C. Mueller-Eckhardt

Laboratory diagnosis of heparin‐associated thrombocytopenia and comparison of platelet aggregation test, heparin‐induced platelet activation test, and platelet factor 4/heparin enzyme‐linked immunosorbent assay

BACKGROUND: As clinical diagnosis of heparin‐associated thrombocytopenia (HAT) is often difficult, confirmation by sensitive laboratory assays is desirable.

INTRAVENOUS IMMUNOGLOBULIN VERSUS ORAL CORTICOSTEROIDS IN ACUTE IMMUNE THROMBOCYTOPENIC PURPURA IN CHILDHOOD

In a randomised, multicentre study intravenous IgG was compared with oral corticosteroids in 108 children with untreated acute immune thrombocytopenic purpura. IgG was an efficient treatment with no severe untoward reactions. The effects of corticosteroids and IgG were identical for rapid responders, who accounted for 62% of all patients. In contrast, patients requiring more than initial treatment responded better if randomised to IgG. The serum IgG level increased two-fold after IgG. A significant rise in IgM levels was observed after both IgG and corticosteroids. The platelet-associated IgG index was high in 75% of all patients. No significant differences between the two treatment groups were found, but rapid responders had a smaller mean initial platelet-associated IgG index which returned more rapidly and more permanently to normal than that of slow responders.

EFFECT OF INTRAVENOUS IMMUNOGLOBULIN IN IMMUNE THROMBOCYTOPENIA: Competitive Inhibition of Reticuloendothelial System Function by Sequestration of Autologous Red Blood Cells?

The rapid rise in platelet count after immunoglobulin treatment in acute and chronic forms of idiopathic thrombocytopenic purpura (ITP), autoimmune neutropenia, and post-transfusion purpura is well documented. It is suggested that the rise in platelet count is due to competitive inhibition of the macrophage binding of platelets by preferential sequestration of immunoglobulin-coated red blood cells. Measurement of haptoglobin levels, a sensitive indicator of haemolysis, suggests that clinically inapparent haemolysis occurs during immunoglobulin therapy of ITP patients. In-vitro experiments confirm that there is immunoglobulin coating of red blood cells. The hypothesis is further supported by the findings that immunoglobulin treatment in autoimmune haemolytic anaemia is ineffective, and that platelet counts rise in some ITP patients after induction of a mild haemolytic syndrome by injection of anti-Rho (D).

HPA‐5b (Bra) neonatal alloimmune thrombocytopenia: clinical and immunological analysis of 39 cases

Summary. Maternal alloimmunization against fetal platelets can cause fetal and neonatal thrombocytopenia (NAIT). The HPA‐1a (PIA1, Zwa) antigen is by far the most common antigen implicated in NAIT. However, today another antigen often linked with that affection is HPA‐5b (Bra). This is a report of 39 cases of NAIT involving the HPA‐5b antigen. Thrombocytopenia may be of grave consequence. Three infants developed intracerebral haemorrhages (ICH). Of these, one died presumably as a consequence of ICH. Central nervous system (CNS) sequelae in the neonatal period was observed in two children. The potential hazards of death or disabling neurologic sequelae following intracerebral haemorrhage call for rapid and reliable diagnosis and effective therapy. Because there is high risk that subsequent pregnancies might be also affected by NAIT, the mothers of a previously affected child should be managed similarly to the HPA‐1b mothers (PIA2, Zwb). The antenatal diagnosis of thrombocytopenia should be made and if necessary the in utero therapy instituted.

The Clinical Significance of Platelet-Associated IgG: a Study on 298 Patients with Various Disorders

Summary. Platelet‐associated IgG (PAIgG) was studied by a quantitative platelet radioactive anti‐IgG test (PRAT) in 298 patients. At the time of investigation, 171 patients were thrombocytopenic (platelet count <100 × 109/1), 127 had normal platelet counts. Patients fell into the following disease categories: Idiopathic thrombocytopenic purpura (ITP) (N=81), possible ITP (19), acute ITP (9), systemic lupus erythematosus (22), autoimmune haemolytic anaemia of warm‐type (18), systemic blood disease (65), liver diseases (35), others (49). A significant elevation of PAIgG was found in all disease categories. There was a significant correlation between PAIgG and the reciprocal values of platelet counts for most disease groups. No relationship was discernible between PAIgG and hypergammaglobulinaemic states (serum IgG >1.8 g/l), Platelet survival studies (N=30) revealed that normal and increased values of PAIgG were associated with normal or shortened platelet mean life span. It is concluded that an elevated PAIgG is only one of several factors involved in the development of immunologically mediated thrombocytopenia.

