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Background: Although quantitative evidence is lacking, it is generally believed that the majority of cases of transfusion-related acute lung injury (TRALI) are caused by female blood donors. We aimed to examine the relation between female donors and the occurrence of TRALI., Study Design and Methods: We performed an international, multicenter case-referent study. TRALI patients who were diagnosed clinically, independent of serology or donor sex, and had received transfusions either only from male donors or only from female donors (unisex cases) were selected. The observed sex distribution among the donors of these TRALI patients was compared to the expected sex distribution, based on the relevant donor populations., Results: Eighty-three clinical TRALI cases were included; 67 cases received only red blood cells (RBCs), 13 only plasma-rich products, and three both. Among RBC recipients the relative risk (RR) of TRALI after a transfusion from a female donor was 1.2 (95% confidence interval [CI], 0.69-2.1) and among plasma-rich product recipients the RR was 19 (95% CI, 1.9-191). The p value for the difference between RBCs and plasma was 0.023., Conclusion: Our data support the notion that plasma from female donors is associated with an increased risk of TRALI, while RBCs from female donors are not., (© 2010 American Association of Blood Banks.)

The pathologies of paroxysmal nocturnal hemoglobinuria (PNH) are primarily caused by somatic mutation in the PIG-A gene in hematopoietic stem cells resulting in glycosyl phosphatidylinositol deficiency and accumulation of phosphatidylinositol (PI) in plasma membranes. The mechanism of pathologic clone domination over normal hematopoietic clones in PNH patients is not yet understood. Forty-four PNH patients, including 9 with aplastic anemia traits (AA/PNH), 31 without full aplasia in bone marrow (de novo PNH, or dn/PNH), and 4 with unclassified PNH, and 200 ethnically matched controls were tested for the HLA A, B, C, DRB1, and DQB1 alleles and haplotype associations. The top block association analysis showed the primary association of PNH with 3 haplotype fragments: the class I fragment A*2501-Cw*1203-B*1801 (risk ratio [RR], 6.64; P=.00012), and 2 class II fragments: DRB1*1501-DQB1*0602 (RR, 7.09; P=.0000015) and DRB1*0401-DQB1*0301 (RR, 6.76, P=.0093). The stratified analysis revealed that the A*2501-Cw*1203-B*1801 haplotype associated preferentially with AA/PNH, and its component HLA molecule showed immunodominant antiapoptotic peptides derived from PI-activated phospholipase D; whereas the DRB1*1501-DQB1*0602 haplotype was associated strongly with dn/PNH and presented immunodominant class II-derived autopeptides. We concluded that certain HLA haplotypes were associated with PNH much more strongly than their allelic components. At least 3 HLA haplotype blocks (A*2501-Cw*1203-B*1801, DRB1*1501-DQB1*0602, and DRB1*0401-DQB1*0301) were primarily associated with PNH. Our results supported the hypothesis of the roles in AA/PNH of antiapoptotic and in dn/PNH of autoimmune mechanisms., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

The Colton (Co(a)) antigen is of high frequency; its incidence in Caucasians is about 99.8%. Reports on haemolytic transfusion reactions and haemolytic disease of the foetus/newborn (HDFN) due to anti-Co(a) are rare. We report a severe HDFN due to anti-Co(a). The first child of the mother was healthy. The second died a few hours after delivery because of hydrops fetalis, likely due to HDFN; anti-Co(a) in the maternal serum, the father typed as Co(a+). The third pregnancy was followed up by the measurements of anti-Co(a) titre (additional antibodies were excluded), its functional activity by the chemiluminescence test (CLT) and the Doppler flow in the middle cerebral artery of the foetus. Increased values of antibody titre up to 128, the CLT to 30% and multiplex of median of the peak systolic velocity to 1.71 indicated haemolytic disease and the necessity for an intrauterine transfusion. The foetus received the maternal red blood cells (RBCs). Delivery had to be by Caesarean section for obstetrical reasons at 34-week gestation. The newborn (anti-Co(a) on red cells and in plasma, the rise of the bilirubin concentration up to 333 micromol L(-1)) had four exchange transfusions: the first of maternal RBCs, the remaining of donor's Co(a+) cells and one top-up transfusion. The baby was discharged in good health. Anti-Co(a) was responsible for severe HDFN. Proper monitoring during pregnancy and antenatal and post-natal therapy were successful. This is the second severe published HDFN due to anti-Co(a).

