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.

Anti-Co(a) implicated in severe haemolytic disease of the foetus and newborn.

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).

Leucine33-proline33 substitution in human platelet glycoprotein IIIa determines HLA-DRw52a (Dw24) association of the immune response against HPA-1a (Zwa/PIA1) and HPA-1b (Zwb/PIA2).

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.

Evaluation of human blood monocytes in erythrophagocytosis assay with anti-Rh antibodies.

Results of the phagocytosis assay with monocytes and erythrocytes sensitized with subclass specific anti-Rh(D) antibodies (IgG1, IgG3, IgG1 + 3) are dependent on the selection of sera used for sensitization. Phagocytic abilities expressed as the percentage of active monocytes as well as the number of interacting red cells requires a certain degree of the sensitization of erythrocytes and is higher if serum contains IgG3 antibodies.

Lymphocyte subpopulations in healthy newborns and in immunohemolytic anemia due to Rh incompatibility.

The subpopulations of lymphocytes in cord blood were analyzed in 50 healthy newborns and in 25 with hemolytic disease of the newborns (HDN) using E, EA, EAC rosette tests with sheep erythrocytes, EA rosette test with human erythrocytes and SmIg+ test. A statistically significant decrease of the percent of T lymphocyte and increase in the absolute values of all lymphocyte subpopulations were found in healthy newborns as compared with adults. In the newborns with HDN a correlation was observed between the severity of the disease and the results of rosette tests. Three groups were distinguished: 1) very low values of all rosette tests, severe anemia in newborns, and high titer of antibodies in their mothers, 2) low values of EA rosette tests, less severe anemia antibody titer in the mothers lower than in the first group, 3) rosette tests within normal range, newborns usually without anemia, low titer of maternal antibodies.

DRB polymerase chain reaction fingerprinting, DQA oligotyping and mixed lymphocyte culture results in related bone marrow donors and recipients.

Donor and recipient are usually regarded as well matched for bone marrow transplantation when they are compatible in HLA class I and II antigens and in mixed lymphocyte culture (MLC). However, results of serological typing of class II antigens may be unreliable. Hence, polymerase chain reaction fingerprinting of HLA DRB (PCR FP) was introduced for the screening of related donors for 29 patients awaiting bone marrow transplantation. In addition, the sequence-specific oligonucleotide (SSO) typing of DQA alleles was performed. In 18 pairs the results of DNA analysis methods were compared with the results of MLC. 72% of pairs were HLA DQA compatible and 59% showed compatibility in PCR FP. MLC compatibility was found in 61%. A higher correlation of PCR FP and MLC results was observed.

Moderate hemolytic disease of the newborn due to anti-Hr0 in a mother with the D--/D-- phenotype.

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.

A gel microtyping system for diagnosis of paroxysmal nocturnal hemoglobinuria.

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.

[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.

[Vaccination against hepatitis B in children with chronic hematologic diseases treated with immunosuppression]. / Szczepienia przeciw wirusowemu zapaleniu watroby typu B u dzieci z przewleklymi chorobami krwi leczonych immunosupresyjnie.

Prevention of hepatitis B infection is an important factor in the successful management of cancer and aplastic anaemia cases. Our result suggested that children with Hodgkin's disease and solid tumors vaccinated during early stage of immunosuppressive therapy are good responders to hepatitis B vaccine. Active immunisation with hepatitis B vaccine (Engerix B), was also effective in children with leukaemia after completing immunosuppressive therapy. Protective levels of antibodies remained 6 years after vaccination. Vaccination according to shortened schedule (0-10-20 days) was not effective in these children. Passive immunisation is indicated in children with chronic neoplastic haematological diseases during immunosuppressive therapy. In 6 children the lack of seroconversion after vaccination was due to immune disorders.

[Diagnosis of human parvovirus B19 infection in nonimmune hydrops fetalis]. / Diagnostyka zakazenia ludzkim parwowirusem B19 w nieimmunologicznym obrzeku plodu.

Parvovirus B19 (PV B19) infection was investigated in 29 pregnant women with fetal hydrops, after exclusion of feto-maternal incompatibility within red blood cell antigens, TORCH infections, feto-maternal hemorrhage and genetics reasons. The active viral infection was detected in 9 women (31%) by PCR amplification of DNA B19; in 2 of them IgM and IgG, in 1 IgM and in 4 IgG antibodies were also present. In 6 women (20%) IgG antibodies were only found, but not IgM and DNA B19, which confirmed infection in the past. In addition in 9 cases DNA B19 was evaluated in the fetal blood. The results in the mothers and their fetuses were concordant (4 positive, 5 negative). Our conclusion is that in nonimmune hydrops fetalis, PV B19 infection should be based on the viral DNA evaluation in the blood of mother (or fetus). IgM antibodies, in time of fetal disorders, might not be detected.

Analysis of 15 cases with platelet EDTA-dependent antibodies.

In 15 patients with thrombocytopenia EDTA-dependent platelet antibodies (IgM agglutinins active in 20 degrees C) were detected. These antibodies were found in patients with autoimmune diseases, infections and neoplasma as well as in healthy persons (including pregnant woman). In 10 persons pseudothrombocytopenia (PTCP) was diagnosed since the low platelet counts were found only in EDTA-blood, and the patients did not have bleeding symptoms. In 5 other cases EDTA-dependent antibodies were not the only cause of thrombocytopenia since the low platelet counts were also observed in the citrate-blood and occassionally the bleeding symptoms occurred; in 3 of them in addition autoantibodies were suspected. Due to the proper diagnosis of PTCP, in five patients unnecessary corticotherapy was discontinued, one person avoided splenectomy, in two patients previously postponed surgery was performed and one person could be passed for employment.

