Patricia Fromont

Specific antiplatelet glycoprotein autoantibodies are associated with the thrombocytopenia of primary antiphospholipid syndrome

Thrombocytopenia is a frequent complication of primary antiphospholipid syndrome (PAPL) and has been attributed to antibodies directed against platelet glycoproteins (Gp) and also to antiphospholipid antibodies. We tested patients with PAPL with and without thrombocytopenia for specific antiplatelet autoantibodies. Platelet autoantibodies were detected by means of platelet immunoassays which included MAIPA with a panel of monoclonal antibodies directed against all the platelet Gps known to be possible targets for platelet autoantibodies. A high prevalence of serum platelet antibodies was found in patients with thrombocytopenia (73%, 11/15 patients) whereas antiplatelet antibody was detected in only one of the 10 control patients (P < 0.01). The antibodies mainly recognized GpIIbIIIa (n = 7), but also CD9 (n = 5), GpIaIIa (n = 4), GpIbIX (n = 3) and GpIV (n = 3). Platelet‐Gp antibodies eluted from the platelet surface had the same reactivity as those found in the original sera from three of the four patients tested, whereas no anticardiolipin activity was found in the platelet eluates, suggesting the absence of cross‐reactivity between anticardiolipin and antiplatlet antibodies. The MAIPA assay was also performed with F(ab′)2 fragments obtained by pepsin digestion of serum IgG from four patients. The same results were obtained with F(ab′)2 fragments and the original serum, demonstrating that platelet antibodies specifically bind in vivo to platelet Gps via their F(ab′)2 fragments. Our results suggest a link between specific platelet antibodies and the thrombocytopenia of PAPL.

Platelet alloimmunization after multiple transfusions: a prospective study of 50 patients

Fifty polytransfused patients were prospectively studied to determine the frequency of post‐transfusion alloimmunization and its influence on the response to platelet transfusion. Platelet‐ and HLA‐specific antibodies were detected by means of the standard and antiglobulin‐augmented lymphocytotoxicity techniques (LCT), the platelet suspension indirect immunofluorescence test (PSIIFT), and monoclonal antibody immobilization of platelet antigens (MAIPA). HLA antibodies were detected in 13 patients (26%) (IgM = 6; IgG = 6; IgM + IgG = 1). The standard LCT was positive in 12 of these 13 patients. Complement‐independent HLA antibodies, only detectable in the PSIIFT and the antiglobulin‐augmented LCT, were documented in two patients and were associated with poor post‐transfusion platelet recovery in the patient who could be evaluated. All the HLA antibodies were detected in the PSIIFT, while only four were detected in the MAIPA. Platelet‐specific alloantibodies were found in two patients by means of PSIIFT or MAIPA and may have led to poor post‐transfusion platelet recovery in one patient. Platelet autoantibodies were detected in two patients but were not associated with platelet refractoriness. Paraformaldehyde‐dependent platelet antibodies were detected in 11 patients but were not associated with platelet refractoriness.

CD36 deficiency is frequent and can cause platelet immunization in Africans

BACKGROUND: CD36 is expressed on several cell lineages. About 5 to 10 percent of Asians lack platelet membrane CD36 (pCD36), but the frequency of pCD36 deficiency in other ethnic groups is not known. Persons who are pCD36‐negative are apparently healthy but can develop CD36 isoimmunization.

Immunochemical analysis of platelet autoantibodies in HIV-related thrombocytopenic purpura: a study of 68 patients

Serum antiplatelet IgG and platelet‐associated IgG (PAIgG) were studied in 68 AIDS‐free human immunodeficiency virus (HIV)‐infected patients with severe immunologic thrombocytopenic purpura (ITP), for the presence of platelet autoantibodies. Serum IgG with antiplatelet activity was found in 72% of the sera. However, the presence of autoantibodies against platelet surface glycoproteins was not found in these sera by means of Western blot and immunoprecipitation procedures. Nevertheless, an immunoblot immuno‐assay and an indirect immunofluorescence test against semi‐permeabilized platelets demonstrated the presence of antibodies in the patient sera, that reacted with intracyto‐plasmic platelet components, and which might participate in the elimination of platelet fragments. Direct immunofluorescence tests demonstrated an increased amount of PAIgG in 75% of the patients; the bound antibodies could be eluted with ether in 44% of the cases. These eluates were found to bind to normal platelets but not to Glanzmann type I platelets. Finally, immunoprecipitation procedures demonstrated the presence of platelet autoantibodies in six of the 35 eluates studied. These antibodies recognized GPIIb in two cases. GPIIIa in one case, and an unidentified platelet protein of 150 kDa in the three other cases. The discrepancy between sera and platelet eluates was interpreted as being due to the low titre of the antibodies and to their dilution in polyclonal hypergammaglobulinaemia.