Background and Objectives: Transfusion-related acute lung injury (TRALI) is underdiagnosed and underreported. This is why we present cases suspected for TRALI, in which leucocyte antibodies were examined., Material and Methods: We analysed 44 patients with respiratory insufficiency, related to transfusion, who met criteria of acute lung injury (ALI). Lymphocyte and granulocyte antibodies were examined in donors and patients by six methods., Results: Based on recent trends, we divided patients into two groups: TRALI (without risk factors for ALI) and possible TRALI (with probable risk factors). The incidence of antibodies was 68.2%, the majority were human leucocyte antigen (HLA) class I and/or II, the minority were non-specific granulocyte antibodies; half of all detected antibodies, however, reacted with granulocytes. Antibodies were found in 17 donors (more often in TRALI than in possible TRALI) and in 19 patients (in four - suspected to be of the donor origin, which would diminish the number of antibodies to 15). In seven available cases, we observed cognate antigen and/or positive cross-match. In the majority of patients, TRALI occurred after transfusion of red cells, in 56.2%- stored above 14 days; all the units were non-leucoreduced. Lookback in two donors showed that transfusions in 20 patients did not result in reported TRALI, even in the patient with cognate antigen., Conclusions: Our clinical observations suggest that to distinguish between TRALI and possible TRALI is difficult and the results are equivocal - it is worth considering whether it can be omitted. We have confirmed that antibodies are involved in TRALI, although their role is very complex. The role of stored red blood cells in the development of TRALI requires further observations in comparison with a control group of patients without TRALI.

The pathogenesis and tissue damage that accompanies destruction of platelets in immune thrombocytopenias (IT) is still not understood very well and in addition to platelets, other cells (e.g. endothelial cells, CD34+ hematopoietic stem/progenitors) may also become affected. Based on our previous work that platelet antigens (e.g., CD41) may be transferred by platelet-derived microvesicles (PMV) to the surface of other cells, we asked if platelet derived-antigens, especially those that are involved in the formation of anti-platelet antibodies in IT (e.g., against antigen HPA 1 a) could be also transferred by similar mechanism. To address this issue normal human CD34+ cells, human umbilical vein-endothelial cells (HUVEC) and monocytic cell line THP-1 were incubated with PMV derived from HPA1a+ donors. We noticed that the HPA1a antigen is highly expressed on PMV-derived from the HPAla positive platelets and is transferred in PMV-dependent manner to the surface of CD34+ cells, HUVEC and monocytic THP-1 cells. These cells covered with HPA1a positive PMV but not by PMV derived from HPAla negative platelets reacted with anti-HPA1a antibodies derived from the alloimmunized pregnant women. More importantly, human hematopoietic cells that were preincubated with HPA1a+ PMV and subsequently exposed to anti-HPA 1 a serum and human NK cells, become subject to elimination by antibody dependent cell cytotoxicity ADCC. Thus, we postulate that PMV-dependent transfer of antigens may playing an important role in "expanding" the population of target cells that may be affected by anti-platelet antibodies and explain several pathologies that accompany IT (e.g. damage of endothelium, cytopenias).

Background: Cell-free fetal DNA in maternal plasma opens the way for routine risk-free diagnosis of fetal D status of D- mothers. The focus was on accuracy of RHD typing and confirmation of fetal DNA in maternal plasma while RHD was not detected., Study Design and Methods: Plasma DNA was extracted (by manual and/or automatic method) from 255 D- pregnant women and amplified in exons 7 and 10 and intron 4 of RHD gene with real-time polymerase chain reaction. The presence of fetal DNA was confirmed by testing SRY and, when negative, by one of 11 different polymorphisms found in the father but not in the mother. The results were compared with the D status of the newborns., Results: After exclusion of 25 cases (10%) because of material shortage, in 230 cases (90%) available for complete study, the predictive value of the procedure of fetal RHD testing (RHD genotyping plus confirmation of fetal DNA) was 99.6 percent. SRY detection confirmed fetal DNA presence in maternal plasma in all boys, whereas the detection of various polymorphisms in all girls but one., Conclusions: Fetal RHD genotyping from maternal plasma may be used with confidence, although additional polymorphisms for confirmation of fetal DNA should be included for 100 percent predictive value (instead of 99.6%).

