S. Santoso

Autoantibodies against platelet glycoprotein Ib/IX: a frequent finding in autoimmune thrombocytopenic purpura

Summary Many autoantibodies involved in the pathogenesis of autoimmune thrombocytopenic purpura (AITP) are directed against epitopes on platelet glycoproteins (GP). These autoantibodies are a specific diagnostic characteristic of patients with AITP. In this study, the relative frequency of antibodies against GPs IIb/IIIa and Ib/IX was assessed in sera from 81 AITP patients with a glycoprotein‐specific enzyme immunoassay (MAIPA assay) using monoclonal antibodies against these platelet GPs. All sera contained platelet‐specific antibodies which had been detected by platelet immunofluorescence. Of the 81 antibodies tested, 58 (72%) reacted with at least one of the platelet GPs studied. Autoantibodies against GPIb/IX were as common as antibodies against the GPIIb/IIIa complex. The same ratio of specificities was observed on autologous platelets of an independent cohort of 29 patients. The epitope of three autoantibodies against GPIb/IX and of mab Gi10, a monoclonal antibody, which inhibits binding of these autoantibodies, was further characterized. Severity of thrombocytopenia was not related to the GP specificity of the autoantibody. The observation that in 23 (28%) of these sera the antigenic determinants could not be assigned to the glycoproteins under investigation suggests that platelet autoantibodies may react with other GPs or other membrane constituents, e.g. glycolipids.

Prospective evaluation of PF4/heparin immunoassays for the diagnosis of heparin-induced thrombocytopenia.

Summary.  Background: Heparin‐induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies that recognize platelet factor 4‐heparin (PF4/hep) complexes leading to platelet activation. Several methods are available for the identification of HIT antibodies. Objectives: To evaluate the clinical usefulness of different antigen‐binding assays for detection of antibodies against PF4/hep complexes in a prospective study. Patients/methods: A prospective cohort of 500 surgical and medical patients suspected of having HIT was evaluated. The laboratory assessment included particle gel immunoassay (PaGIA), polyspecific ELISA recognizing IgG, IgM and IgA antibodies (Poly‐ELISA), IgG‐specific ELISA (IgG‐ELISA) and the HIPA test. The pretest probability of HIT was determined using the 4T’s model. Positive and negative predictive values (PPV, NPV) of each immunoassay were determined depending upon the heparin‐induced platelet activation (HIPA) results and the clinical scoring. The operating characteristics of each immunoassay were determined using the receiver‐operation characteristic (ROC) curve. Results: Platelet‐activating antibodies were identified in 35/500 patients, all of whom had intermediate to high clinical probability. PF4/hep antibodies were detected in 124, 86 and 90 sera using Poly‐ELISA (PPV = 28), IgG‐ELISA (PPV = 40.6) and PaGIA (PPV = 36.6). NPV was 100% for Poly‐ and IgG‐ELISA, but only 99.5% for PaGIA. ROC analysis revealed that PaGIA is less informative than ELISA. The IgG‐ELISA provides better diagnostic information than the other assays. In addition, there is a clear correlation between optical density (OD) value and the probability of having HIT. Conclusions: Our observation indicates that an IgG‐ELISA provides the best diagnostic information of all antigen‐binding assays.

Clinical Aspects and Typing of Platelet Alloantigens

Platelet alloantigens can induce the formation of corresponding alloantibodies when exposed to phenotypically negative individuals. These antibodies are responsible for fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, passive alloimmune thrombocytopenia and transplantation‐ associated thrombocytopenia and may contribute to platelet transfusion refractoriness together with HLA antibodies. Besides antibody detection laboratory diagnosis of the clinical syndromes requires alloantigen typing. Furthermore, typed platelet donors are a prerequisite for effective platelet transfusion therapy. Different techniques for phenotyping are well established and easy to perform but they rely on the availability of antisera. Since the molecular genetic background of the clinically most relevant alloantigens has been elucidated during the last years various genotyping methods have been applied to the platelet membrane polymorphisms and thus facilitated widespread platelet alloantigen typing. Generation of antibodies from phage display libraries and of lymphoblastoid cell lines from donors with all genetic variants will allow further developments.

A New Platelet-Specific Alloantigen Bra

Abstract. Sera obtained from 4 mothers of children with neonatal alloimmune thrombocytopenia contained a platelet‐specific alloantibody, anti‐Brawhich defined an antigen apparently different from all known platelet alloantigens. All 4 fathers were Brapositive, whereas all mothers were Branegative. The minimal postnatal values of platelet counts ranged from 19×109to 75×109/1. Family studies showed that the Braantigen is inherited as an autosomal, codominant trait. Its antigen frequency in the German population is 20% (21 of 105 unrelated donors were positive). The estimated gene frequency is 0.11. The antibodies were identified by a glycoprotein‐specific enzyme immunoassay using monoclonal antibodies for antigen immobilization, while they could not reliably be detected by binding assays employing whole platelets (platelet immunofluorescence, indirect competitive ELISA).

Genomic RFLP typing of human platelet alloantigens Zw(PlA), Ko, Bak and Br (HPA-1, 2, 3, 5).