Posttransfusion purpura-like syndrome associated with CD36 (Naka) isoimmunization

BACKGROUND: CD36 deficiency, which could lead to CD36 isoimmunization, has been reported in the Japanese population. CD36 isoantibody has been involved in platelet transfusion refractoriness. CASE REPORT: A 50‐year‐ old woman originally from Corsica developed severe acute thrombocytopenia after massive transfusion. She was found to be CD36 deficient, and platelet immunoassays revealed a CD36 (Naka) platelet isoantibody. Although the involvement of another mechanism could not be entirely ruled out, the thrombocytopenia was attributed to posttransfusion purpura‐like syndrome. The antibody was also involved in platelet transfusion refractoriness. CD36 deficiency was present in two members of the patients family as well. Flow cytometry studies demonstrated the absence of CD36 expression on the surface of blood monocytes and cultured erythroblasts and megakaryocytes from one of the two CD36‐deficient family members studied, but, in the absence of previous immunization, these CD36‐deficient patients were not isoimmunized. In contrast, CD36 deficiency was not found in a population of 808 healthy blood donors in the Paris, France, area.

Platelet autoantibodies and lupus-associated thrombocytopenia

Summary. To characterize the antigenic targets of anti‐platelet antibodies (APA) found in systemic lupus erythematosus (SLE)‐associated thrombocytopenia, 48 patients with immune thrombocytopenia and SLE were compared with 20 patients with SLE who had never been thrombocytopenic. Both cases and controls were tested for circulating APA by an indirect platelet suspension immunofluorescence assay (PSIIFT) and by indirect monoclonal antibody specific immobilization of platelet antigens (MAIPA). A direct platelet suspension immunofluorescence assay (PSIFT) was also used for antibodies bound to platelets in vivo in thrombocytopenic patients; 13 of them with high titres of platelets‐bound APA were investigated by direct and indirect MAIPA and platelet eluate analysis. Circulating APA were detected by PSIIFT in 88% of cases and 55% of controls (P = 0·0066) and platelet‐bound antibodies were detected by PSIFT in 90% of cases. Indirect MAIPA detected specific APA (mainly directed against GpIIbIIIa) in 36% of cases and only 5% of the controls (P = 0·0076). Nine out of the 13 fully investigated thrombocytopenic patients (69%) had a positive direct MAIPA and/or APA detected in platelet eluates. In conclusion, the production of specific anti‐platelet autoantibodies, mainly directed against GpIIb/IIIa, and their binding to platelet membrane plays an important role in the pathogenesis of SLE‐associated thrombocytopenia.

