V. Kiefel

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.

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.

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.

Platelet alloantibodies in transfused patients

BACKGROUND: Patients receiving cellular blood components may form HLA antibodies and platelet‐specific alloantibodies.

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.

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).

International study to compare antigen‐specific methods used for the measurement of antiplatelet autoantibodies

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.