Biomedical subjects
Publications and source records attributed to V Kiefel.
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Post-transfusion purpura (PTP) is a rare transfusion reaction almost exclusively observed in female patients. Affected patients develop severe immune-mediated thrombocytopenia in the course of a strong anamnestic alloimmune reaction against a platelet-specific antigen. The pathophysiology of thrombocytopenia has remained elusive. Immunological analysis in the HPA-1a-alloimmunized patient described in this report revealed an antibody with features considered typical of PTP: not only was anti-HPA-1a detectable in plasma, but it could also be eluted from the patients' (alloantigen negative) platelets, and anti-HPA-1a could be detected in eluates from both antigen positive and negative test platelets, which had been incubated in the patient's serum. This is in contrast to two sera with HPA-1a alloantibodies obtained from mothers of children with neonatal alloimmmune thrombocytopenia which were strictly HPA-1a specific. It is proposed that alloantibodies with HPA-1a-like specificity explain the patient's immune thrombocytopenia. The technique described in this report is proposed for further investigation, as it might be useful for discrimination of alloantibodes in PTP and alloantibodies of transfused thrombocytopenic patients.
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Antibody formation against alloantigens of the human platelet membrane is responsible for clinical syndromes and transfusion related conditions as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), platelet transfusion refractoriness (PTR) and passive alloimmune thrombocytopenia. Moreover, rare cases of alloimmune reactions involving platelets have been observed after transplantation of hematopoietic stem cells. Among alloantigens of the platelet membrane shared with other cells (type I alloantigens) are the glycoconjugates of the ABO system and class I human leukocyte antigen (HLA) antigens. Antibodies against these structures are responsible for PTR and for febrile nonhemolytic transfusion reactions. Antibodies against type II antigens (formerly termed "platelet specific antigens") have been observed in NAIT, PTP and passive alloimmune thrombocytopenia. ABH antigens have been identified on intrinsic platelet membrane glycoproteins. Moreover, it is now clear that HLA class I antigens are an integral part of the platelet membrane. The quantity of both HLA and ABH-antigen expression on the platelet membrane varies considerably. Single point mutations account for almost all platelet specific alloantigens, but most antigenic determinants seem to depend upon glycoprotein conformation: generally, platelet specific alloantibodies fail to recognize synthetic peptides encompassing the polymorphic residues. Restriction fragment polymorphism analysis and allele-specific PCR have been implemented for genotyping of platelet alloantigens in many laboratories. Antigen specific assays using monoclonal antibodies (MAIPA, immunobead assay) became de facto standard for diagnosis of platelet antibodies in serum/plasma samples. It can be expected that innovative techniques as human alloantibody fragments produced by phage display technique and the production of recombinant antigens will allow rapid and reliable phenotyping and antibody detection in the future.
Thrombocytopenia is a frequent comorbid condition in many in hospital patients. In some patients, drugs are the cause of low platelet counts. While cytotoxic effects of anti-tumor therapy are the most frequent cause, immune mechanisms should also be considered. This review addresses thrombocytopenias in four groups. Heparin-dependent thrombocytopenia (HIT), by far the most frequent drug-induced immune-mediated type of thrombocytopenia, has a unique pathogenesis and clinical consequences. HIT is a clinicopathological syndrome in which antibodies mostly directed against a multimolecular complex of platelet factor 4 and heparin cause paradoxical thromboembolic complications. The mechanisms through which heparin can enhance thrombin generation are discussed and treatment alternatives for affected patients are presented in detail. It is of primary importance to recognize these patients as early as possible and to substitute heparin with a compatible anticoagulatory drug, such as hirudin, danaparoid or argatroban. Patients seem to benefit from therapeutic doses of alternative treatment rather than from low-dose prophylactic doses. With the increasing use of glycoprotein (GP) IIb/IIIa inhibitors in patients with acute coronary syndromes, thrombocytopenias are increasingly recognized as an adverse effect of these drugs. Up to 4% of treated patients are affected. Most important, pseudothrombocytopenia, a laboratory artefact, is as frequent as real drug-induced thrombocytopenia and must be excluded before changes in treatment are considered. The pathogenesis of these thrombocytopenias is still debated; an immune mechanism involving preformed antibodies is likely. However, since these antibodies are also detectable in a high percentage of normal controls and of patients not developing thrombocytopenia, their impact is still unclear. Patients with real thrombocytopenia are at an increased risk of bleeding; treatment consists of cessation of the GP IIb/IIIa inhibitor and platelet transfusions in cases of severe hemorrhage. Classic immune thrombocytopenia can be induced by some drugs, e.g. gold, which trigger anti-platelet antibodies indistinguishable from platelet autoantibodies found in autoimmune thrombocytopenia. Drug-induced and drug-dependent immune thrombocytopenia is induced by antibodies recognizing an epitope on platelet GP formed after binding of a drug to a platelet glycoprotein. Still unresolved is whether antibody binding is the consequence of a conformational change of the antigen, the antibody, or both. These antibodies typically react with monomorphic epitopes on platelet GP, but only in the presence of the drug or a metabolite. Although several platelet GP have been identified as antibody target (GPIb/IX, GPV, GP IIb/IIIa), antibodies in an individual patient are highly specific for a single GP. Clinically, these patients present with very low platelet counts and acute, sometimes severe, hemorrhage. Treatment is restricted to withdrawal of the drug and symptomatic treatment of bleeding.
