Thrombocytopenia induced by vancomycin-dependent platelet antibody.

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 This was followed by vegetative endocarditis, whereupon antibiotics were withdrawn so as to isolate the organism.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.

[Intrauterine transfusion in fetal alloimmunothrombocytopenia: comparison of maternal and fetal weight-adjusted IgG therapy with exclusive fetal thrombocyte transfusion]. / Intrauterine Hämotherapie bei fetaler Alloimmunthrombozytopenie: Vergleich der maternalen und fetalgewichtadaptierten IgG-Therapie mit ausschliesslicher Thrombozytentransfusion des Feten.

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.

Diclofenac-induced immune haemolytic anaemia: simultaneous occurrence of red blood cell autoantibodies and drug-dependent antibodies.

During the last 5 years we have identified a total of 17 patients (nine females and eight males aged between 53 and 85 years) with immune haemolytic anaemia related to diclofenac (a nonsteroidal anti-inflammatory drug). All patients developed acute intravascular haemolysis. Two patients died, and eight patients developed temporary renal failure that required haemodialysis. The direct antiglobulin test was positive with anti-IgG and anti-C3d in all cases, with anti-IgA in 4/10 cases tested, and negative with anti-IgM. The indirect antiglobulin test was moderately or weakly positive in 11 cases, and IgG autoantibodies could be eluted from the red blood cells (RBCs) of all patients. Initially, the diagnosis of autoimmune haemolytic anaemia of warm type was suggested in all cases. All patients had simultaneously developed autoantibodies and drug-dependent antibodies. The majority of drug-dependent antibodies (n = 13) reacted with urine containing the drug and its metabolites (ex vivo antigen), the native drug, and diclofenac-treated RBCs. The antibodies in the remaining four cases were detectable only in the presence of ex vivo antigen. Diclofenac appears to bind only weakly to RBCs in the absence of the drug-dependent antibodies. We conclude that diclofenac forms neoantigens with RBCs that may stimulate the production of autoantibodies and drug-dependent antibodies. The resulting haemolytic syndrome is very similar to autoimmune haemolytic anaemia of warm type.

Therapy with intravenous immunoglobulin G (ivIgG) during pregnancy for fetal alloimmune (HPA-1a(Zwa)) thrombocytopenic purpura.

We have evaluated the effect of maternal intravenous immunoglobulin G (ivIgG) treatment on platelet counts in fetal alloimmune thrombocytopenia. Seven patients were studied. All of them were multiparous women who had been immunized against the HPA-1a antigen during previous pregnancies and had given birth to at least one severely thrombocytopenic infant. In this study, umbilical blood collection was performed first at the 20th week of gestation and repeated 2-13 times (mean 6 times), depending on the degree of fetal thrombocytopenia. Fetal platelet counting was combined with intrauterine transfusion of 20-30 ml of HPA-1a-negative platelet concentrates to prevent bleeding following umbilical cord puncture. Initial fetal platelet counts ranged from 10,000 to 91,000 per microliters. Maternal treatment with ivIgG (1 g per kg body weight; mean dose 70 g) was given once a week over 7 weeks. In five of seven cases, the basal platelet count did not rise and in two of these cases, it decreased during maternal ivIgG treatment. In one fetus, the baseline platelet count increased from 10,000 to 35,000 per microliters during ivIgG, and in another fetus from 23,000 to 64,000 per microliters. Our observations suggest that ivIgG has no definite benefit for fetal alloimmune thrombocytopenia. Since platelet counts can be very low, careful fetal monitoring by umbilical blood sampling is required. Frequent platelet transfusions in short intervals may be necessary to increase platelet counts in extremely thrombocytopenic fetuses.

Platelet autoantibodies (IgG, IgM, IgA) against glycoproteins IIb/IIIa and Ib/IX in patients with thrombocytopenia.

