Analysis of leucocyte antibodies, cytokines, lysophospholipids and cell microparticles in blood components implicated in post-transfusion reactions with dyspnoea.

BACKGROUND AND OBJECTIVES: Post-transfusion reactions with dyspnoea (PTR) are major causes of morbidity and death after blood transfusion. Transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO) are most dangerous, while transfusion-associated dyspnoea (TAD) is a milder respiratory distress. We investigated blood components for immune and non-immune factors implicated in PTR. MATERIAL AND METHODS: We analysed 464 blood components (RBCs, PLTs, L-PLTs, FFP) transfused to 271 patients with PTR. Blood components were evaluated for 1/antileucocyte antibodies, 2/cytokines: IL-1ß, IL-6, IL-8, TNF-α, sCD40L, 3/lysophosphatidylcholines (LysoPCs), 4/microparticles (MPs) shed from plateletes (PMPs), erythrocytes (EMPs) and leucocytes (LMPs). RESULTS: Anti-HLA class I/II antibodies or granulocyte-reactive anti-HLA antibodies were detected in 18.2% of blood components (RBC and FFP) transfused to TRALI and in 0.5% of FFP transfused to TAD cases. Cytokines and LysoPCs concentrations in blood components transfused to PTR patients did not exceed those in blood components transfused to patients with no PTR. Only EMPs percentage in RBCs transfused to patients with TRALI was significantly higher (P < 0.05) than in RBCs transfused to patients with no PTR. CONCLUSION: Immune character of PTR was confirmed mainly in 1/5 TRALI cases. Among non-immune factors, only MPs released from stored RBCs are suggested as potential mediators of TRALI. Our results require further observations in a more numerous and better defined group of patients.

The relevance of HPA-15 antigen expression for anti-HPA-15 antibody detection.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level.

Monitoring and treatment of anti-D in pregnancy.

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.

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.

Transfusion-related acute lung injury and leucocyte-reacting antibodies.

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.

Leucocyte antibodies in blood donors and a look back on recipients of their blood components.

BACKGROUND AND AIM: 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. MATERIAL AND METHODS: Antibodies were examined in 1043 donors by five methods, look back performed in 26 recipients. RESULTS: 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. CONCLUSION: Detection of antibodies in donors cannot predict TRALI, even in recipients with cognate antigen(s).

The risk of antibody formation against HNA1a and HNA1b granulocyte antigens during pregnancy and its relation to neonatal neutropenia.

The evaluation of immunization by the HNA1a and 1b antigens during pregnancy was based on (i) their genotyping in 1038 unselected mothers and newborns of homozygous mothers, (ii) granulocyte counting in all born infants and (iii) examination of granulocyte antibodies in maternal sera if an HNA1 incompatibile child was born. A total of 548 (52.8%) mothers were heterozygous--thus further examinations were not done. Four hundred and ninety (47.2%) were homozygous, of whom 203 (41.3%) delivered an incompatible child, i.e. 19.6% of all the infants. Among available sera from 195 mothers with feto-maternal incompatibility, the granulocyte-specific antibodies were found in nine (4.5%); six of these (3%) were HNA1 (four anti-1a, two anti-1b), and in three others the specificity was not determined. In the remaining 28 sera, the only antibodies detected were HLA. Hence, six out of 1000 pregnant women can be expected to develop anti-HNA1. In none of the newborns was the cord neutrophil count below 1.5 x 109 L-1 and signs of infection found, thus the incidence of NAIN seems to be lower than 1 per 1000 infants. A comparison with our previous, unpublished data suggests that the incidence of severe NAIN is roughly 1 per 6000 (four cases among 24101 newborns).

Isoimmune neonatal neutropenia due to anti-Fc(gamma) RIIIb antibody in a mother with an Fc(gamma) RIIIb deficiency.

A rare case of neutropenia in a newborn due to anti-Fc(gamma) RIIIb antibody is described. The newborn, born from the 5th pregnancy, had severe infection and no neutrophils. Full clinical and neutrophil count recovery was observed when the child was 5 weeks old. In maternal serum, panreactive granulocyte alloantibodies were detected. The mother's and her two sisters' granulocytes appeared to be Fc(gamma) RIIIb deficient as found using pheno- and genotyping methods. All of them were healthy. The anti-Fc(gamma) RIIIb specificity of antibodies was identified by the monoclonal antibody immunobilization of neutrophil antigen assay. Such antibodies were not found in both sisters with the Fc(gamma) RIIIb deficiency, although they were pregnant, one of them on the seventh occasion.