Paroxysmal nocturnal hemoglobinuria (PNH), an acquired stem cell defect, is underdiagnosed because of its atypical symptoms in some patients and because available methods, which are time consuming and complicated, are not widely used. The hemolysis of PNH red blood cells (RBCs) is attributed to their enhanced susceptibility to complement lysis caused by a deficiency in glycosylsphosphatidylinositol (GPI)-anchored complement regulatory membrane proteins, especially membrane inhibitor of reactive lysis (MIRL [CD59]). We evaluated the diagnostic value of a simple hemagglutination test using the gel microtyping system by comparing it with lytic tests (the Ham test and the sucrose lysis test) and with flow cytometry (FC) assessment of expression of GPI-anchored proteins (CD59 and CD55). Examining 51 blood samples from 48 patients, we found that the gel test is useful as a screening test for PNH diagnosis and can replace the Ham test and the sucrose lysis test. The threshold of the gel test is about 10 percent of defective RBCs detected by FC. It should, however, be supplemented with FC so as to analyze precisely the defective RBCs and granulocytes in patients with positive gel test results, and, in case of negative results, to detect a small clone of defective cells in atypical cases. Due to the simplicity of the gel test, its wide use can facilitate the diagnosis of PNH.

Hemolytic disease of the newborn (HDN) due to anti-Hr0 antibody is typically severe and often fatal. We report a case of moderate HDN due to anti-Hr0 in a woman with the D--/D-- phenotype. A 33-year-old woman delivered her second child who was mildly jaundiced. The highest level of bilirubin was 26.1 mg/dL on the third day postpartum and the hemoglobin concentration was 14.0 g/dL. The newborn recovered after phototherapy and no mental retardation was noticed after 1 year of follow up. An exchange transfusion was excluded due to the lack of a compatible donor and the physical condition of the mother precluded blood donation. The maternal RBCs were D+C-c-E-e-; only G and Rh29 of the Rh system were expressed. Thus, her probable phenotype was D--/D--. Her alloantibody was identified as anti-Hr0 (anti-Rh17) as it reacted with all red blood cells (RBCs) but not her own, other D--- RBCs, and Rhnull RBCs. The results of the antibody titer (64) and activity in a chemiluminescense test (CLT; 34%) were consistent with a moderate HDN. Family studies were negative for the D--/D-- phenotype and consanguinity was not proved. This is the first described case of moderate HDN due to anti-Hr0. The result of antibody activity in the CLT might be helpful in predicting the severity of HDN in other rare HDN cases.

Mild courses of haemolytic disease of the foetus or newborn (HDN) due to Rh (D) blood group antibodies are associated with and may therefore be ameliorated by maternal antibodies reacting with human leucocyte antigens (HLA) of the child, an observation drawn from our own earlier data (Neppert J, Kissel K. Lancet 1992;339:1481). This study (i) corroborates this association; (ii) reveals shortcomings in the published data; and (iii) examines the characteristics of HDN cases when these shortcomings have been rectified. Samples from 51 women with antibodies against their child's blood group antigens of the Rh system were analysed for HLA A, B, C and DR antibodies during parturition. The mothers were divided into two groups, either severe or mild, dependent upon the clinical course of the HDN, and the incidence of HLA antibody production was determined for each group. HLA A, B, C and/or DR antibodies were detected in 85.2% of those women whose children had a mild course of HDN prenatally or perinatally (n=27). This is statistically greater than the incidence of 50.0% (Fisher's exact test: p=0.014) found in the group of women whose children had a severe HDN either pre- or perinatally (n=24) and is greater than the 35% (n=20; p=0.0001) found in women without Rh or other irregular antibodies. HLA DR antibodies were detected in three cases. The findings support our hypothesis that maternal anti HLA A, B and C antibodies may protect against a potential severe HDN. We therefore assume that those women will benefit who have already had a child with a severe HDN and in whom HLA antibodies were not previously detected, if HLA antibody production is provoked by subcutaneously inoculating with the father's leucocytes before or at the beginning of the new pregnancy.

In this study, platelet counts were determined from the cord blood of consecutive 9142 newborns. Neonates with known autoimmune thrombocytopenia were not included. The platelet count < 100 x 10(9)/L was found in 64 newborns. In five of them, neonatal alloimmune thrombocytopenia (NAIT) was diagnosed. The overall incidence of neonatal thrombocytopenia was 0.7%, the incidence of NAIT was about 10 times less. Serological and clinical observations are summarized from 238 thrombocytopenic newborns (54 from the above group and 184 previously referred to serological investigations). All of the newborns were divided into two groups: NAIT (46 cases) and other thrombocytopenias (192 cases). Among platelet-specific antibodies in NAIT, 91.4% were anti-HPA-1a, the rest were anti-HPA-1b and anti-HPA-5b. In the majority of the cases, antibodies were detectable by the platelet suspension immunofluorescence test (PSIFT) and monoclonal antibody immobilization of platelet antigens (MAIPA) assay. In 19.6% cases, antibodies were detectable by MAIPA only. In 10.9% of these cases, antibodies were undetectable. Thrombocytopenia < 50 x 10(9)/L and hemorrhagic diathesis were more often observed in NAIT than in other thrombocytopenias, whereas associated disorders that could contribute to thrombocytopenia, here observed almost only in the latter group. We also report certain other observations, such as the presence of anti-HLA antibodies, a rise in the anti-HPA-1 a antibody titer after infection without pregnancy, and a higher incidence of petechiae in nonimmune thrombocytopenia as compared with the incidence of low platelet counts.