Summary. The diallelic human platelet alloantigen systems 1–5 have been found to result from single base pair substitutions in the encoding genes of platelet membrane glycoproteins IIIa, Ib, IIb and Ia. This is the basis of DNA methods for determination of platelet alloantigens. In this study, 98 blood donors were typed in the HPA‐1, 2, 3 systems and, for the first time, in the HPA‐5 system. Serologically obtained data (MAIPA and platelet agglutination) were compared with results from analysis of restriction fragment length polymorphisms (RFLP). Discordances were found in the HPA‐2 and 3 systems and can be ascribed to false typing results in both the serological and genomic methods. In the HPA‐1, 2 and 5 systems, all samples were typed correctly with RFLP analysis. Serologically, two donors were falsely typed positive with anti‐HPA‐2b in platelet agglutination and one donor with anti‐HPA‐3a in MAIPA assay. In the HPA‐3 system, another four donors were misinterpreted to be HPA‐3 a negative in RFLP analysis. Possible technical problems in PCR‐RFLP‐typing are discussed and another strategy of HPA‐1 typing using the restriction enzyme Scr FI is evaluated.

Human Platelet-Specific Alloantigens: Update

Platelet alloantigens and their respective alloantibodies play an important role in immune-mediated platelet disorders. There are essentially three clinical conditions caused by platelet alloantibodies: Neonatal alloimmune thrombocytopenia (NAIT), posttransfusion purpura (PTP) and platelet transfusion refractoriness (1). In addition, two rare alloimmune thrombocytopenia syndromes were described: passive alloimmune thrombocytopenia (2) and transplantation-associated thrombocytopenia (3). In the past two decades considerable progress has been made in the characterization of platelet alloantigens. The use of serological antigen capture assays such as the monoclonal antibody immobilization of platelet antigens (4) and immunochemical methods led to a rapid increase in the number of newly recognized platelet alloantigens. The introduction of PCR technology for amplification of platelet specific mRNA (5 ) made the characterization of platelet alloantigens possible at the molecular level. Based on the molecular genetic basis of platelet alloantigens a number of genotyping techniques could be developed. In this chapter, we will briefly review the progress of the molecular genetics and the structure of human platelet alloantigens. Platelet alloantigenslantibodies

Metabolite‐specific (IgG) and drug‐specific antibodies (IgG, IgM) in two cases of trimethoprim‐sulfamethoxazole‐induced immune thrombocytopenia

Two cases of trimethoprim‐sulfamethoxazole (TMP‐SMX)‐induced immune thrombocytopenia are reported in which unusual drug‐dependent platelet antibodies were demonstrated by immunofluorescence and enzyme‐linked immunosorbent assay. Whereas two distinct sulfamethoxazole‐dependent antibodies of the IgG and IgM class were detectable in the serum of one patient, the serum of the other patient contained a platelet antibody exclusively reactive with N‐4‐acetyl‐sulfamethoxazole, a metabolite of sulfamethoxazole. Urine from a healthy volunteer collected after administration of therapeutic doses of TMP‐SMX proved to be an appropriate source of ex vivo metabolites for antibody testing. The results of this study stress the role of metabolite‐specific antibodies in drug‐dependent immune thrombocytopenia and underscore the necessity of including metabolite preparations of drugs in serologic analyses.

Frequency of platelet-specific antigens among Indonesians.

This study reports the frequencies of the alloantigens of four major platelet‐specific alloantigen systems among Indonesians. One hundred and sixty‐eight unrelated Indonesian blood donors were phenotyped for the alloantigens of the Zw (PIA, HPA‐1), Bak (HPA‐3), Yuk (Pen, HPA‐4), and Br (HPA‐5) systems by use of a glycoprotein‐specific immunoassay. All were positive for the alloantigens Zwa, Yukb, and Brb. Three (1.79%) and 1 (0.59%) of the 168 donors were positive in testing for Zwb and Yuk(a) antigens, respectively. Fifteen (9.26%) of 162 Indonesians had Br(a) antigens. Of the 166 donors tested, 121 (72.89%) were Bak(a) positive and 134 (80.72%) were Bakb positive. In addition, the phenotype frequency of Nak(a) was determined by using monoclonal antibody OKM5 in a platelet enzyme‐linked immunosorbent assay. Its frequency in the present cohort was 95.83 percent (161/168). This study confirms the differences in platelet antigen distributions in Asians and whites. Both glycoprotein IV deficiency and the Yuk polymorphism are also found among Indonesians.

Neonatal alloimmune thrombocytopenia due to fetomaternal Zwb incompatibility.

Abstract. A case of neonatal alloimmune thrombocytopenia due to fetomaternal incompatibility against the platelet‐specific antigen Zwb (P1A2) is described. The antibody was of the IgG class and did not fix complement in vitro. Its specificity was established by panel identification and by comparison to a Zwb antiserum. Gene dosage determinations of Zwa antigens on platelets of parents and child revealed that the mother was homozygous Zwa Zwa while father and child were heterozygous for this antigen.

Platelet transfusion refractoriness associated with two rare platelet-specific alloantibodies (anti-Baka and anti-PlA2) and multiple HLA antibodies

A 65‐year‐old patient with pancytopenia resulting from osteomyelosclerosis became refractory to platelet transfusions during long‐time transfusion support. He developed two rare, platelet‐specific antibodies (anti‐PlA2 and ‐Baka) disguised by strong, multispecific HLA antibodies. The specificity of the platelet‐specific antibodies was detected by a newly designed enzyme‐linked immunosorbent assay using glycoprotein‐specific monoclonal antibodies for immobilization of platelet antigens.