[Monoclonal origin of autoantibodies of the warm type in hemolytic autoimmune anemia]. / Zagadnienie monoklonalnosci autoprzeciwcial typu cieplego w niedokrwistosciach autoimmunohemolitycznych.

In 59 patients with warm-type autoimmunohaemolytic anaemia classes and types of immunoglobulins, presence of complement, serological specificity of autoantibodies, and in 14 selected cases also IgG subclasses were determined. It was found that warm autoantibodies differed from anti-Rh alloantibodies. In 27 patients only one type of L chain was found but, in the light of complex investigations, the authors reached the conclusion that this result cannot be sufficient for accepting autoantibodies as monoclonal immunoglobulins, since in some of them 2 or 3 IgG subclasses and more than one serologic specificity were demonstrated. These observations indicate the need for more precise investigation methods for explaining the problem of monoclonal origin of warm autoantibodies.

[Light chains of warm autoantibodies in patients with autoimmune hemolytic anemia. Long-term observation]. / Wieloletnia obserwacja lancuchów lekkich autoprzeciwcial typu cieplego u chorych z niedokrwistoscia autoimmunohemolityczna.

In 18 patients with autoimmunohaemolytic anaemia presence of light chains of IgG autoantibodies was south for by the method of direct antiglobulin reaction. In 11 patients variability was observed in the detection of light chains. The authors discuss the possible causes of this observation: variable participation of different cell lines in the production of autoantibodies and changes in the conformation of immunoglobulin molecule in the erythrocyte.

[Anti-U antibodies in autoimmune hemolytic anemia]. / Przeciwciala anty-U w niedokrwistosciach autoimmunohemolitycznych

The results of serological investigations in 23 patients with autoimmunohaemolyts anaemia (AIHA) and warm-type autoantibodies belonging to IgG immunoglobulins are presented. The serological specificity of autoantibodies was demonstrated in 8 cases, and in 4 of them specificity to single antigen of the Rh system was demonstrated, in the remaining 4 cases they were specific against U antigen. The method of identification of these autoantibodies and their characteristic features are described. Analysing the clinical course of AIHA in three cases with anti-U autoantibodies it was stated that was chronic, intermittently recurring and was not associated with proliferative disorders of the haematopoietic system.

[Immunization in the range of HL-A antigen system in volunteers immunized with small doses of blood incompatible in the Rh system]. / Immunizacja w zakresie antygenów ukladu HL-A u ochotników uodpornianych malymi dawkami krwi niezgodnymi w zakresie ukladu Rh

Thirty-six healthy men Rh-negative were immunized with whole Rh-positive blood for production of anti-D antibodies were observed for development of anti-leucocyte antibodies determined by incidental incompatibility in HL-A antigens. Small doses of blood containing 25-35 X 10(6) white blood cells were found to be sufficient for production of weak anti-leucocyte antibodies in 91.7% of immunized subjects. The ability to produce anti-leucocyte antibodies was nearly twice as high as the ability to produce anti-Rh antibodies. No correlation was observed between the ability of immunization in the range of erythrocyte or leucocyte antigens.

[Effect of HL-A system antigen differences on the specificity of produced anti-leukocyte antibodies]. / Wplyw róznic antygenowych ukladu HL-A na swoistosc wytwarzanych przeciwcial anty-leukocytarnych

In Rh-negative volunteers immunized for development of anti-D antibodies the specificity of developed anti-leucocyte antibodies was determined. The specificity of these antibodies was analysed in relation to HL-A antigenic differences determined in donors and recipients. The antigenic power of sublocus II was found to be superior to that of sublocus I. Another observation was more frequent production of cross-reacting antibodies. A trial of analysis of these volunteers in whom no antileucocyte antibodies developed during immunization with blood was undertaken.

[HL-A antigens in patients with Hodgkin's disease]. / Wystepowanie antygenów ukladu HL-A u osób z ziarnica zlosliwa

HL-A antigens were determined in 46 patients with Hodgkin's disease and a statistical analysis was carried out by the chi square test to compare the incidence of these antigens in patients with that in healthy people. Statistically significant differences were found in the first place in the HL-A5 antigen (chi square = = 32.2) (p less than 0.005). This antigen was much more frequent in patients (43%) than in the healthy population (15%). A slightly lower frequency of antigen W5 was found in the group of patients (healthy controls -- 13.5%, patients -- 4.5%, chi square = 7.08, p less than 0.05).

[Autoimmune hemolytic anemia with presence of 3 types of autoantibodies to erythrocytes]. / Niedokrwistosc autoimmunohemolityczna z obecnoscia trzech rodzajów autoprzeciwcial dla krwinek czerwonych.

In a patient with chronic lymphatic leukaemia severe autoimmunohaemolytic anaemia (NAIH) was observed with coexistence of cold incomplete IgM, warm incomplete IgG and warm haemolysins active against papainized erythrocytes in low pH medium. The serologic and immunochemical characteristics of autoantibodies are presented and the mechanism of their action in vitro is tentatively explained.