| 1. |
- Forsberg, B, et al.
(författare)
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The platelet-specific alloantigen PlA1 (HPA-1a): a comparison of flow cytometric immunophenotyping and genotyping using polymerase chain reaction and restriction fragment length polymorphism in a Swedish blood donor population.
- 1995
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Ingår i: Transfusion. - 0041-1132. ; 35:3, s. 241-6
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: There is an increasing interest in the development of rapid and reliable techniques for platelet alloantigen typing. STUDY DESIGN AND METHODS: By use of standardized flow cytometry and a specific human alloantiserum, 236 Swedish blood donors were immunophenotyped for the platelet-specific alloantigen, PlA1 (HPA-1a). RESULTS: Ten individuals (4.2%) had low fluorescence intensities and were considered PlA1-negative (HPA-1a-negative); all of them also demonstrated a PlA2/PlA2 (HPA-1b/1b) genotype in a polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay of the underlying DNA polymorphism. The remaining population had clear positive fluorescence and was regarded as PlA1-positive (HPA-1a-positive). The fluorescence distribution histogram among PlA1-positive (HPA-1a-positive) individuals was dome-shaped, and those individuals who were homozygous for PlA1 (HPA-1a) could not be distinguished from those who were heterozygous. This finding was further substantiated by PCR-RFLP analysis of the PlA1/PlA2 (HPA-1a/1b) genotype; a heterozygous genotype was found among those having a medium fluorescence intensity as well as among those having a strong fluorescence intensity. CONCLUSION: Flow cytometry is a valuable tool for large-scale detection of PlA1 (HPA-1a). However, flow cytometry based on only one antiserum cannot distinguish between homozygous and heterozygous carriers of PlA1 (HPA-1a). For zygosity testing and when platelets are difficult to obtain, the PCR-RFLP technique is the assay of choice.
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| 2. |
- Stockelberg, Dick, 1950, et al.
(författare)
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Evidence for a light chain restriction of glycoprotein Ib/IX and IIb/IIIa reactive antibodies in chronic idiopathic thrombocytopenic purpura (ITP).
- 1995
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Ingår i: British journal of haematology. - 0007-1048. ; 90:1, s. 175-9
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Tidskriftsartikel (refereegranskat)abstract
- To address the assumption of clonally restricted antibodies in immune thrombocytopenias we studied sera from 19 patients with chronic ITP known to possess antibodies reactive with glycoprotein (GP) Ib/IX and/or GPIIb/IIIa. These sera were re-analysed using the standard monoclonal antibody immobilization of platelet antigens (MAIPA) assay and 16 patients exhibited IgG antibodies reactive with GPIIb/IIIa; seven patients showed also a reactivity with GPIb/IX. Employing a light-chain-specific MAIPA assay, 75% (12/16) of these sera displayed GPIIb/IIIa-specific antibodies that were light chain restricted; only 13% (2/16) of the GPIIb/IIIa reactive sera showed a mixed kappa and lambda phenotype. A light-chain-restricted phenotype was also seen for the GPIb/IX reactive antibodies. To further substantiate these findings, the MAIPA assay was modified in order to avoid interference from human anti-mouse antibodies. A high frequency of light-chain restricted platelet antibodies was also found using the modified MAIPA technique. These results support the hypothesis of a clonal B-cell expansion in immune thrombocytopenias, producing antibodies with a restricted idiotype repertoire and reacting with a limited number of epitopes.
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| 3. |
- Hou, M, et al.
(författare)
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Antibodies against platelet GPIb/IX, GPIIb/IIIa, and other platelet antigens in chronic idiopathic thrombocytopenic purpura.
