A. E. G. Kr. Borne

A Simple Immunofluorescence Test for the Detection of Platelet Antibodies

Immunofluorescence tests on platelets have always been hampered by nonspecific fluorescence caused by non‐immunological binding of plasma proteins to the platelet membrane. It was found that this could be easily overcome by fixation of the cells with paraformaldehyde (PFA). By using PFA‐fixed platelets, a simple method for the detection of platelet antibodies, the platelet suspension immunofluorescence test (PSIFT) was developed.

Vasculitis and antineutrophil cytoplasmic autoantibodies associated with propylthiouracil therapy

Vasculitis is a rare complication of propylthiouracil therapy. Antineutrophil cytoplasmic antibodies (ANCA) have been described in association with several vasculitic disorders. We report detection of ANCA against human neutrophil elastase, proteinase 3, and myeloperoxidase in serum from six patients who developed evidence of vasculitis during propylthiouracil treatment of hyperthyroidism. On withdrawal of the drug ANCA concentrations fell and clinical symptoms resolved completely.

Autoimmune thrombocytopenia : Detection of platelet autoantibodies with the suspension immunofluorescence test

Summary An immunofluorescence test on paraformaldehyde‐fixed platelets in suspension was used for the detection of antibodies on platelets and in the sera of patients with idiopathic or secondary thrombocytopenia.

Megakaryoblastic differentiation of proerythroblastic K562 cell-line cells.

The human proerythroblastic leukemia cell-line K562 was induced to differentiate into megakaryocytic cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). Megakaryocytic differentiation was detected when lineage-specific monoclonal antibodies were used to monitor the effect of TPA on K562 cells. A monoclonal anti-platelet antibody (C17) directed against an epitope present on GP IIIa appeared to react with K562 cells after induction. This was observed together with the disappearance of glycophorin A, the erythrocyte-specific lineage antigen. The induced megakaryocytic cells were also detected by ultrastructural platelet peroxidase (PPO). Immunoprecipitation, after ectolabeling of the cells with the C17 antibody and SDS-polyacrylamide gel electrophoresis, proved that TPA-induced K562 expressed both GP IIIa and GP IIb. However, the monoclonal antibody C15 directed against another epitope of platelet GP IIIa reacted only partially, or not at all, indicating that GP IIIa expressed on TPA-induced K562 differs structurally from that on normal platelets. K562 clones, expressing glycophorin A in all cells, were obtained by limiting dilution and culture. When these clones were treated with TPA, again megakaryocytic cells were obtained. These findings are discussed in relation to normal megakaryocytopoiesis.

Monoclonal antibodies against human platelet glycoprotein IIIa

Summary. Two murine monoclonal antibodies specific for human platelets were prepared and characterized by immunofluorescence, immunoprecipitation and by studying their effect on platelet function.

Autoimmune Granulocytopenia: the Detection of Granulocyte Autoantibodies with the Immunofluorescence Test

Neutropenia may be caused by neutrophil autoantibodies. The detection of such antibodies has always been difficult. Recently, we developed a sensitive indirect immunofluorescence technique applicable to granulocytes which proved to be of value in the detection of granulocyte alloantibodies. We have now used this method to investigate the serum and cells of 29 patients with idiopathic or secondary neutropenia.

Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons

BACKGROUND: Qualitative RHD variants are the result of the replacement of RHD exons by their RHCE counterparts or of point mutations in RHD causing amino acid substitutions. For RHD typing, the use of at least two RHD typing polymerase chain reaction (PCR) assays directed at different regions of RHD is advised to prevent discrepancies between phenotyping and genotyping results, but even then discrepancies occur. A multiplex RHD PCR based on amplification of six RHD‐specific exons in one reaction mixture is described. STUDY DESIGN AND METHODS: Six RHD‐ specific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7, and 9. DNA from 119 donors (87 D+, 14 D‐ and 18 with known D variants; whites and nonwhites) with known Rh phenotypes was analyzed. RESULTS: All six RHD‐specific exons from 85 D+ individuals were amplified, whereas none of the RHD exons from 13 D‐ individuals were amplified. Multiplex PCR analysis showed that the genotypes of two donors typed as D+ were DIVa and DVa. Red cell typing confirmed these findings. From all D variants tested (DIIIc, DIVa, DIVb, DVa, DVI, DDFR, DDBT) and from RoHar, RHD‐specific exons were amplified as expected from the proposed genotypes. CONCLUSION: The multiplex PCR assay is reliable in determining genotypes in people who have the D+ and partial D phenotypes as well as in discovering people with new D variants. Because the multiplex PCR is directed at six regions of RHD, the chance of discrepancies is markedly reduced. The entire analysis can be performed in one reaction mixture, which results in higher speed, higher accuracy, and the need for smaller samples. This technique might be of great value in prenatal RHD genotyping.