Deposition of Terminal C5b–9 Complement Complexes on Erythrocytes and Leukocytes during Cardiopulmonary Bypass

Hemolysis, leukopenia, a hemostatic deficit, and nonspecific systemic reactions collectively known as the postperfusion syndrome develop in patients who undergo cardiopulmonary bypass. We now report that terminal C5b-9 complement complexes are deposited on erythrocytes and polymorphonuclear neutrophilic leukocytes during cardiopulmonary bypass. Plasma samples taken from 48 unselected patients during and at the end of cardiopulmonary bypass contained raised levels of fluid-phase SC5b-9 complement complexes, indicating that the complement sequence had been activated to completion. Various degrees of overt intravascular hemolysis were observed in all the patients, and lysed erythrocyte membranes were recovered from the blood samples. Immunoassays performed with use of antibodies to C5b-9 neoantigens demonstrated the presence of C5b-9 on red-cell ghosts but not on intact erythrocytes. The appearance of ghosts carrying C5b-9 always coincided with hemolysis. Furthermore, granulocytes isolated from 20 patients during bypass were all found to carry C5b-9 complexes, whereas cells isolated before or 24 hours after surgery carried no C5b-9. The neoantigen-positive material present in detergent extracts of granulocytes sedimented in a broad peak (25 to 40 sedimentation coefficient [S]) in sucrose-density gradients, exactly as did pore-forming C5b-9 complexes. Deposition of C5b-9 on blood cells during cardiopulmonary bypass may be partly responsible for the hemolysis and may augment granulocyte activation by the stimulation of arachidonate metabolism in those cells.

Analysis of granulocyte‐reactive antibodies using an immunoassay based upon monoclonal‐antibody‐specific immobilization of granulocyte antigens

Summary. To detect human granulocyte‐reactive antibodies, a glycoprotein‐specific enzyme immunoassay for platelet antibodies was adapted for the use of granulocytes as target cells. Peripheral blood granulocytes were simultaneously incubated with a monoclonal antibody (mAb) and the serum to be investigated. After solubilization, aliquots of the cell lysate were transferred to plastic tubes coated with goat anti‐mouse antibodies. Following immobilization of the trimolecular (mAb‐glycoprotein‐human antibody) complex it was detected by addition of enzyme‐labelled goat anti‐human antibodies using a luminescence technique. This assay allowed identification of different granulocyte‐reactive antibodies present in the same sample without the need for complicated absorption studies. Alloantibodies against HLA and the granulocyte‐specific NA antigens as well as isoantibodies against the Fc‐gamma‐receptor III (FcRIII) were detectable using mAb‐specific immobilization of granulocyte antigens (MAIGA). Binding of autoantibodies to the FcRIII and to the CD 11b/CD 18 complex could be shown.

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.

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.

The human platelet alloantigens Br(a) and Brb are associated with a single amino acid polymorphism on glycoprotein Ia (integrin subunit alpha 2).

The human GPIa/IIa complex, also known as integrin alpha 2 beta 1, serves as a major receptor for collagen in platelets and other cell types. In addition to its role in platelet adhesion to extracellular matrix, GPIa/IIa is also known to bear the clinically important Br(a) and Brb alloantigenic determinants, which can result in antibody-mediated platelet destruction. Immunochemical studies showed that the Br antigenic epitopes reside solely on the GP Ia subunit and do not depend on sialic acid residues. To define the polymorphism responsible for the Br alloantigen system platelet RNA PCR technique, was used to amplify GPIa mRNA transcripts. Nucleotide sequence analysis of the amplified platelet GPIa cDNA from Br(a/a) and Brb/b individuals revealed a single A<-->G polymorphism at base 1648. MnlI RFLP analysis of cDNA from serologically determined individuals confirmed that this polymorphism segregates with Br phenotype. This single base change results in a substitution of Lys (AAG) in Br(a) to Glu (GAG) in Brb at amino acid residue 505 In spite of the reversal in charge at this position, however, we found no difference in the ability of Bra and Brb homozygous platelets to adhere to collagens types I, III, or V, nor did anti-Bra or anti-Brb alloantibodies interfere with platelet adhesion to any of these fibrillar collagens. The identification of the nucleotide substitution that defines the Bra/Brb alloantigen system will now permit both pre- and postnatal diagnosis for Br phenotype.