Diminished presentation of complement regulatory protein CD55 on red blood cells from patients with hereditary haemolytic anaemias.

INTRODUCTION: Hereditary haemolytic anaemias (HHA) encompass a heterogeneous group of anaemias characterized by decreased red blood cell survival. The aim of this study was to evaluate the status of red blood cell (RBC) surface molecules known or previously proposed to participate in preventing premature RBC clearance, analysing erythrocytes from patients with two types of HHA: hereditary spherocytosis (HS) and microcytosis. MATERIAL/METHODS: Relative binding of five monoclonal antibodies (mAbs), anti-CD55, anti-CD59, anti-CD44, anti-CD47 and anti-CD58, was evaluated in erythrocytes of patients with HS and hereditary microcytosis, using flow cytometry. The amount of CD55 protein was assessed by semi-quantitative Western blots densitometry analysis. RESULTS: The majority of both HS and microcytic patients demonstrated significant reduction of anti-CD55 binding by erythrocytes (average 23% and 19%, respectively, P < .001), with no concomitant anti-CD59-binding deficiency. Anti-CD44, anti-CD47 and anti-CD58 binding was within the healthy control range or was slightly decreased. CONCLUSIONS: This study provides evidence supporting the presence of erythrocytes deficient in CD55 presentation in HS and hereditary microcytosis. Moreover, deficiency of CD55 antigen presentation on RBC does not correlate with the amount of CD55 in RBC membrane. Further studies using molecular techniques will clarify the exact participation of CD55 deficiency in premature RBC clearance in HHA.

Quantitative fetomaternal hemorrhage assessment with the use of five laboratory tests.

INTRODUCTION: In various countries, standard doses of anti-D IgG used for postpartum immunoprophylaxis of hemolytic disease of fetus and newborn (HDFN) vary from 100 µg to 300 µg. There are also different regulations concerning FMH assessment, and opinions about applicable tests are inconclusive. METHODS: Three flow cytometry tests (FCTs) with anti-D, anti-HbF, anti-HbF+CA antibodies, and two modifications of microscopic Kleihauer-Betke test (KBT) were used. RESULTS: In all artificial mixtures with known concentrations, FCTs and KBT with counting 10 000 RBCs had similar satisfying sensitivity and specificity. KBT with counting 2000 RBCs had to be disqualified because of significant discrepancies between expected and measured values of FMH. The test procedure with anti-D was easier and shorter than the remaining tests, but it can be only used for FMH assessment in RhD-negative mothers with RhD-positive newborns. In one clinical sample, it was impossible to distinguish fetal RBCs from maternal F cells in KBT and FC with anti-HbF but other tests were useful. CONCLUSION: In the four tests, correlation between expected and obtained results was appropriate (CCC Ì´1). Each test had some advantage and limitation in any clinical situation. Therefore, it is best to have opportunity to perform two or three assays in the laboratory.

Quantitation of red cell-bound IgG by an enzyme-linked antiglobulin test in the patients with warm-type autoimmune haemolytic anaemia.

We have previously reported the evaluation of a gel-direct antiglobulin test and enzyme-linked antiglobulin test (ELAT) in the laboratory diagnosis of autoimmune haemolytic anaemia (AIHA). We now report our experience with quantitative ELAT performed on a large group of patients under long-term observation. The number of IgG molecules/red blood cell was determined in 658 blood samples from 268 randomly selected patients with warm-type AIHA. Eighty-six patients were tested every 2-4 weeks for several months. Laboratory signs of haemolysis were present in 65.7% of blood samples with a small amount of red cell-bound autoantibody (< 200 IgG molecules/red blood cell) and in 70.4% of blood samples with moderately coated red blood cells (200-1000 molecules/red blood cell). Haemolysis was demonstrated in 87.9% samples with > 1000 IgG molecules/red blood cell, which were predominantly IgG3 and C3 complement, the qualitative factors that may increase haemolysis. In 79% of periodically tested patients, the number of IgG autoantibody molecules/red blood cell decreased and this correlated with the improvement of haemolysis parameters. The number of IgG molecules varied in 21% of AIHAs and was associated with poor prognosis.

B cells CD19+ in patients with B-cell chronic lymphocytic leukaemia and autoimmune haemolytic anaemia.

