenic patients or patients with platelet dysfunction. There are many controversies that surround platelet-transfusion practices today. 1,2 Platelet transfusions are associated with a number of adverse events, like fever, bacteremia, exanthema, and, rarely, GVHD. A photochemical treatment (PCT) method, which utilizes the psoralen (S-59) and long wavelength ultraviolet light, has been developed to inactivate infectious pathogens and WBCs in PCs (Cerus Corporation, Concord, CA, and Baxter Healthcare Corporation, Deerfield, IL). 3-6 A

BACKGROUND: RH genotyping assays are mainly based on research in whites. These assays may not be reliable in a multiracial society because of the genetic variation in RH among ethnic groups. STUDY DESIGN AND METHODS: Five groups from different ethnic backgrounds were serologically typed for C and c and were genotyped on nucleotide C48 and intron 2 for RHC and RHc on nucleotides C178 and C307. RESULTS: RHc genotyping with both methods proved to be reliable. RHC genotyping on C48 is not reliable because of a 48G>C mutation in the RHce allele (false-positive prediction of C). This mutation was found in every ethnic group and does not affect c or e expression. RHC genotyping on intron 2 is unreliable because of r′s (Cdes) alleles (a false-negative prediction of C). This allele was found in whites and blacks from Curacao and South Africa. Reactions of r′s cells with anti-C are weaker, but no negative reactions with various MoAbs were found. A new method (RHC/c/hex3-intron 4/exon 7 multiplex PCRs) was developed based on intron 2 and r′s hybrid exon 3 characteristics (RHC) and C307 (RHc). CONCLUSIONS: Reliable RHC and RHc genotyping is possible in different ethnic groups with the RHC/c/hex3-intron 4/exon 7 multiplex PCR approach.

BACKGROUND: In the human Rh blood group system, c is, after D, the most immunogenic antigen. STUDY DESIGN AND METHODS: The background of a new partial c phenotype (Dc), identified on the RBCs of two unrelated white persons, was studied. This was done by analyzing the reactivity of the RBCs from the donors with anti-c reagents, by performing sequence analysis, and by carrying out transduction studies. RESULTS: Serologic results suggested the existence of a new partial c phenotype. Genomic DNA and cDNA analysis revealed a normal RHCe allele, a normal RHD allele, and an RHD allele that carried two point mutations: 307T>C and 329T>C (the latter known to be associated with the DVII, Tar-positive phenotype). No normal RHc allele was found. Thus, it was most likely that c is encoded by the mutated RHD allele (phenotype DDcCCee). Indeed, subsequent transduction of K562 erythroleukemic cells with an RHD cDNA carrying the 307T>C point mutation (leading to S103P) resulted in the expression of c. CONCLUSION: In the human Rh system, P103 is involved in the expression of c. Moreover, c can be expressed in vivo on the D polypeptide.

BACKGROUND: A new citrate-acetate-NaCl platelet additive solution, identified as PAS 2, was developed to prepare platelet concentrates (PCs) from pooled 0.5 CPD buffy coats (BCs). STUDY DESIGN AND METHODS: A study was undertaken to evaluate PAS 2 in vitro (n = 8) and in vivo (n = 9) against a commercially available solution (Plasma-Lyte A). In a paired in vitro study, a comparison was made of platelet and white cell concentration; blood gases and bicarbonate; glucose and lactate concentration; total intracellular concentration of adenine nucleotides and beta-thromboglobulin release. RESULTS: A lower platelet yield (p < 0.0001) and a higher beta-thromboglobulin release (p < 0.01) are observed with Plasma-Lyte A. For this reason, half-strength (0.5) CPD was changed to full-strength CPD in the clinical study with Plasma-Lyte A. In a clinical evaluation of nine patients with bone marrow failure, all received PCs with both PAS 2 and Plasma-Lyte A that had a shelf life of at least 4 days. Corrected count increments (CCls) were as follows, on average (95% Cl): the CCl at 1 to 4 hours was 22.4 (95% Cl, 15.2-29.4) for PAS 2 and 24.0 (95% Cl, 16.9-31.2) for Plasma-Lyte A; that at 12 to 24 hours was 11.3 (95% Cl, 4.1-18.4) for PAS 2 and 14.2 (95% Cl, 7.1-21.3) for Plasma-Lyte A; and that at 36 to 48 hours was 4.2 (95% Cl, -3.0-11.3) for PAS 2 and 8.7 (95% Cl, 1.1-16.2) for Plasma- Lyte A. No significant difference between the two solutions was found. CONCLUSIONS: PAS 2 and Plasma-Lyte A make important contributions to platelet transfusion quality improvement and give an excellent CCl even after 4 days of storage.