[Feto-maternal incompatibility due to the platelet specific antigens: frequency, diagnosis and general rules of management]. / Konflikt matczyno-plodowy w zakresie swoistych antygenów krwinek plytkowych--wystepowanie, diagnostyka i zasady postepowania.

In this review actual problems of the frequency, diagnosis and general rules of management of the neonatal alloimmune thrombocytopenia (NAIT) due to platelet antigens are presented.

Neonatal and maternal thrombocytopenia: incidence and immune background.

We performed a prospective study on the incidence of thrombocytopenia (t-penia) and its immunological origin in unselected 26,275 mothers and 24,101 newborns. Platelet antibodies were examined by the platelet immunofluorescence test (PIFT) and the monoclonal antibody immobilisation of platelet antigens assay (MAIPA). T-penia (platelet count < 100 x 10(9)/I) was found in 124 (0.5%) mothers (in 0.04%) severe, <50x 10(9)/l) and in 116 (0.5%) newborns (in 0.15% severe); 90 (72.6%) and 112 (96.6%), respectively, were available for further studies. In both groups non-immune t-penia was diagnosed about 4.5 times more often than the immune t-penia. Among 90 mothers, t-penia was severe in 11.1%, antibodies were detected in 17.8%; both factors were not prognostic for delivering thrombocytopenic newborns. Among 112 babies, 21 were delivered by thrombocytopenic mothers and 91 by mothers with normal platelet count; among newborns with immune t-penia the proportion of alloimmune (NAIT) to autoimmune was equal (10 with NAIT, 10 with autoimmune, 4 of them born by mothers with hidden autoimmune t-penia). In 33% of the neonates t-penia was severe, most often among NAIT. In conclusion, although t-penia in mothers as well as in infants is not frequent and severe, and an immune origin not often found, the search for antibodies, in particular alloantibodies, should be done. Even if the serological results are not helpful at the moment, they can be of importance in subsequent pregnancy and for related pregnant women.

Transfusion-related acute lung injury due to granulocyte-agglutinating antibody in a patient with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is usually reported after the transfusion of blood components from donors with white cell (WBC) antibodies, but only very rarely if the patient has these antibodies. The pathogenesis of TRALI is not fully understood. Not all transfusion recipients develop TRALI, even though WBC antibodies are present in the donor or the recipient. CASE REPORT: A patient with paroxysmal nocturnal hemoglobinuria (PNH) who developed TRALI after the transfusion of non-WBC-reduced red cells is described. Granulocyte-agglutinating anti-5b was detected in his serum, and the crossmatch with the donor granulocytes was positive. The patient also developed a severe exacerbation of hemolysis with renal failure; serologic results excluded an immune hemolytic posttransfusion reaction. The patient recovered from both events after about 1 week. CONCLUSION: Granulocyte-agglutinating antibodies present in the recipient play an important role in TRALI, and also other factors may contribute to its pathogenesis. The reaction between the PNH patient's antibody (anti-5b) and transfused WBCs was found not only to be responsible for the respiratory distress but also to have triggered, through the innocent-bystander mechanism of complement activation, an intensive hemolysis, which was very likely a contributing factor in the development of TRALI.

Neonatal thrombocytopenia: incidence, serological and clinical observations.

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.

Platelet-specific antibodies in transfused patients.

Sera of 104 patients with haematological diseases, transfused with platelets and sometimes also with red cells, were prospectively investigated in: the lymphocytotoxicity test (LCT), the platelet suspension immunofluorescence test (PSIFT) and the monoclonal antibody immunobilization of platelet antigens (MAIPA) technique. All tests on admission were negative. The overall incidence of alloantibodies after transfusions was 30.8%. Platelet-specific antibodies were, however, detected much more rarely (9.6%) than anti-HLA (21.3%). The specificity against HPA antigens was defined in 4 (3.9%) patients: anti-HPA-la, 2b, 3a and 5b. In the remaining 6 (5.7%) cases only the glycoproteins on platelets were identified (GPIb and GPIIIa) which gave positive reaction with the antibodies. Our observations indicate a rather low incidence of platelet-specific antibodies after transfusions.

Effects of human neutrophil elastase and cathepsin G on the reactivity of platelets with antiplatelet antibodies.