Autoimmune thrombocytopenic purpura (AITP) is most frequently induced by platelet-specific autoantibodies against epitopes on platelet GP Ib/IX or GP IIb/IIIa. These antibodies are reliably detected on the patients' autologous platelets. So far, studies on the characterization of platelet autoantibodies have been restricted to IgG antibodies. We used the monoclonal antibody immobilization of platelet antigens assay (MAIPA) in a modified version to detect GP-specific IgG, IgM, and IgA antibodies. Platelets of 46.2% of patients carried elevated amounts of IgG antibodies. IgM and IgA antibodies were observed less frequently and showed only weak OD signals in the MAIPA assay. Circulating IgG antibodies in serum were found in 11.5% of patients. Circulating IgM autoantibodies were observed in 8.9% and IgA antibodies in no patient with AITP. Results of direct MAIPA assay were compared with the reactivity of eluates in the platelet adhesion immunofluorescence assay and were found to be highly concordant. Patients with AITP in remission carried high percentages of anti-GP IIb/IIIa. Findings made in this study suggest that autoantibodies of the IgM and IgA classes play only a minor role in the pathogenesis of AITP.

[Prenatal management of fetuses with alloimmune thrombocytopenia]. / Pränatale Betreuung von Feten mit Alloimmunthrombozytopenie.

Fetal alloimmune thrombocytopenia is caused by maternal immunization against a fetal platelet alloantigen and transplacental transfer of the antibody into the fetal circulation. Since 10-20% of the fetuses or newborns are threatened by intracranial hemorrhages (ICH) early management is required. Intensive prenatal monitoring should be performed if a maternal HPA-1a antibody is known and a previous infant suffered from thrombocytopenia and/or ICH. Fetal blood sampling (FBS) should be started at 20th to 22nd weeks of gestation to assess fetal phenotype and platelet count. Different concepts to elevate the fetal platelet count have been discussed: corticosteroids, maternal intravenous immunoglobulins (ivIgG), fetal ivIgG and repeated fetal platelet transfusions. In a European survey with data from five centres maternal corticoid treatment and ivIgG infusion were accompanied by increasing fetal platelet counts in only 20 and 24% of the cases, respectively. In fetuses with very low platelet counts only transfusions of compatible platelets in short intervals are able to sustain a safe platelet count. Fetuses with mild thrombocytopenia should be monitored by subsequent FBS since it could be shown that platelet counts tend to decline during gestation. To avoid bleeding complications during and after FBS which was observed in about 5% of the cases every cord vessel puncture should be covered by a platelet transfusion. As no safe and non-invasive therapy exists for fetal alloimmune thrombocytopenia the value of prenatal screening programs in unaffected pregnancies is questionable.

[Fetal weight-adjusted intrauterine IgG therapy in neonatal alloimmune thrombocytopenia]. / Fetalgewichtadaptierte intrauterine IgG-Therapie bei neonataler Alloimmunthrombozytopenie.

Fetal alloimmune thrombocytopenia is caused by materno-fetal transfer of platelet antibodies. Since the thrombocytopenic fetus is threatened by intracranial hemorrhage, prenatal observation and, if necessary, treatment is required. However, the benefit of therapeutic options, including intravenous IgG (ivIgG), platelet transfusions or fetal IgG transfusions is still controversial. In this study we have evaluated the effect of intrauterine IgG and intraumbilical platelet transfusions on fetal platelet counts. All patients were multiparous women who were immunized against the Zwa antigen during previous pregnancies and had given birth to at least one severely thrombocytopenic infant. First umbilical blood was sampled at the 20th week of gestation. Fetal treatment of IgG was given, on av erage, over 9 weeks. In all cases, fetal IgG levels rose significantly whereas platelet counts did not increase following fetal IgG treatment. We conclude that fetal IgG infusions have no detectable effect on fetal allo-immune thrombocytopenia. Since platelet counts can be very low as early as 20 weeks of gestation, careful fetal monitoring by umbilical blood sampling is essential. Platelet transfusions in short intervals appear to be the only effective regimen to increase platelet counts in thrombocytopenic fetuses at risk.