Pathogen reduction (PR) of platelet products increases costs and available clinical studies are equivocal with respect to clinical and haemostatic effectiveness. We conducted a multicentre, open-label, randomized, non-inferiority trial comparing the clinical effectiveness of buffy-coat derived leukoreduced platelet concentrates (PC) stored for up to 7 d in plasma with platelets stored in platelet additive solution III (PASIII) without and with treatment with amotosalen-HCl/ultraviolet-A (UVA) photochemical pathogen reduction (PR-PASIII). Primary endpoint of the study was 1-h corrected count increment (CCI). Secondary endpoints were 24-h CCI, bleeding, transfusion requirement of red cells and PC, platelet transfusion interval and adverse transfusion reactions. Compared to plasma-PC, in the intention to treat analysis of 278 evaluable patients the mean difference for the 1-h CCI of PR-PASIII-PC and PASIII-PC was -31% (P < 0.0001) and -9% (P = n.s.), respectively. Twenty-seven patients (32%) had bleeding events in the PR-PASIII arm, as compared to 19 (19%) in the plasma arm and 14 (15%) in the PASIII arm (P = 0.034). Despite the potential advantages of pathogen (and leucocyte) inactivation of amotosalen-HCl/UVA-treated platelet products, their clinical efficacy is inferior to platelets stored in plasma, warranting a critical reappraisal of employing this technique for clinical use.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a serious, sometimes fatal complication of transfusion, attributed to white blood cell (WBC)-reactive antibodies present in the blood product. This study investigated incidence and etiology in the Netherlands. STUDY DESIGN AND METHODS: From January 2005 through July 2007, all TRALI cases reported to the Sanquin Blood Banks were evaluated. Only cases meeting the Canadian Consensus Conference criteria for TRALI were included and investigated for patient, donor, and product characteristics. Patients and donors were screened for HLA Class I, HLA Class II, and granulocyte antibodies. RESULTS: A total of 56 TRALI cases were reported of which 49 were completely evaluated. Seventy-eight percent of the patients needed monitoring or mechanical ventilation on the intensive care unit, 10 patients died. In 61% of cases large-volume plasma products were involved. WBC-reactive antibodies in donors were found in 73% of cases, with proven incompatibility in 21 of 44 (48%) investigated cases. Possible TRALI cases, as defined by the Canadian Consensus Conference, had significantly lower incompatibility rates compared to TRALI cases, 18% versus 58% (p = 0.036). In the 21 alloimmune cases, a total of 31 implicated donors were found, of which 26 were female, including 12 fresh-frozen plasma (FFP) products. CONCLUSION: TRALI is the most serious transfusion complication in the Netherlands, causing severe morbidity and mortality. Antibodies were found in the majority of the cases, but causality with proven incompatibility could be established in 21 cases (48%). Female FFP products were involved in 57% of proven alloimmune cases and would theoretically be prevented using male FFP only.

