[Show abstract][Hide abstract] ABSTRACT: The HPA-15 antigen system is characterized by a low antigen expression on platelets. The antibodies against this antigen are implied in fetal/neonatal alloimmune thrombocytopenia (F/NAIT), post-transfusion purpura, and refractoriness to platelet transfusions. Detection of these antibodies appears to be related to the level of HPA-15 expression on the platelets used in the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay.
We performed genotyping of 300 healthy blood donors for HPA-15 by TaqMan real-time PCR technology, and the HPA-15 antigen expression was investigated in 13 HPA-15aa and 19 HPA-15bb individuals. We also investigated the relevance of HPA-15 antigen expression on donor platelets used in MAIPA for antibody detection in 223 multitransfused hematological patients and 271 women with suspected F/NAIT.
In Polish donors, the HPA-15a allele frequencies were lower than the HPA-15b (0.480 vs. 0.515). We identified three HPA-15 expression groups: high (36.7 ± 8.36 MFI - eight cases), medium (19.5 ± 6.2 MFI - 21 cases), and low (6.5 ± 5.9 MFI - three cases). The HPA-15 expression was stable over time. The HPA-15aa and HPA-15bb platelets with high antigen expression were used for anti-HPA-15 antibody detection; anti-HPA-15 antibodies were detected in 4/223 (1.8%) patients receiving multiple transfusions but in none of the 271 women with suspected F/NAIT. Further examination of the four sera by MAIPA with various platelets revealed the optical density in the assay to be closely related to the level of HPA-15 antigen expression.
Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level.
Full-text · Article · Jul 2011 · International journal of laboratory hematology
[Show abstract][Hide abstract] ABSTRACT: The deficiency of glycosyl-phosphatidylinositol (GPI)-anchored proteins in plasma membranes of PIG-A gene mutated hematopoietic stem cells (HSCs) is so far insufficient to explain the domination of paroxysmal nocturnal hemoglobinuria (PNH) clone over the normal HSC. We attempted to elucidate possible link between MHC and initial severe aplastic anemia (ISAA/PNH) type and non-aplastic (n/PNH) outcome of PNH. In 50 PNH patients assigned as ISAA/PNH (n = 13), n/PNH (n = 33) or nonassigned (n = 4) and 200 ethnically matched controls we analyzed MHC associations. Our data confirmed strong associations of DRB1*15:01 (RR = 3.51, p = 0.0011) and DQB1*06:02 (RR = 7.09, p = 0.000026) alleles, especially with n/PNH subtype. B*18:01 allele was associated with increased risk of ISAA/PNH subtype (RR = 5.25, p = 0.0028). We conclude that both class II and class I MHC alleles are associated with different subsets of PNH. Clonal selection of PIG-A mutated cells with cognate metabolic block is associated with MHC class II alleles DRB1*15:01 and DQB1*06:02 independent from initial severe AA clone selection. MHC class I molecule B*18:01 can additionally influence the domination of PNH clone in PNH subjects with initial severe aplastic anemia.
Full-text · Article · Mar 2011 · Archivum Immunologiae et Therapiae Experimentalis
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Oct 2010 · Transplantation Proceedings
[Show abstract][Hide abstract] ABSTRACT: Prophylactic anti-D is a very safe and effective therapy for the suppression of anti-D immunization and thus prevention of haemolytic disease of the foetus and newborn. However, migration from countries with low health standards and substantial cuts in public health expenses have increased the incidence of anti-D immunization in many "developed" countries. Therefore, this forum focuses on prenatal monitoring standards and treatment strategies in pregnancies with anti-D alloimmunization. The following questions were addressed, and a response was obtained from 12 centres, mainly from Europe.
[Show abstract][Hide abstract] ABSTRACT: Background. Pseudothrombocytopenia (PTCP) involves a spuriously low platelet count associated with in vitro platelet agglutination due to the presence of antiplatelet antibodies reacting with platelets in blood drawn into ethylenediaminetetraacetic acid (EDTA). Objectives. Clinical and serological analysis as well as follow-up of 217 PTCP cases diagnosed in Poland in 1989-2007. Material and Methods. EDTA-dependent antiplatelet antibodies were tested by the platelet immunofluorescence test (PIFT) using FITC-labeled antiglobulin conjugates against human IgG and IgM. PIFT results were evaluated by flow cytometry and microscopy. Results. Of the group of 217 PTCP cases, 39% were healthy subjects and 61% were patients with mostly vascular, infectious, autoimmune, liver, and metabolic diseases. EDTA-dependent antibodies were detected in 97% of the cases, IgG antibodies in 19%, IgM in 34%, and IgG+IgM in 44%. IgG antibodies were more frequent in the healthy subjects than in the patients (p < 0.01) and IgM more frequent in patients (p < 0.025). Follow-up was performed in 88 PTCP patients (mean follow-up time: 4 years, range: 1-18 years). New diseases were determined in 31% of the cases (5 initially healthy, 22 previously ill subjects). The frequencies of the new diseases (autoimmune, metabolic, neoplastic, vascular, infectious) did not differ from those in the Polish population (p > 0.05). EDTA-dependent antibodies were still present in 81% of the PTCP subjects. Pseudoleukocytosis was determined in 34% of the healthy persons with PTCP. Conclusions. These observations indicate that 1) PTCP does not predispose to the occurrence of the above-mentioned diseases; it is therefore likely that EDTA-dependent antibodies have no clinical significance, 2) it is extremely important to differentiate pseudothrombocytopenia and thrombocytopenia to avoid unnecessary treatment, 3) in EDTA-dependent PTCP cases, platelet count should be performed in blood collected in an anticoagulant other than EDTA or immediately following blood collection in a Bürker chamber.