The study presents results of B and T lymphocytes population analysis in patients with chronic lymphocytic leukaemia B cells and autoimmune haemolytic anaemia (CLL-B + AIHA). We evaluated the following groups of patients: (1) with newly recognized CLL-B and co-existent AIHA (untreated), (2) after short-term treatment with corticosteroids, (3) after treatment with chemotherapy and corticosteroids. The control groups were made of patients with CLL-B without AIHA. The populations of lymphocytes and determination of cells immunophenotype were performed by means of flow cytometry. The analysed data were obtained from 25 patients. The untreated patients with CLL-B + AIHA presented significantly more numerous population of neoplastic cells CD19+ CD5+ in comparison with patients without AIHA. The patients with AIHA showed a reduced percentage of B CD19+ CD22+ cells in comparison with those without AIHA. Untreated patients with AIHA or after a short-term corticosteroid treatment showed a higher ratio of the number of CD19+ CD5+ cells to the number of T CD4+ and T CD8+ lymphocytes than CLL-B patients without AIHA. It can be presumed that the differences found may be related to the pathogenesis of the autoimmune haemolysis syndrome in patients with CLL-B.

Glycoconjugate abnormalities in patients with congenital dyserythropoietic anaemia type I, II and III.

Congenital dyserythropoietic anaemia type II (CDA II) is well known for glycosylation abnormalities affecting erythrocyte membrane glycoconjugates that encompass hypoglycosylation of band 3 glycoprotein and accumulation of glycosphingolipids: lactotriaosylceramides, neolactotriaosylceramide and polyglycosylceramides. These abnormalities were not observed in erythrocytes from patients with CDA of either type I or III. Recently, however, we have described a CDA type I patient in Poland with identical, though less pronounced, glycoconjugate abnormalities to those observed in patients with CDA type II. The abnormalities included partial unglycosylation of O-linked glycosylation sites in glycophorin A. These abnormalities are now reported in three Bedouin patients from Israel with CDA type I. In addition, the erythrocyte membranes of these patients exhibited highly increased globotetraosylceramide content. Glycoconjugate abnormalities were also present in erythrocyte membranes from three patients from Northern Sweden with CDA type III but they almost exclusively affected glycosphingolipids. In erythrocytes of all patients examined including one with CDA type II, polyglycosylceramides were significantly hypoglycosylated although, on a molar basis, their contents in erythrocyte membranes were increased. Thus, glycoconjugate abnormalities of varying intensity occur in erythrocyte membranes from all patients with CDA that were investigated.

Evaluation of the microcolumn technology for pretransfusion testing in multiple myeloma patients.

OBJECTIVE: Microcolumn tests are useful for serological investigations, although because of their high sensitivity, false-positive results might be expected, e.g. in hypergammaglobulinemia. The aim of this study was to evaluate these tests in multiple myeloma. METHODS: Pretransfusion testing was done in 80 patients with multiple myeloma using microcolumn and traditional tube tests. RESULTS: All sera were negative in microcolumn indirect antiglobulin test and enzyme test, positive in 58% of samples in the enzyme tube test. The microcolumn direct antiglobulin test was positive in about 40% of samples but never in the tube direct antiglobulin test. This was not due to the presence of autoantibodies but to nonspecific binding of immunoglobulins related to their concentration in sera. CONCLUSION: Microcolumn tests appeared to be useful for pretransfusion testing in multiple myeloma in spite of positive autocontrols.

Gel test application for IgG subclass detection in auto-immune haemolytic anaemia.

BACKGROUND AND OBJECTIVES: Commercially available gel test microtubes are not available with antisera for the determination of IgG subclasses. The aim of this study was to adapt the gel technique for this purpose and apply it to the investigation of patients with autoimmune haemolytic anaemia (AIHA). MATERIALS AND METHODS: We studied 66 red cell samples from 49 patients with AIHA of the warm-active IgG type. Standard serologic and haematologic methods were used. We adapted the DiaMed gel test by using IgG-subclass antisera. RESULTS: We found the adapted test useful in determining the subclass of autoantibodies in eluates. We could identify the IgG subclass in all the AIHA patients, even those that were 'Coombs-negative', with less than 200 IgG molecules bound in vivo per red cell. Comparison of the gel test with the standard spin tube test and the microtiter plate test showed the superiority of the gel test, i.e., detection of IgG subclasses was much better than with the tube test and the results were more clearcut than those of the microplate test. The gel test is also the simplest and least time-consuming and permits a later reading of results. Application of the test in our 66 samples confirmed that IgG1 was the most frequent (96%). In 59% of the cases it was accompanied by IgG of other subclasses. Multiple subclasses were most common in the cases with stronger in vivo IgG red cell sensitization and severe haemolysis. Accompanying IgG3 was detected only in patients with obvious haemolysis. CONCLUSION: The gel test is more sensitive than other procedures for the determination of IgG subclass and has the advantages of simplicity, rapidity, low cost, and stability of the agglutinates.

The value of gel test and ELAT in autoimmune haemolytic anaemia.