Neutrophil-specific antigen and gene frequencies in the French population

To the Editor: Ohto and Matsuol recently reported the antigen and gene frequencies of the neutrophil-specific antigens NA1, NA2, NB1, and NC1 in the Japanese population. Studies were done on 303 random-donor granulocyte suspensions with a monoclonal anti-NAl and on 78 with polyclonal anti-NA2, -NBl, and -NC1. Two main conclusions were reached: first, that NA1 is more common than NA2 in the Japanese (contrasting with the respective frequencies in the white American population) and, second, that the total gene frequency at the NA locus was less than 1.00, which suggests the existence of a silent allele. We wish to comment on these findings in the light of our results from tests on the French population (Paris area). Random healthy blood donors were phenotyped using polyclonal antisera specific for NA1 and NA2 (n=2668) and NB1 (n = 2193) in the EDTA-dependent granulocyte microagglutination test (GAT). The antisera were locally identified by comparison with reference antisera supplied by Dr. P. Lalezari (New York, NY). No significant differences were observed between antigen or gene frequencies in our population and those reported in a white North American population (Table 1). The frequencies of the NA phenotype are shown in Table 2. The existence of double-blank phenotypes (ix., NA1( ) NA2(-); see Table 2) and the fact that the total NA gene frequency did not reach 1.00 suggest the existence of other alleles at the NA locus in our population, as described elsewhere.lJ To test this hypothesis, new samples from 6 of the 12 donors with the double-blank phenotype were tested with a larger panel of polyclonal antisera in the GAT and in the granulocyte indirect immunofluorescence test (GIIFT),4 and with monoclonal anti-NAl and/or -NA2. These monoclonal antibodies were directed against the NA1 and NA2 antigens present on IgG Fc receptors, as reported a t the Fourth International Workshop and Conference on Human Leucocyte Differentiation Antigens (Vienna 19895) and were supplied by Dr. Albert von dem Borne (Amsterdam, The Netherlands). In these tests, the phenotypes of five of the six donors were shown to be NA1(+) or NA2(+), or both. Only one of the six donors remained NA1( -), NA2( -), but this null phenotype could not be confirmed, as the monoclonal anti-NA2 was not available at that time. We conclude, first, that there are no significant differences between the NA and NB antigen frequencies in the French and white American populations, as determined using polyclonal antisera, and second, that the reactivity of NA antigens varies Table 2. Phenotype frequency (NA system)*

Granulocyte antibody screening: evaluation of a bead-based assay in comparison with classical methods

BACKGROUND: Granulocyte antibodies have been implicated in allo‐ and autoimmune neutropenia and in transfusion reactions.

Early Immunization against Platelet Glycoprotein IIIa in a Newborn Glanzmann Type I Patient

Abstract. Alloimmunization against platelet glycoprotein lib and/or IIIa is a complication rarely observed during the evolution of type I Glanzmanns thrombasthenic patients [1,2]. The occurrence of such alloantibodies is usually due to repeated blood transfusions [2, 3] and greatly complicates the treatment of these patients since they prevent effective platelet transfusion and might, theoretically, cause posttransfusion purpura. We describe the case of a newborn thrombasthenic patient who developed an IgG platelet allo‐antibody 1 month after birth. The diagnosis of Glanzmanns thrombasthenia was complicated by the rare platelet phenotype (PLAt‐negative PLA2‐positive) of the healthy mother, which was probably heterozygous for the abnormal thrombasthenic gene. Immunofluorescence and immunoblotting techniques demonstrated that the patient antibody was principally directed against the platelet glycoprotein IIIa. Surprisingly, this patient had only received four blood transfusions (fresh frozen plasma on days 1 and 2, and standard red blood cell concentrates on days 5 and 6) before the discovery of the antibody, suggesting prior in utero sensitization. This study emphasizes the need for early diagnosis of the disease. Thrombasthenic patients should be transfused with deleukocyted platelet‐free blood products.

Brb, a platelet alloantigen involved in neonatal alloimmune thrombocytopenia.

Abstract. Serum from a pregnant woman with the May‐Hegglin anomaly contained a platelet‐specific antibody. The serum reacted in the platelet indirect immunofluorescence test (PIIFT) with 97.6% of random donor platelets and those of the father but not with the mothers own platelets. This antibody induced a moderate thrombocytopenia in the infant that responded to infusion of intravenous immunoglobulin concentrates. The platelet phenotypes were PLA1+, Baka+, Bra+/Brb‐ for the mother, PLA1+, Baka+, Bra‐/Brb+ for the father, and PLA1+, Bra+/Brb+ for the neonate. Analysis of the maternal serum with an immunoassay based on monoclonal antibody immobilization of platelet antigens (MAIPA) and immunoprecipitation techniques demonstrated the absence of antibodies directed against HLA class I antigens and that the antigen recognized was located on the platelet‐GpIa/IIa complex. This antigen was present on 113/115 random donor platelets, in 7 of the 7 unrelated May‐Hegglin platelets, and only absent in 3/24 Bra+ individuals, including the mother. No platelet‐specific antibody was present in the serum of the 7 unrelated May‐Hegglin subjects. The antigen recognized by this platelet‐specific antibody thus meets the criteria defining the antithetic allele of Bra, i.e. the Brb alloantigen.