Characterization of the structural requirements for a carbohydrate based anticoagulant with a reduced risk of inducing the immunological type of heparin-associated thrombocytopenia.

HAT is the most frequent drug induced immune-thrombocytopenia. We recently identified multimolecular PF4/heparin complexes as the major antigen. In order to evaluate the structural requirements for formation of the antigenic complex, we chemically synthesized 13 glucan sulfates and used 5 heparin fractions (2.4-4.8 kD) and a synthesized pentasaccharide, representing the antithrombin III binding sequence of heparin, for further characterize the HAT antigen. In the presence of glucan sulfates and heparin, HAT antibodies caused platelet activation typically at low but not at high concentrations, as measured by 14C-5HT release. The concentration range giving the activation pattern depended on the degree of sulfation (DS) and molecular weight (MW) of the glucan sulfates but not on the type of glycosidic linkage of a polysaccharide. With linear glucan sulfates with a chain length of 35 monosaccharides, the critical DS to form the HAT antigen ranged between 0.60 and 1.20. Glycosidic branched glucan sulfates were able to form the HAT antigen at a lower DS and a lower MW than linear glucan sulfates. Platelet activation by HAT-antibodies in the presence of linear curdlan sulfate fractions was dependent on their MW. At a low concentration (0.01 microM) medium-size fractions (60 kD) caused platelet activation but neither small (12 kD) nor large fractions ( > 150 kD) did. At higher concentrations (2 microM) the opposite reaction pattern was observed. In the case of heparin, the optimal chain length for forming the HAT antigen is a hexadecasaccharide (4.8 kD). Antigen generation decreased with larger and smaller fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Discovering objects in a blood recipient information system.

Application of object-oriented (OO) methodologies has been generally considered as a solution to the problem of improving the software development process and managing the so-called software crisis. Among them, object-oriented analysis (OOA) is the most essential and is a vital prerequisite for the successful use of other OO methodologies. Though there are already a good deal of OOA methods published, the most important aspect common to all these methods: discovering objects classes truly relevant to the given problem domain, has remained a subject to be intensively researched. In this paper, using the successful development of a blood recipient information system as an example, we present our approach which is based on the conceptual framework of responsibility-driven OOA. In the discussion, we also suggest that it may be inadequate to simply attribute the software crisis to the waterfall model of the software development life-cycle. We are convinced that the real causes for the failure of some software and information systems should be sought in the methodologies used in some crucial phases of the software development process. Furthermore, a software system can also fail if object classes essential to the problem domain are not discovered, implemented and visualized, so that the real-world situation cannot be faithfully traced by it.

Genotyping of the granulocyte-specific NA antigens from small quantities of blood or serum.

To avoid the well-known shortcomings of phenotyping granulocytes for the NA antigens using NA-specific human sera, a DNA-based method to determine the NA genotype was developed. Genomic DNA was isolated from blood cells or serum, amplified by polymerase chain reaction (PCR), immobilized on nylon membrane and genotyped using digoxigenin-labeled, sequence-specific oligonucleotides (SSO). The genotyping results of whole blood samples from 54 and of serum from 20 individuals correlated perfectly with our phenotyping using the antigen capture assay MAIGA. In three cases with the phenotype "NA-null" no hybridization of the NA-specific oligonucleotides occurred. These data show that SSO is a reliable method for NA genotyping especially if only small volumes of blood or even only serum probes are available.

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

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-3a 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.

NA gene frequencies in the German population, determined by polymerase chain reaction with sequence-specific primers.