Two human neutrophil serine proteases, elastase (HNE) and cathepsin G (CathG), are known to change the structure and hemostatic function of platelet surface membrane. The platelet membrane contains glycoproteins (GPs) which function as alloantigens, autoantigens and targets of drug-induced antibodies. The aim of this study was to investigate whether proteolysis of platelet GPs by HNE and CathG is associated with changes in the reactivity of platelets to antiplatelet antibodies. The platelet immunoreactivity was examined using the MAIPA (monoclonal antibody-specific immobilization of platelet antigens) assay and PSIFT (platelet suspension immunofluorescence test). The treatment of platelets with HNE led to a moderate increase in their reactivity to quinidine-dependent (anti-GP Ib) antibody and to a slight decline in the expression of HPA-1a. In contrast, CathG did not provoke any significant changes in platelet reactions with quinidine dependent and anti-HPA-1a antibodies. Both enzymes had no significant effect on the expression of HLA-A2, HLA-A3, HLA-B7 and HLA-B8 on platelets.

[Practical usefulness of a donor registry with determined HLA class I antigens--current personal experience]. / Praktyczne znaczenie rejestru dawców z okreslonymi antygenami HLA klasy I--dotychczasowe doswiadczenie wlasne.

The experience in organizing the HLA class I typed donors registry and practical application for platelet transfusions in immunized patients is presented. It appears that the registry containing 3461 donors gives quite a high possibility of finding a compatible or partly compatible HLA donor for a patient with relatively high frequency antigens. A discrepancy is, however, observed between finding a donor in the registry and the transfusion of the matched platelets. Better collaboration between physicians and persons organizing the registry is required to improve transfusions of the matched platelets in immunized patients. Preliminary experience is also described concerning the usefulness of the same registry for finding the HLA class I compatible donors for bone marrow transplantation.

[Diagnostic evaluation of HLA sera obtained as a result of cooperation between the Institute of Hematology and blood donation stations]. / Ocena diagnostyczna surowic ukladu HLA uzyskiwanych we wspólpracy Instytutu Hematologii ze stacjami krwiodawstwa.

The aim of the cooperation between the Laboratory of Leucocyte and Platelet Immunology, Institute of Haematology and Blood Donation Stations was typing of anti-HLA sera from the blood of pregnant women. During 18 months of this cooperation 477 sera were studied. In 255 sera (53.1%) lymphocytotoxic antibodies were demonstrated and their specificity was determined. The types anti-HLA sera will be used for determination of HLA antigens in blood donors donating blood in the Stations. The results of these determinations, technical problems and conclusions drawn from this cooperation are discussed. Continuation of this cooperation will contribute to further limitation of the import of anti-HLA sera.

[Histocompatibility antigens in childhood myasthenia]. / Badanie antygenów zgodnosci tkankowej w miastenii dzieciecej.

In a group of 55 myasthenic children 21 HLA antigens locus A and B were determined. The frequency of the HLA-B8 antigen was found to be significantly higher in childhood myasthenia as compared with control group. This correlation was not found in ocular myasthenia in children. Determination of HLA antigens in childhood myasthenia may facilitate the diagnosis and prognosis and predict the further course of the disease in cases in which the disease begins with symptoms of fatigability of oculomotor muscles.

HIV antibody status and immunological abnormalities in Polish haemophiliacs.

Sera of 520 multitransfused haemophiliacs were examined for antibody to HIV; 447 patients had haemophilia A and 73 had haemophilia B. In 382 patients with haemophilia A and in 62 with haemophilia B solely Polish-made blood products were used for replacement therapy. The remaining haemophiliacs had also received imported clotting factor concentrates prior to the investigation. Only 8 patients (haemophilia A - 7, haemophilia B - 1) developed anti-HIV and all of them had been exposed to commercial concentrates. The analysis of T-cell subsets demonstrated an inverted T4/T8 ratio (less than 1.0) in 7 (30%) of the 23 haemophiliacs treated solely with domestic cryoprecipitate and in 3 (37%) of the 8 seropositive recipients of commercial concentrates. The most frequent alteration in both subgroups was a reduced ratio with either normal absolute numbers or an increase in T8 cells. Increased serum IgG levels were found in 82% of the users of cryoprecipitate and in 75% of the seropositive patients. Serum beta-2-microglobulin level was elevated in 69 and 62% of each subgroup, respectively. The observed immunological abnormalities, at least in the cryoprecipitate treated subgroup, may be causally related to factors other than HIV infection.