Platelet antibodies in immune thrombocytopenia.

Several tests to detect antibodies to platelets in patients with immune thrombocytopenia have been developed over the 40 years since it was first noted that this disorder was mediated by antiplatelet factors. Early tests were crude and not reproducible. A major landmark was the quantitation of IgG immunoglobulin on platelet membrane and the observation by several workers that marked increases of all classes of immunoglobulin occurred on platelets in patients with immune thrombocytopenia. Although at one time accepted as a diagnostic characteristic of autoimmune thrombocytopenia (AITP), the initial euphoria was tempered over the last decade by the realisation that elevation of platelet associated immunoproteins was not pathognomonic of this disorder and that raised levels were seen in several other disease processes, sometimes even when platelet counts were normal. The nature of these immunoproteins needs careful understanding. True platelet autoantibodies will manifest as increased platelet immunoproteins but not all such platelet proteins are platelet antibodies. There is speculation about the existence and the mode of activity of IgA and IgM immunoglobulins, both commonly found on platelets in AITP. It is sometimes almost inconceivable that the extremely large amounts of PAIgG could possibly represent true autoantibody. Immune complexes are found in plenty in these and other disorders in which thrombocytopenia manifest. In such situations it is likely that 'amplified' immune complexes are bound by Fc receptors, as may be found in viral mediated ITP or in septicaemic states. There is now sound evidence that several glycoprotein such as GP IIb/IIIa, GP Ib, GP V, found on platelet membrane, act as target antigen sites for the attachment of antibodies to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnosis of alpha-thalassemia trait from Coulter Counter 'S' indices.

A number of patients of Mediterranean and Asian origins were found to have unexplained microcytic hypochromic red blood cells. Iron deficiency and beta-thalassaemia trait were both satisfactorily excluded in all of them. The haematological indices of these patients, obtained on a Coulter Model 'S' Counter, were found to be very similar to those seen in obligatory heterozygotes for alpha-thalassaemia. It is postulated that these patients were also carriers for alpha-thalassaemia. Subsequent investigation of some of these patients showed the characteristically reduced rates of alpha-chain synthesis seen in this condition. The discriminant function of England and Fraser (1973) may be of help in diagnosing this state. alpha-Thalassaemia trait should be considered in all patients of 'high-risk' ethnic origins with a blood picture suggestive of beta-thalassaemia trait but in whom the levels of Hb A2 and Hb F are within normal limits.

A new IVIgG preparation (IVIgG pH 4.25): preliminary results for the treatment of immune thrombocytopenia.

Fifteen patients comprising ten with chronic idiopathic autoimmune thrombocytopenic purpura, three with acute autoimmune thrombocytopenic purpura, and two with secondary chronic autoimmune thrombocytopenic purpura were treated. All patients received 5-day courses of intravenous immunoglobulin (pH 4.25) at a dose of 400 mg kg body weight daily and five patients received maintenance treatment with double dose single infusions. Responses were seen in 12 patients which were in general of brief duration although one patient had a sustained remission. Five patients were treated with maintenance therapy of 800 mg/kg given at approximately 3-weekly intervals and maintained satisfactory platelet counts.

The effect of storage in different anticoagulants on platelet associated IgG.

Blood was collected from nine normal volunteers into each of four different anticoagulants and in order to simulate transport conditions, was stored at room temperature for 96 h. The platelet associated IgG (PAIgG) was determined from aliquots thereof over this period. Blood in EDTA gave slightly higher initial values (day 0) than in other anticoagulants. PAIgG levels increased at different rates in all anticoagulants thereafter. In contrast to recent reports which suggested that falsely elevated levels were likely to be seen after storage in EDTA, we found little difference in these values in blood in different anticoagulants at 72 h and all PAIgG measurements remained within our quoted normal range at this time. After 96 h storage however, one of nine (11%) in CPD-A, two of nine (22%) in EDTA, two of nine (22%) in Na-citrate and six of nine (66%) in EDTA paraformaldehyde gave falsely elevated results. The possible mechanisms of these changes are discussed. The assay system employed in this study measures 'total' platelet IgG in a platelet extract in contrast to some other assays which quantify surface platelet IgG alone and it is possible that this difference in technique is responsible for the relatively smaller percentage changes after storage than reported by others. All anticoagulants tested in this study proved satisfactory both for handling and for the measurement of PAIgG at 48 h, with the proviso that normal ranges should be established for each.

Platelet associated complement components (PAC3c and PAC3d) in patients with autoimmune thrombocytopenia.

Platelet associated C3c and C3d (PAC3c and PAC3d) were quantitated by enzyme linked assay in 105 patients with idiopathic autoimmune thrombocytopenia (AITP) in whom elevated platelet associated immunoglobulins (IgG and IgM) had previously been documented. Increased levels of complement components were demonstrated in 46 of 105 patients (43.8%). In 11 of these patients, PAC3d alone was abnormal implying that C3b had been inactivated after cleavage by C3 inactivator in vivo. Complement binding was seen in two of 16 patients (12.5%) with raised PAIgG alone, four of 19 patients (21.0%) with raised PAIgM alone and in 40 of 70 patients (57.1%) in whom PAIgG and PAIgM were raised together. This difference was highly significant (P = 0.01). The clinical implication of these findings are discussed.

Platelet antibodies in thrombocytopenic patients.