A biannual external quality assessment (EQA) scheme for flow cytometric lymphocyte immunophenotyping is operational in the Benelux countries since 1996. We studied the effects of the methods used on assay outcome, and whether or not this EQA exercise was effective in reducing between-laboratory variation. Eighty test samples were distributed in 20 biannual send-outs. Per send-out, 50-71 participants were requested to enumerate CD3+, CD4+, and CD8+ T cells, B cells, and NK cells, and to provide methodological details. Participants received written debriefings with personalized recommendations after each send-out. For this report, data were analyzed using robust multivariate regression. Five variables were associated with significant positive or negative bias of absolute lymphocyte subset counts: (i) platform methodology (i.e., single-platform assays yielded lower CD4 + and CD8+ T-cell counts than did dual-platform assays); (ii) sample preparation technique (i.e., assays based on mononuclear cells isolation yielded lower T-cell counts than those based on red cell lysis); (iii) gating strategies based on CD45 and sideward scatter gating of lymphocytes yielded higher CD4+ T-cell counts than those based on "backgating" of lymphocytes guided by CD45 and CD14); (iv) stabilized samples were generally associated with higher lymphocyte subset counts than nonstabilized samples; and (v) laboratory. Platform methodology, sample stabilization, and laboratory also affected assay variability. With time, assay variability tended to decline; this trend was significant for B-cell counts only. In addition, significant bias and variability of results, independent of the variables tested for in this analysis, were also associated with individual laboratories. In spite of our recommendations, participants tended to standardize their techniques mainly with respect to sample preparation and gating strategies, but less with absolute counting techniques. Failure to fully standardize protocols may have led to only modest reductions in variability of results between laboratories.

Randomized studies testing the clinical efficacy of platelet additive solutions (PASs) for storage of platelets are scarce and often biased by patient selection. We conducted a multicenter, randomized study to investigate clinical efficacy of platelets stored in PAS II versus plasma, also including patients with clinical complications associated with increased platelet consumption. A total of 168 evaluable patients received pooled buffy coat–derived platelet concentrates (PCs) suspended in either plasma (n = 354) or PAS II (n = 411) stored up to 5 days. Both univariate as well as multivariate analysis showed a significant effect of used storage medium in regard to 1- and 24-hour count increments and corrected count increments, in favor of plasma PCs. However, there were no significant differences between the groups regarding bleeding complications and transfusion interval. Adverse transfusion reactions occurred significantly less after transfusions with PAS II PCs (P = .04). Multivariate analysis showed no significant effect of the used storage medium on the incidence of 1- and 24-hour transfusion failure. We showed safety and efficacy of PAS II PCs in intensively treated patients; however, plasma PCs show superior increments.

BACKGROUND: The RHD phylogeny in humans shows four main clusters of which three are predominantly observed in (African) black persons. Each of the African clusters is characterized by specific amino acid substitutions relative to the Eurasian RHD allele. RH phylogeny defines the framework for identification of clinically relevant aberrant alleles. This study focuses on the weak D type 4 cluster (characterized by RHD(T201R, F223V) (602C>G 667T>G)) in five ethnic groups. STUDY DESIGN AND METHODS: A total of 1702 samples were screened for the presence of 602C>G and 667T>G by sequence-specific polymerase chain reaction (PCR-SSP). Eighty samples were assigned to the weak D type 4 cluster and were molecularly characterized by PCR-SSP and RHD sequencing. Antigens of aberrant alleles were characterized with monoclonal anti-D according to the 37-epitope model when possible. RESULTS: Five new aberrant alleles, DIII type 6, DIII type 7, DARE, RHD(T201R, F223V) (without 819G>A), and RHD(F223V), were identified and DIII type 6, DARE, and RHD(F223V) were serologically characterized with monoclonal anti-D. Both the DARE and RHD(F223V) showed epitope loss. It is postulated that the 1136C>T nucleotide substitution (characteristic for the DAU allele cluster) is present on the DVa(KOU) allele. CONCLUSION: Identification of the new variant alleles refines the phylogeny of RHD in humans. The proposed DVa(KOU) allele with 1136C>T (DVa(KOU)T379M) is probably caused by conversion of the DAU0 allele and the DVa(KOU) allele, forming a phylogenetic link between the DV allele and the DAU cluster. By describing the RHD(F223V) (602C>G) and RHD(T201R, F223V) (602C>G and 667T>G) alleles formal proof is given for the origin of the non-Eurasian cluster.