No preview · Article · Jul 2009 · Advances in Clinical and Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 13 anti-Rh sera were compared for their usefulness in the detection of Fc-receptor-bearing lymphocytes (EAhum test). IgG subclasses of anti-Rh antibodies were determined by the antiglobulin test with monospecific sera and by the detection of Gm allotypic markers in the haemagglutination inhibition test. Six sera with IgG1 + IgG3 or IgG1 + IgG2 + IgG3 antibodies and one with pure IgG3 antibodies were found to be useful, whereas six other sera with only IgG1 were unsuitable for the EAhum test. G3m markers were detected only on the anti-Rh antibodies which were capable of forming rosettes with lymphocytes. The data show that human peripheral lymphocytes possess Fc receptors for IgG3 immunoglobulins.
[Show abstract][Hide abstract] ABSTRACT: 1) We have presented our experiment conducted to detect anti-K antibodies from the Kell-system in pregnant women and their connection with potential destruction of foetal red cells, which may result in haemolytic disease of the foetus and the newborn (HDFN). 2) We have also indicated serological and molecular methods important for a proper diagnosis.
27 women with anti-K. Serological diagnosis of K antigen in fathers and children. KEL1 gene examination in foetuses from DNA isolated from foetal cells contained in amniotic fluid, using discrimination of alleles--real-time PCR method.
Anti-K were detected in most women after blood transfusion, the fathers were usually K negative. Foetomaternal incompatibility was found in 6 out of 27 women. Haemolytic disease was observed in 5 cases: 3-severe, 1-fatal, 1-mild. Foetal genotyping allowed us to avoid cordocentesis in two pregnant women, it appeared that both foetuses have not received KEL1 gene from heterozygous (Kk) fathers--in the previous pregnancies the children had died because of HDFN.
1) In every pregnant woman with anti-K, the K antigen should be examined in the father. 2) In every K+ father the phenotype should be evaluated and if he is heterozygote (Kk), the fetal KEL1 gene must be examined, to avoid unnecessary cordocentesis. 3) If KEL1 gene is not detected in the foetus, HDFN will not occur. 4/Foetal KEL1 genotyping may be performed in all mothers with anti-K and heterozygous father in our Department, after providing the material from the amniocethesis.
No preview · Article · Jun 2008 · Ginekologia polska
[Show abstract][Hide abstract] ABSTRACT: Two women with post-transfusion purpura (PTP) are presented, one with anti-HPA1a and the other with anti-HPA3a antibodies. Platelet-specific antibodies were identified using the platelet immunofluorescence test (PIFT) and the monoclonal antibody immobilization of platelet antigens (MAIPA) assay. Lymphocytotoxic and red cell antibodies were also detected in both patients, the latter being responsible for a delayed haemolytic transfusion reaction (DHTR). In the patient with anti-HPA1a antibody, red cell anti-c alloantibody was found in the serum and in the eluate from red cells; it was active in the monocyte monolayer assay (MMA). The patient with anti-HPA3a antibody had the red cell alloantibodies anti-D, -M and -S detected in the serum, the last being responsible for DHTR.
No preview · Article · Apr 2008 · Transfusion Medicine
[Show abstract][Hide abstract] ABSTRACT: Anti-Rhc antibodies may be the reason for the hemolytic disease of the newborn, therefore, noninvasive Rhc determination is important for pregnancy monitoring. For this purpose, we decided to introduce real-time polymerase chain reaction (PCR) method.
Blood from 200 donors, plasma and whole-blood from 11 Rhc-negative mothers, as well as blood from fathers and newborns were examined. Rhc sensitivity and specificity were first determined by real-time PCR using genomic DNA from donors. The same Rhc genotyping method was used for fetal Rhc detection in maternal plasma. To confirm the fetal Rhc-negative result, plasma was tested with a panel of biallelic insertion/deletion polymorphisms for the presence of fetal DNA.
The c allele assay showed full specificity. The mean Ct value for one copy of c allele diluted in C-negative DNA was determined from extrapolating the correlation curve as 39.9. Full concordance was observed between the fetal Rhc genotypes from maternal plasma and the newborn phenotypes.
Preliminary results show that it is possible to examine fetal c allele of RHCE gene in the plasma of pregnant women with anti-c by means of a noninvasive method. The diagnostic accuracy of the procedure, however, has yet to be confirmed in a larger group.
No preview · Article · Apr 2008 · Prenatal Diagnosis
[Show abstract][Hide abstract] ABSTRACT: 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).