BACKGROUND: The granulocyte antigens NA1 and NA2 are often targets of granulocyte antibodies causing immune neutropenia. Currently, NA typing relies on the properties of the typing sera or antibodies and the techniques used. Therefore, the technique of polymerase chain reaction with sequence-specific primers (PCR-SSP) was adapted for DNA-based NA typing and was used for determining the NA gene frequencies in the German population. STUDY DESIGN AND METHODS: The genomic DNA of 160 unrelated healthy individuals was typed for NA1 and NA2 by PCR-SSP. In 60 granulocyte samples, the NA phenotype was additionally determined by the antigen capture assay and the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results. RESULTS: Results of the antigen capture assay and PCR-SSP correlated precisely, whereas nine individuals were typed heterozygous only by GIFT. The gene frequencies were 0.35 for NA1 and 0.65 for NA2. CONCLUSION: The NA2 gene is more frequent in the German population than the NA1 gene, as determined by genotyping using PCR-SSP. In contrast to GIFT, which showed an error rate for NA typing of 15 percent, PCR-SSP and the antigen-capture assay are more reliable methods of NA typing of granulocytes.

Evidence that the granulocyte-specific antigen NC1 is identical with NA2.

The neutrophil-specific antigen NC1 is defined by an antibody in the serum of a mother who gave birth to a child with alloimmune neonatal neutropenia. NC1 has been reported to be associated with the neutrophil-specific antigen NA2, but the precise relation of NC1 and NA2 remained unclear. Therefore, we investigated the serum using the antigen capture assay MAIGA and the granulocyte (GIFT) and lymphocyte (LIFT) immunofluorescence tests. In GIFT, no NA association was observed. In LIFT, serum antibodies bound preferably to lymphocytes with the HLA antigens HLA-B7 and cross-reacting antigens. In MAIGA, an antibody specific for the NA2 variant of the granulocyte Fc gamma-receptor III was observed. The NA2 specificity was confirmed by testing granulocytes from 40 further different donors. This indicates that the NC1 and NA2 antigens are identical. A positive GIFT result but a negative one in LIFT using cells of an NA2-negative typed individual suggest the presence of an additional, non-NA2-specific granulocyte antibody.

Alloimmunization against Iy, a low-frequency antigen on platelet glycoprotein Ib/IX as a cause of severe neonatal alloimmune thrombocytopenic purpura.

Neonatal alloimmune thrombocytopenia (NAIT) is usually induced by platelet-specific antibodies against HPA-1a (Zwa) or HPA-5b (Bra). Recently, low-frequency alloantigens on the platelet glycoprotein (GP) IIb/IIIa complex have been discovered as a cause for NAIT. In this report, a new low-frequency platelet-specific alloantigen, Iy, is described which induced severe NAIT. The corresponding antigen was detected in 1/249 unrelated German blood donors. Antibody binding assays with trypsin-digested platelets (ELISA, immunoprecipitation with biotin-labelled platelets) indicate that the antigen is not localized on the glycocalicin moiety of GP Ib alpha, but may be situated on the remnant moiety of GP Ib alpha, GP IX or GPIb beta. Apparently, Iy is not related to the HPA-2 (Ko) antigen system.

Maternal intravenous immunoglobulin treatment does not prevent intracranial haemorrhage in fetal alloimmune thrombocytopenia.

In fetal alloimmune thrombocytopenia (FAIT) the fetus is threatened by intracranial haemorrhage (ICH); therefore early diagnostic and therapeutic intervention is required. We followed the clinical course of a 30-year-old woman during her fifth pregnancy after she had given birth to a child with alloimmune thrombocytopenia due to anti-Zwa. The fetus was monitored by 13 fetal blood samplings (FBS) always followed by transfusion of either maternal or compatible donor platelets. Intravenous immunoglobulin (ivIg) treatment of the mother was begun at 20 weeks of gestation when the fetal platelet count was 36 x 10(9)/l. The fetal platelets were typed Zwa positive by DNA analysis. Despite 11 weeks of maternal ivIg treatment fetal platelet counts progressively declined to 6 x 10(9)/l and ICH occurred. Subsequently, the fetus was successfully managed by intrauterine platelet transfusions at shorter intervals (3-5 days) and elective Cesarean section was carried out at 35 weeks of gestation. We conclude that maternal ivIg treatment does not prevent ICH in FAIT. The treatment of choice for severely affected cases is serial FBS combined with transfusion of compatible platelets.