A murine monoclonal IgM antibody specific for blood group P antigen (globoside)

A murine monoclonal IgM erythrocyte antibody appeared to have anti‐P (anti‐globoside) specificity. The antibody was a relatively weak cold agglutinin, but a strong haemolysin and its reactivity with red cells was markedly enhanced by enzyme treatment. This antibody was used to study the cell and tissue distribution of globoside. Globoside was not only detectable on red cells and erythroblasts, but also on endothelial cells and on subsets of platelets, megakaryocytes and fibroblasts. It was not detectable on granulocytes, monocytes and most peripheral blood lymphocytes. Neither was it present on erythroblast precursors (CFU‐E, BFU‐E), pro‐erythroblasts or on the cells of the pro‐erythroblastic cell lines K562 and HEL. However, K562 cells expressed globoside when induced to mature into erythroblasts by sodium butyrate. Cells of patients with various leukaemias were also tested. A significant number of positively reacting cells was frequently (six out of 18) seen in cases with a CML blast crisis (CML‐BC) and rarely in AML (four out of 37 cases). In CML‐BC the P‐positive cells were probably erythroblasts and/or megakaryoblasts. Thus, globoside appeared to be an interesting marker in CML‐BC of the erythroblastic or mixed erythroblastic‐megakaryoblastic type.

International study to compare antigen‐specific methods used for the measurement of antiplatelet autoantibodies

Platelet‐associated and plasma autoantibodies against platelet glycoproteins (GP) have been demonstrated in patients with autoimmune thrombocytopenia (AITP) using various methods. Eight laboratories in seven countries participated in this international study to evaluate the interlaboratory agreement using glycoprotein‐specific immunoassays for these autoantibodies. The participating laboratories received blind samples of frozen washed platelets and plasma from 22 normal donors and 22 AITP patients. Platelet‐associated and plasma autoantibodies against GPIIb–IIIa and GPIb–IX were measured by MAIPA, immunobead assay or modified antigen capture assay. Of the control samples, 96.0% and 97.2% of all results for platelet‐associated and plasma autoantibodies to GPIIb–IIIa/GPIb–IX, respectively, were negative. The mean variation coefficient of the control samples of platelet‐associated and plasma autoantibodies was 89.5% (range 11.1–272.9%) and 46.5% (range 21.0–78.0%), respectively. In 20/22 patient samples, platelet‐associated autoantibodies to either glycoprotein were noted by at least two laboratories. The mean degree of agreement in these samples was 74.0%. There was a significant correlation in the individual antibody measurements between all laboratories (Kendall coefficient of concordance 0.60 and 0.38, P < 0.001; Spearman rank order test, range of correlation coefficient 52.3–94.0% and 42.2–85.0%, P < 0.05, for anti‐GPIIb–IIIa and anti‐GPIb–IX, respectively). In contrast, plasma autoantibodies to either glycoprotein were noted by at least two laboratories in only 13/22 patient samples. Moreover, the degree of agreement was poor (50.1%) and a significant correlation was noted between only six pairs of laboratories. We conclude that methods used in this study yield good interlaboratory agreement in measuring platelet‐associated autoantibodies against GPIIb–IIIa and GPIb–IX. In contrast, poor agreement was found in detecting plasma autoantibodies to the same glycoproteins.

Autoimmunity against blood cells in human immunodeficiency-virus (HIV) infection.

Summary. In persons with AIDS or at risk from AIDS, autoantibodies against platelets and granulocytes were frequently detected. Platelet‐bound immunoglobulins were demonstrated by immunofluorescence in all 16 patients with AIDS, in five out of seven patients with AIDS‐related complex/persistent generalized lymphadenopathy (ARC/PGL) and even in seven of 10 healthy sexually active homosexual men. Granulocyte‐bound immunoglobulins were found by immunofluorescence in 12 of the 16 AIDS patients, five of the seven patients with ARC/PGL and two of the 10 symptomless men. Red cell bound immunoglobulins were not detected. All patients with AIDS and ARC/PGL and three of the symptomless men were seropositive for human immunodeficiency virus (HIV).