No preview · Article · Mar 2008 · Transfusion Medicine
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: The role of leucocyte antibodies in donors is poorly understood in pathogenesis of transfusion-related acute lung injury (TRALI). We examined antibodies in donors and traced recipients transfused with their blood components.
Antibodies were examined in 1043 donors by five methods, look back performed in 26 recipients.
Anti-human leucocyte antigen detected by enzyme-linked immunosorbent assay in 9.8% women but none in men. Specificities identified using FlowPRA, antibodies detected after several months. TRALI reported in one recipient from immunized donor. In 11 of 26 recipients without TRALI, cognate antigens were identified.
Detection of antibodies in donors cannot predict TRALI, even in recipients with cognate antigen(s).
[Show abstract][Hide abstract] ABSTRACT: Post-transfusion purpura (PTP), a delayed post-transfusion event, is underdiagnosed, not only in Poland. Thrombocytopenia results from the destruction of patients' and transfused platelets by platelet specific antibodies (anti-HPA). The incidence of PTP is not estabilished.
Analysis of 10 cases with PTP diagnosed in Poland during last 25 years.
9 women and 1 man with normal platelet count who developed thrombocytopenia with diathesis haemorrhagica after transfusion of red cells or/and platelets. Platelet specific antibodies (anti-HPA) were examined by the platelet immunofluorescence test and by the monoclonal immobilization of platelet antibodies (MAIPA) assay; leucocyte antibodies by standard lymophocytotoxicity test (LCT).
In all 10 patients the PTP was diagnosed because: 1) thrombocytopenia with diathesis haemorrhagia occured 3-10 days after the transfusion (in 5 cases blood was transfused during the operation), 2) in all the patients anti-HPA were detected (in 9- anti-HPA-1a, in 1 case anti-HPA-3a), 3) a primary HPA alloimmunization was very probable. A recovery of platelets count occured in 6 patients within 8-34 days after corticosteroids and/or IVIG, however, therapeutic effect of them was difficult to assess due to a great probability of spontaneous remission up to 1 month. Four patients died due to their basic disease although the impact of the PTP cannot be excluded.
The diagnosis of PTP was possible because of the detection of anti-HPA antibodies in patients who developed thrombocytopoenia after blood transfusion.
No preview · Article · Jul 2006 · Polski merkuriusz lekarski: organ Polskiego Towarzystwa Lekarskiego
[Show abstract][Hide abstract] ABSTRACT: Transfusion-Related Acute Lung Injury (TRALI) has been diagnosed very rare until recent years. However, the growing interest in TRALI allows to asses, that it is the second commonest cause of transfusion association death. In Poland only single cases of TRALI have been published.
We analyzed 34 cases with dyspnea reported as a post-transfusion event and examined leukocyte antibodies, which are supposed to be an important pathogenic factor in TRALI.
34 patients were classified into: the group A--patients with a pulmonary oedema after exclusion of other reasons (TRALI, n= 11); the group B-- patients with pulmonary oedema, but with difficulties to exclude other reasons (possible TRALI, n=15); the group C--post transfusion dyspnea without pulmonary oedema (patients where not classified as the TRALI, n=8). In all the groups other clinical symptoms were also analyzed. The leukocyte antibodies were most often detected in the group A (91%), less often in the group B (53%) and C (37.5%).
Transfusion-related dyspnea should be individually analyzed before the final diagnosis of TRALI. If in the donor of transfused blood the leukocyte antibodies are detected, the "trace back" procedure should be started to see whether in other patients a transfusion-related dyspnea was diagnosed. This procedure is important for potential exclusion of a given donor from blood donation.
No preview · Article · Jun 2006 · Polski merkuriusz lekarski: organ Polskiego Towarzystwa Lekarskiego
[Show abstract][Hide abstract] ABSTRACT: We have recently developed and published a noninvasive determination of fetal RhD status by examination of cell-free DNA in maternal plasma. The predictive value of the procedure of fetal testing, already published by us, was 99,6%.
To assess the necessity of RhD fetal testing in immunized Rh(-) mothers with Rh(+) partners.
Rh(-) mothers with anti-D antibodies, their partners and children.
Molecular: RHD gene examination by real-time polymerase chain reaction; analysis of control genes present in the father but not in the mother. Serological: titre of anti-D antibodies, Rh phenotypes.
Among 53 Rh(+) partners of immunized Rh(-) pregnant women, 56,6% were homozygous and 43,6%--heterozygous. The latter ones might have had either Rh(+) or Rh(-) children; in fact, in 52,2% of fetuses the D gene was not detected. Among fetuses of homozygous fathers (based on their phenotypes) 2 fetuses, to our surprise, occured to be Rh(-); however the subsequent genotyping showed that both fathers were heterozygous. The titres of anti-D in both groups of mothers with Rh(-) and Rh(+) fetuses were very similar.
The examination of fetal D gene by noninvasive method should be performed in each alloimmunised Rh(-) mother if her partner is Rh(+). The prediction of RhD fetal status based on the fathers phenotype can be misleading, thus may result in unnecessary invasive method.
No preview · Article · Jun 2006 · Ginekologia polska