BACKGROUND: Patients receiving cellular blood components may form HLA antibodies and platelet-specific alloantibodies. STUDY DESIGN AND METHODS: Serum samples from a cohort of 252 patients with hematologic or oncologic diseases who are receiving cellular blood components were studied for platelet-reactive antibodies. Specificity of platelet alloantibodies was determined with a panel of typed platelets RESULTS: Platelet-reactive antibodies were detected in the sera of 113 patients (44.8% of 252), HLA antibodies in the sera of 108 (42.9%), and platelet-specific antibodies in the sera of 20 (8%). The following platelet-specific antibodies were identified: anti-HPA-5b (n = 10), anti-HPA-1b (n = 4), anti-HPA-5a (n = 2), anti-HPA-1a (n = 1), anti-HPA-2b (n = 1), anti-HPA-1b+5b (n = 1), and anti-HPA-1b+2b (n = 1). Fifteen sera from the 108 patients with anti-HLA (13.9%) contained additional platelet-specific alloantibodies, while in 5 sera, platelet-specific alloantibodies only were detected: anti-HPA-5b (n = 4) and anti-HPA-1a (n = 1). Of the 108 sera with HLA antibodies, 29 (26.9%) showed discordant results when studied with the lymphocytotoxicity test and the glycoprotein-specific immunoassay. Ten sera contained panreactive antibodies against platelet glycoproteins (GP) IIb/IIIa, GPIa/IIa, and/or GPIb/IX. Alloimmunization occurred in 58.3 percent of female patients with previous pregnancies, but in only 23.3 percent of those without previous pregnancies (p = 0.0049). CONCLUSION: Platelet alloantibody specificities in transfused patients (predominantly anti-HPA-5b and -1b with antigen frequencies <30% among whites) differ significantly from those observed in patients with neonatal alloimmune thrombocytopenia or posttransfusion purpura, in whom anti-HPA-1a (antigen frequency >95%) is the most prevalent specificity. HLA antibody detection yields discordant results when the lymphocytotoxicity assay and a glycoprotein-specific immunoglobulin-binding assay are used.
We recently described a new low-frequency platelet alloantigen on the human platelet glycoprotein (GP) Ib-IX complex, termed Iy(a), which was implicated in a severe case of neonatal alloimmune thrombocytopenia. Immunoprecipitation studies with trypsin-treated platelets indicated that the Iy(a) alloantigenic determinants are formed by the membrane-associated remnant moiety of GP Ibalpha (GP Ibalpha(r)) together with GP Ibbeta and GP IX. To elucidate the molecular basis underlying the Iy(a) alloantigen, we amplified GPIbalpha(r), GPIbbeta, and GPIX genes by polymerase chain reaction (PCR). Nucleotide-sequence analysis of these 3 genes showed a G to A transition at position 141 on GPIbbeta gene in a subject positive for Iy(a). This transition resulted in a Gly(15)Glu dimorphism on the N-terminal domain of GPIbbeta. This finding was confirmed by genotyping analysis of 6 Iy(a)-positive subjects by restriction fragment length polymorphism (RFLP) studies using NarI endonuclease. In 300 randomly selected healthy blood donors, one Iy(a)-positive individual was found. Phenotypes determined by monoclonal antibody-specific immobilization of platelet antigens assay and genotypes determined by RFLP were identical in this population. Analysis of Iy(a)-positive platelets showed that the point mutation affected neither the degree of surface expression nor the function of the GP Ibalpha-GP Ibbeta-IX complex on the platelet surface. Transient expression of the GP Ib-IX complex in CHO cells using wild-type GP Ibbeta (Gly(15)) or mutant GP Ibbeta (Glu(15)) allowed us to demonstrate that this single amino acid substitution is sufficient to induce Iy(a) epitope(s). (Blood. 2000;95:1849-1855)
A new platelet-specific alloantigen, termed Sit(a), was identified in a severe case of neonatal alloimmune thrombocytopenia. The Sit(a) alloantigen is of low frequency (1/400) in the German population. Immunochemical studies demonstrated that the Sit(a) epitopes reside on platelet glycoprotein (GP) Ia. Nucleotide sequence analysis of GPIa cDNA derived from Sit(a)-positive platelets showed C(2531)-->T(2531) point mutation, resulting in Thr(799)Met dimorphism. Analysis of genomic DNA from 22 Sit(a)-negative normal individuals showed that the Thr(799) is encoded by ACG(2532) (90.9%) or ACA(2532) (9.1%). To establish a DNA typing technique, we elucidated the organization of the GPIa gene adjacent to the polymorphic bases. The introns (421 bp and 1.2 kb) encompass a 142-bp exon with the 2 polymorphic bases 2531 and 2532. Polymerase chain reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme Mae III showed that the Met(799) form of GPIa is restricted to Sit(a) (+) phenotype. Analysis of stable Chinese hamster ovary transfectants expressing allele-specific recombinant forms of GPIa showed that anti-Sit(a) exclusively reacted with the Glu(505)Met(799), but not with the Glu(505)Thr(799) and the Lys(505)Thr(799) isoforms. In contrast, anti-Br(a) (HPA-5b) only recognized the Lys(505)Thr(799) form, whereas anti-Br(b) (HPA-5a) reacted with both Glu(505)Thr(799) and Glu(505)Met(799) isoforms. These results demonstrated that the Met(799) is responsible for formation of the Sit(a) alloantigenic determinants, whereas amino acid 505 (Lys or Glu) specifically controls the expression of Br(a) and Br(b) epitopes, respectively. Platelet aggregation responses of Sit(a) (+) individuals were diminished in response to collagen, indicating that the Thr(799)Met mutation affects the function of the GPIa/IIa complex.