- 1995
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Ingår i: European journal of haematology. - 0902-4441. ; 55:5, s. 307-14
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies involved in the pathogenesis of chronic idiopathic thrombocytopenic purpura (ITP) react most frequently with platelet glycoprotein (GP) Ib/IX and GPIIb/IIIa. However, uncertainty as to the specificity, frequency, and clinical significance of such antibodies still remains. By using a modified antigen-capture ELISA (MACE), an immunoprecipitation assay, and an immunoblot assay, sera from 60 patients with chronic ITP were analyzed. GP-specific antibodies were found in 50% (30/60) of the patients, with 14 patients having antibodies directed solely to GPIIb/IIIa, 8 holding antibodies specific only for GPIb/IX, and 8 possessing antibodies against both antigens. Serum antibodies were more frequently (p < 0.01) detected in either active and/or non-splenectomized ITP patients. Moreover, in patients displaying antibodies against GPIb/IX, significantly (p < 0.05) lower platelet counts were observed. Using the immunoblot assay, antibodies specific for a 30 kD platelet antigen were detected in 12 of 60 patients. This antigen could not be immunoprecipitated from surface labelled platelet membranes, indicating an intracellular location. We conclude that in chronic ITP, (1) the frequency of anti-GPIIb/IIIa antibodies is close to that of anti-GPIb/IX antibodies, (2) anti-GP antibodies are more likely to be detected in patients with an active disease status and, (3) a 30 kD internal platelet protein is another frequent antigen.
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| 4. |
- Hou, M, et al.
(författare)
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Fab-mediated binding of glycoprotein Ib/IX and IIb/IIIa specific antibodies in chronic idiopathic thrombocytopenic purpura.
- 1995
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Ingår i: British journal of haematology. - 0007-1048. ; 91:4, s. 944-50
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Tidskriftsartikel (refereegranskat)abstract
- The antibody domain responsible for the interactions between platelet glycoproteins (GP) and serum IgG autoantibodies in patients with chronic idiopathic thrombocytopenic purpura (ITP) was studied. Sera from nine non-transfused ITP patients and 20 normal controls and a serum containing an anti-PlA1 antibody were employed. Serum, purified IgG and F(ab')2 fragments were prepared and their binding to platelet GPIb/IX and GPIIb/IIIa were analysed using a modified MAIPA assay and an antigen capture ELISA. In all experiments most of the autoantibodies studied behaved identically to the anti-PlA1 antibody in that the IgG-F(ab')2 fragments retained their ability to bind to the respective glycoprotein. Substituting the enzyme-conjugated secondary antibody (Fab specific), in the MAIPA assay, with an Fc specific antibody removed all reactivities observed against platelet GPs, produced by IgG-F(ab')2 fragments. Furthermore, in an antigen-capture ELISA, IgG autoantibodies against platelet GPIb/IX and/or GPIIb/IIIa were blocked preferentially by pre-incubating the ITP sera with a goat anti-human IgG (F(ab')2 specific) antibody, but not with an anti-Fc antibody. We conclude that these ITP patients produced antibodies specific for platelet GPIb/IX and/or GPIIb/IIIa, and that the autoantibody-platelet interaction was mediated by the classic Fab binding.
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| 5. |
- Hou, M, et al.
(författare)
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Glycoprotein IIb/IIIa autoantigenic repertoire in chronic idiopathic thrombocytopenic purpura.
- 1995
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Ingår i: British journal of haematology. - 0007-1048. ; 91:4, s. 971-5
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Tidskriftsartikel (refereegranskat)abstract
- The objective of the present study was to further disclose the autoantigenic repertoire carried by the platelet glycoprotein (GP) IIb/IIIa complex. IgG-F(ab')2 fragments were prepared from two prototype ITP patients, and their ability to block the binding of GPIIb/IIIa reactive antibodies derived from other patients with ITP was evaluated using a modified MAIPA asay; a PlA1 alloantiserum and 20 normal sera were included as controls. It was found that the two prototype IgG-F(ab')2 fragments were each able to significantly block the binding of serum IgG to GPIIb/IIa in six (55%) and seven (64%) out of 11 patients with chronic ITP, respectively. No significant blocking effect was observed for IgG-F(ab')2 fragments prepared from normal subjects. Also, the binding of the PlA1 alloantiserum to its epitope on GPIIIa was not affected by any of the blocking IgG-F(ab')2 fragments exploited in the study. These data substantiate that in chronic ITP at least half of the GPIIb/IIIa reactive sera bind to homogenous autoepitopes.
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