Alloimmune neonatal neutropenia resulting from immunization to a high-frequency antigen on the granulocyte Fc gamma receptor III.

BACKGROUND: Alloimmune neonatal neutropenia is mainly caused by NA- or NB1-specific alloantibodies. An antibody in the serum of a Turkish mother who had given birth to an infant with alloimmune neonatal neutropenia showed no NA or NB specificity and was therefore investigated further. STUDY DESIGN AND METHODS: The number of antibody-binding sites was calculated by determination of elutable IgG from granulocytes using a quantitative sandwich enzyme-linked immunosorbent assay. Complement activation was tested by immunofluorescence (C3d) and cytotoxicity tests. The antigen was identified using the antigen-capture assay, monoclonal antibody-specific immobilization of granulocyte antigens, and a modified immunoprecipitation method based upon biotinylation of proteins and visualization by luminescence (luminoimmunoprecipitation). Family study and determination of antigen frequency were done by immunofluorescence and agglutination tests. RESULTS: A noncytotoxic, granulocyte-specific alloantibody that recognized the Fc gamma receptor III, independent of the NA phenotype, was detected, and 242,000 binding sites per cell were calculated. Of granulocytes from 150 randomly selected German blood donors, the alloantibody bound to all. The maternal cells were typed NA1/NA2- and NB1-positive. CONCLUSION: These data reveal the presence of a previously unrecognized, high-frequency epitope on the granulocyte Fc gamma receptor III. Luminoimmunoprecipitation proved to be a simple, nonradioactive technique that was useful in identifying the molecule involved.

New polymorphism on platelet glycoprotein IIIa gene recognized by endonuclease Msp I: implications for PlA typing by allele-specific restriction analysis.

BACKGROUND: Five human platelet alloantigen systems have been shown to result from single base pair substitutions in encoding regions of platelet glycoprotein genes IIIa, Ib, IIb, and Ia. For each of the diallelic systems, at least one restriction enzyme is known to cut only one of the two haplotypes. In the PlA system, restriction endonucleases Nci I and Msp I both recognize the PlA2 allele. STUDY DESIGN AND METHODS: A causal observation of an unexpected Msp I restriction pattern of a PlA2/PlA2 individual was made. Samples from 261 blood donors were then typed for antigens of the PlA system by restriction fragment length polymorphism analysis using the Nci I and Msp I restriction enzymes. RESULTS: Applying both enzymes, concordant restriction patterns were found in 258 of 261 blood donors. Three donors had a base pair mutation on the PlA2 allele, which creates an additional restriction site for Msp I 20 base pairs downstream from the PlA polymorphic site. Nucleotide sequence analysis revealed a CT217-->CG217G base exchange resulting in a Leu40-->Arg40 polymorphism of glycoprotein IIIa. CONCLUSION: Presuming that the mutation is not a singular phenomenon and also occurs with the PlA1 haplotype, it could lead to false interpretations of restriction analysis with Msp I. To exclude that possibility, Nci I is preferred for restriction fragment length polymorphism typing in the PlA system.

Typing of the granulocyte-specific NA antigens by restriction fragment length polymorphism analysis.

An RNA-based method has been developed to genotype donors for the granulocyte-specific alloantigens NA1 and NA2. mRNA was isolated from granulocytes, reversely transcribed into cDNA and amplified using an Fc-gamma-receptor III-1 sequence-specific primer in the polymerase chain reaction (PCR). PCR products were analysed by restriction fragment length polymorphism (RFLP) using the restriction endonuclease Taq I, which provided a distinct restriction fragment pattern corresponding to the NA alleles. 17 donors were typed by PCR-RFLP and the results were in close accordance with those obtained by serological phenotyping by granulocyte immunofluorescence and the antigen capture assay MAIGA.