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Heparin-induced thrombocytopenia (HIT), a severe complication of heparin treatment, can be associated with new thrombotic complications. HIT antibodies activate platelets via the platelet Fcgamma-receptor (FcgammaRIIa), which carries a functionally relevant polymorphism (FcgammaRIIa-R-H131). The effect of this polymorphism on the clinical manifestations of HIT is controversial. We determined prospectively the FcgammaRIIa-R-H131 genotypes in 389 HIT patients, in 351 patients with thrombocytopenia or thrombosis due to causes other than HIT and without detectable HIT antibodies, and in 256 healthy blood donors. For this purpose, a novel nested sequence-specific primer-polymerase chain reaction (SSP-PCR) was developed. FcgammaRIIa-R/R131 was found to be overrepresented in the HIT patients (27%) compared with the control groups (non-HIT patients [21%] and blood donors [20%]). In a subgroup of 122 well-characterized HIT patients, the genotype distribution in patients presenting with thrombocytopenia only was compared with that of patients who developed thromboembolic complications. The frequency of FcgammaRIIa-R/R131 among patients with thrombotic events was significantly elevated (37% v 17%; P = .036). Our results indicate that genotype distribution can be correlated to the clinical outcome of patients with HIT. We speculate that the reduced clearance of immune complexes in patients with the FcgammaRIIa-R/R131 allotype causes prolonged activation of endothelial cells and platelets, thus increasing the risk for thrombotic complications.
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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.
Immune thrombocytopenia due to passive transfer of anti-PI(A1) alloantibody has been noted as a rare but potentially dangerous complication of plasma transfusions. We report a patient with a preoperative platelet count of 241 x 10(9)/l who developed severe thrombocytopenia within 2 hr following transfusion of 2 U of fresh frozen plasma. The plasma donor was found to be a PI(A1)-negative woman. The platelet count of the PI(A1)-positive patient recovered within 7 days to normal values. In the frozen plasma, excessive antibody binding to GPIIb-IIIa on the recipient's platelets was detected. The antibody was shown to have anti-PI(A1)-specificity. Only 40 min after transfusion of the frozen plasma, no antibody was detected in the plasma of the recipient. This case suggests that passively administered anti-PI(A1) alloantibody is immediately adsorbed onto the recipient's platelets and thus removed from circulation.
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.
BACKGROUND AND OBJECTIVES: Many drugs are associated with thrombocytopenic purpura through immune-mediated platelet destruction. The case of a woman who suffered life-threatening thrombocytopenia during vancomycin treatment for Staphylococcus aureus septicemia is reported. MATERIALS AND METHODS: Conventional clinical and laboratory methods, including flow cytometry. RESULTS: After treatment of septicemia with vancomycin, severe thrombocytopenia and bleeding occurred, without detection of drug-dependent platelet antibodies (DDPA). This was followed by vegetative endocarditis, whereupon antibiotics were withdrawn so as to isolate the organism. The thrombocytopenia was corrected. On day 34, antibiotics including vancomycin were reinstituted, and three days later thrombocytopenia recurred. With a change in antibiotics, the platelet count corrected itself within four days. CONCLUSIONS: Vancomycin may induce potentially severe immunological thrombocytopenia.
Fetal alloimmune thrombocytopenia is caused by maternal immunization against a fetal platelet antigen and transplacental transfer of the antibody into the fetal circulation. Since 10-20% of the fetuses or newborns are threatened by intracranial hemorrhages, early management is required. Fetal blood sampling should be started between the 20th and 22nd week of gestation to assess fetal phenotype and platelet count. Different concepts to elevate the fetal platelet count have been discussed: maternal intravenous immunoglobulins, fetal intravenous immunoglobulins, or only repeated fetal platelet transfusions. Our investigations suggested that platelet transfusions in short intervals appear to be the only effective regimen to increase platelet counts in thrombocytopenic fetuses at risk.
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