A nucleic acid-targeted photochemical treatment (PCT) using amotosalen HCl (S-59) and ultraviolet A (UVA) light was developed to inactivate viruses, bacteria, protozoa, and leukocytes in platelet components. We conducted a controlled, randomized, double-blinded trial in thrombocytopenic patients requiring repeated platelet transfusions for up to 56 days of support to evaluate the therapeutic efficacy and safety of platelet components prepared with the buffy coat method using this pathogen inactivation process. A total of 103 patients received one or more transfusions of either PCT test (311 transfusions) or conventional reference (256 transfusions) pooled, leukoreduced platelet components stored for up to 5 days before transfusion. More than 50% of the PCT platelet components were stored for 4 to 5 days prior to transfusion. The mean 1-hour corrected count increment for up to the first 8 test and reference transfusions was not statistically significantly different between treatment groups (13,100 +/- 5400 vs 14,900 +/- 6200, P =.11). By longitudinal regression analysis for all transfusions, equal doses of test and reference components did not differ significantly with respect to the 1-hour (95% confidence interval [CI], -3.1 to 6.1 x 10(9)/L, P =.53) and 24-hour (95% CI, -1.3 to 6.5 x 10(9)/L, P =.19) posttransfusion platelet count. Platelet transfusion dose, pretransfusion storage duration, and patient size were significant covariates (P

Background: Transfusion of bacterial contaminated platelet (PLT) units can cause serious transfusion reactions. To prevent this complication, bacterial screening by the automated BacTalert® system of platelet concentrates was implemented in the southwest part of the Netherlands since october 2001. Despite this system, two severe transfusion reactions caused by bacterial contaminated PLT units occurred. Therefore, evaluation of the efficacy of this approach was done. Material and Methods: PLT products in the Netherlands are produced by the Sanquin Bloodbanks. In the Southwest part of the Netherlands PLT products mainly consist of pooled products derived from five random donors. Of all produced platelet products, samples were taken and cultured (aerobic and anaerobic) for seven days using the automated BacTalert® system. The PLT products were issued to the hospitals on a "negative-to-date" basis. In case of positive cultures, conformation and identification cultures were taken. At the same time, involved and related products thet were derived from the same donor were also withdrawn. In case of delivery of possibly contaminated products to the hospitals, these were notified and the products were recalled. If the products were already transfused, treating physicians were notified and asked if adverse reactions had occurred in these patients. Results: From October 2001 to 2003, a total of 28,104 produced pooled PLT units were tested for bacterial contamination using the BacTalert® system. Positive signals were found in 203 (0,72%) samples. At the time of a positive signal, 125 PLT products were already issued to the hospitals and of these 125 products, 113 units were already administered to patients. Analysis of all positive cultures revealed mainly skin-derived bacteria, but in 15 sample Bacillus cereus (7%). In 9.4% (=19) of the samples with a postive BacTalert® signal, bacterial growth could not be confirmed. None of the PLT units with a postive bacterial culture that were already administered to patients caused clinically significant transfusion reactions. Nevertheless, two neutropenic patients due to leukemia treatment, suffered from life-threatening B. cereus sepsis after PLT transfusion. In both cases, B.cereus was cultured from the PLT bags, whereas the BacTalert® tested samples remained negative. Conclusions: Issuing pooled PLT units on a negative-to-date basis for bacterial screening resulted in a significant amount of recall procedures. The BacTalert® system failed to detect B. cereus contamination in at least two cases that resulted in sever transfusion reactions. Other strategies, including pathogen reduction, might be applied to guarantee a better degree of bacterial safety.