Three methods for detection of warm type IgG autoantibody were evaluated using 400 blood samples from 147 patients suspected of autoimmune haemolytic anaemia (AIHA). Three direct antiglobulin techniques (DAT) were used: conventional tube DAT, gel-DAT by micromethod and gel-DAT enzyme linked antiglobulin test (ELAT). Eluate examinations confirmed the presence of autoantibodies on red cells. These tests were compared directly using 126 selected blood samples from 85 patients with IgG molecules on their red cells detected by the gel test. In 106 of these samples, collected from 65 patients with clinical symptoms of AIHA, the presence of autoantibody was confirmed by acid elution. The ELAT was positive in 100 samples (94%), 87 samples for tube DAT (82%). The ELAT as well as the tube DAT was negative in 20 samples with non-reactive eluates by gel test. The gel-DAT was therefore not fully specific and detected IgG on red cells of patients with hypergammaglobulinaemia. However, due its higher sensitivity it proved useful as a screening test. The ELAT allowed changes in the number of IgG molecules per red cell to be monitored quantitatively. Both methods play a part in the diagnosis and monitoring patients with warm type IgG auto-antibody.

Enzyme-linked antiglobulin test for the evaluation of the amount of IgG autoantibodies on red blood cells.

An enzyme-linked antiglobulin test (ELAT) was adapted to the assessment of IgG autoantibodies on red cells. Due to the worked out standard curve and the performance of the test on microplates, ELAT became a simple and inexpensive technique. It can be used for the detection of IgG within the range from 70 to 37,500 molecules per red cell. This is a simple quantitative method that can be used in immunohaematological studies of red cells.

[The role of enzyme tests in antibody screening before blood transfusion]. / Znaczenie testów enzymatycznych w wykrywaniu przeciwcial przed przetoczeniem krwi.

In Poland as well as in other European countries there are two obligatory serological tests for antibody screening and crossmatching: indirect antiglobulin test (IAT) and enzyme test. Because of development of some new sensitive laboratory techniques serologists discuss the following questions: should enzyme techniques be still used, should they be replaced by other new methods, may investigations be performed only by the well standardized IAT?

[Use of modified acid elution technique in laboratory diagnosis of autoimmune hemolytic anemia]. / Zastosowanie zmodyfikowanej techniki kwasnej elucji przeciwcial w diagnostyce laboratoryjnej niedokrwistosci autoimmunohemolitycznej.

Three modifications of acid elution method were used for the diagnosis of autoimmune haemolytic anaemia. These procedures were adapted to obtain more potent eluates and to prepare intact antibody-free red blood cells for phenotyping and for autologous absorption of warm-reactive autoantibodies. From the results presented in this paper we conclude that the modifications of the acid elution method are better than the up to now used procedure with a mixture of proteolytic enzyme and a thiol reagent. Moreover, these procedures are quick and easy to perform as well as more efficient. The obtained colourless eluates can be investigated in a gel-lest.

[Anti-S antibodies in autoimmune hemolytic anemia]. / Autoprzeciwciala anty-S w przebiegu niedokrwistosci autoimmunohemolitycznej.

21 year old female with severe symptoms of AIHA developed twice acute haemolytic reactions at the time of blood transfusions compatible in ABO and Rh blood group systems as well as in K antigen, but incompatible by cross-match due to the presence of autoantibody in her serum. High-affinity IgG autoantibody of an unusual anti-S specificity firmly blocked S determinants on the patient red cells. S negative blood was well tolerated. Utero-vaginal infection by human papilloma virus which might have been the cause of AIHA was detected. Long-lasting steroid therapy and specific treatment of infection improved the clinical condition. The patient was under observation for 20 months. Anti-S autoantibody was being detected in the period of 18 months. Then, very weak anti-U autoantibody appeared.

[Use of microtest in gel for compatibility in serological testing before transfusion]. / Zastosowanie mikrometody w zelu dekstranowym w badaniach serologicznych wykonywanych przed transfuzja.

A gel technique for the detection of red blood cell antigen--antibody reaction was evaluated in pretransfusion compatibility tests. The evaluation was undertaken on 917 blood samples of recipients and 2450 samples of donors. Parallel study of microtest in gel and classical techniques revealed that gel technique was more sensitive than the classical one, because some antibodies of Rh, Kell and Kidd systems were found by this method exclusively. It is also more specific because a lot of unspecific reactions could be eliminated. Therefore, it was decided to use the microtest in gel for crossmatching of recipient serum against red cells from each intended donor--particularly when the patient had irregular erythrocyte antibodies. The results presented in this paper revealed that in gel is one of the most potent tests and it is the safest for routine use before transfusion.