Factors governing the functional activity of red cell autoantibodies are poorly defined. Here we report the presence of qualitative differences in the glycosylation of IgG autoantibodies which affect in vitro interactions with Fc gamma RIII. The following antibodies were affinity-purified by adsorption and elution from normal red cells: IgG eluted from the red cells of 27 haemolysing or non-haemolysing patients, anti-D in sera from 11 pregnant women, and IgG1 and IgG3 human monoclonal anti-D. Monoclonal antibodies with differing levels of agalactosyl IgG were produced by culturing cell lines at high or low cell density. The % IgG with oligosaccharides lacking terminal galactose residues (agalactosyl IgG) of antibodies was designated as low, medium or high according to their reactivity with a monoclonal antibody to terminal N-acetylglucosamine. Fc gamma RIII-mediated functional activity was assessed by measuring the K-cell-mediated lysis of red cells in eluates diluted to achieve comparable levels of red cells sensitization. All eluates containing allo-anti-D were lytic (range 74-100%). In contrast, lysis by autoantibodies varied from 0 to 100%; 11/13 eluates from red cells of haemolysing patients promoted > 5% lysis compared to 2/7 eluates from red cells of non-haemolysing patients (P < 0.02). The ability of autoantibodies to promote K-cell-mediated red cell lysis correlated inversely with their level of agalactosyl IgG (r = -0.56, P < 0.01, n = 23). Further, monoclonal anti-D antibodies with very low levels of agalactosyl IgG were comparatively more lytic than the same antibodies containing more agalactosyl IgG. Analysis of the ratio of kappa:lambda light chains suggested that autoantibodies from 6/19 patients were monoclonal or oligoclonal in nature. The data indicate that IgG red cell autoantibodies from different patients are functionally heterogenous, and that this may be due, at least in part, to qualitative differences in the Fc region glycosylation reflected by differences in the proportion of agalactosyl IgG. This heterogeneity is consistent with the clonally-restricted nature of the autoantibodies in some patients.

Monocyte monolayer assays (MMA) were performed using red cells (RBCs) from 159 patients with a positive direct antiglobulin test and monocytes from healthy individuals. The results of the MMA reflected, to a great extent, in vivo haemolysis; 56/63 patients with a positive result, but only 10/96 with a negative one, had evidence of in vivo RBC destruction. Positive MMAs were observed with autoantibodies of all types: warm (42 cases), cold (14 cases), mixed (seven cases), but never in patients with only C3d on their RBCs and no detectable autoantibodies. With warm autoantibodies, the MMA results depended, in part, on the level of IgG sensitization; additional C3d coating acted synergistically to lower the amount of IgG necessary for monocyte interaction to occur. The presence of IgG3 as well as IgG1 on the RBCs was usually associated with a high MMA result; most of these cases, however, also exhibited larger amounts of cell-bound IgG. When the MMA results were analysed in respect of the various types of autoantibodies, differences in the phagocytosis:adherence ratio were observed; in patients with warm autoantibodies, there was a preponderance of phagocytosis, whereas in those with cold and mixed types, adherent RBCs predominated or were found alone.

Alloantibody formation against HPA-1a (Zwa/PIA1) has, to date, only been found in HLA-DRw52(a+) (Dw24) individuals. Alloimmunization against the product of the other HPA-1 allele, HPA-1b, is rare. We have been able to evaluate ten cases of HPA-1b alloimmunization in Europe in order to study whether there is an association between HLA phenotype and anti-HPA-1b antibody formation. HLA typing of these patients was performed with particular attention to the DRw52a specificity using specific T-cell clones. No association with DRw52a or any other known HLA phenotype was found. This finding implies that the amino acid substitution leucine33-proline33 in GPIIIa, responsible for HPA-1a/b, is of primary importance for the association of anti-HPA-1a antibody formation with DRw52a. These data show that the amino acid polymorphism affects the presentation of the immunogenic oligopeptides of HPA-1a and -1b in the HLA class-II groove.