Platelet antibodies either bound to the surface of platelets or free in the serum were sought in patients who had low platelet counts for a variety of reasons. They were detected by finding excess IgG on the surface of washed platelets either directly or after incubation of the serum with normal platelets. The technique used was a modification of that described recently (Dixon et al, 1975) in which the greater the amount of anti-IgG consumed by the reaction with platelets the less the subsequent lysis of sheep red cells coated with IgG. This test could be calibrated by adding known quantities of IgG to the antisera and thus the amount of bound IgG could be measured. Platelets from normal donors and those with thrombocytopenia due to non-immunological causes such as aplastic anaemia or acute leukaemia were found to have 15-70 ng IgG/10(7) platelets (mean 53 ng). 37 out of 38 thrombocytopenic patients in whom immune destruction of platelets was suspected were found to have excess IgG on their platelets ranging from 70 to 720 ng/10(7) (mean 297 ng, P less than 0.001) and there was a significant inverse correlation between this amount and the platelet count (r = 0.85, P less than 0.001). Antibody in the serum was found in 14 of 22 patients with 'idiopathic' thrombocytopenic purpura (ITP), three of four patients with underlying lymphoma and in all five cases of systemic lupus erythematosus (SLE). Four non-thrombocytopenic patients with autoimmune haemolytic anaemia (AIHA) due to IgG on the red cells were also studied and were shown to have no increase in platelet-bound IgG. Our results confirm the work of Dixon et al (1975) that platelet antibody as excess IgG can be readily detected on the surface of platelets in patients with immune thrombocytopenia. The clinical implications of these findings are discussed.

Enzyme linked immunoassay for the detection of platelet associated IgG.

An enzyme linked immunoassay incorporating antihuman globulin coupled with alkaline phosphatase has been developed to measure platelet associated IgG (PAIgG). Using a method in which platelet IgG is extracted into the fluid phase after appropriate procedures, we were able to bind the 'solubilized' PAIgG to commercially obtained antihuman IgG (AHG) which had previously been coated onto polystyrene. The amount of PAIgG thus bound was subsequently measured by the addition of the enzyme reagent using p-nitro phenyl phosphate as substrate. With this technique platelets from normal donors were found to have 2.6-17.4 ng/10(6) platelets (mean +/-2 SD). These values are higher than those obtained when assay systems using intact platelets are employed. Platelets from patients with immune thrombocytopenia had PAIgG values of 8.2-98.0 ng/10(6) platelets. In a few patients with disorders other than autoimmune thrombocytopenia (AITP) increased levels of PAIgG were also demonstrated. The assumption that increased PAIgG always represents platelet autoantibody may not be valid. The relevance of PAIgG as a parameter in the diagnosis and clinical management of patients with AITP is discussed.

Detection of platelet-bound and serum antibodies in autoimmune thrombocytopenia by enzyme-linked assay.

Platelet antibodies were looked for in 47 patients with autoimmune thrombocytopenia using a modification of the enzyme-linked assay previously described. Surface-bound antibodies measured as increased platelet-associated IgG were found in 32 (68%) of the patients. After incubation in test sera, the platelet-associated IgG of normal donor platelets was significantly increased in 27 of the 47 patients (57%), thus demonstrating the presence of platelet antibodies free in their sera. 6 patients had antibodies only in the serum without any elevation of their platelet-associated IgG. When both tests are evaluated together no antibody was detected by either the direct or the indirect test in 9 of the 47 patients (29%) studied. The technique used is described and the interpretation of our results discussed.

Changes in platelet immunoprotein levels during therapy in adult immune thrombocytopenia.

Very early falls in platelet associated IgG, IgM and C3d were demonstrated following therapy with high dose i.v. immunoglobulin and with fresh frozen plasma. This suggests that the changes in platelet immunoproteins may be a primary event and that interference with antibody binding is a possible early mode of action of i.v. IgG.

Platelet associated immunoglobulins (PAIgG and PAIgM) in autoimmune thrombocytopenia.

An enzyme linked assay system was used to quantitate platelet associated IgM (PAIgM) in addition to platelet associated IgG (PAIgG) in normal subjects and in 145 patients with autoimmune thrombocytopenia (AITP). The mean PAIgM level in normals was 1.17 ng/10(6) platelets with a range of 0.01-2.45 ng (mean +/- 2 SD). The corresponding PAIgG values as follows: mean 6.0 ng, range 2.0-10 ng/10(6) platelets. Elevated PAIgG was seen in 67.6% and abnormally raised PAIgM in 79.3% of patients. Both values were raised together in 57.2% and either elevated PAIgG or PAIgM in 89.7%. All patients with PAIgG values greater than 4 times upper limit of normality were found to have abnormal PAIgM. The relevance of elevated PAIgM, the possible interaction between PAIgG and PAIgM and the implication of our results in patients with autoimmune thrombocytopenia are discussed.

Platelet associated IgG and immune thrombocytopenia in lymphoproliferative and autoimmune disorders.

Thrombocytopenia is frequently encountered in patients with lymphoproliferative disorders (LPD) and systemic erythromatosus (SLE) and to a lesser extent in association with other diseases such as rheumatoid arthritis (RA), pernicious anaemia (PA) and autoimmune haemolytic anaemia (AIHA). This report attempts to document the incidence of thrombocytopenia in these disorders, other than that overtly due to malignant infiltration or marrow suppression by drugs and to demonstrate, that in a significant proportion antibody mediated immune destruction of platelets can be confirmed by positive platelet antibody tests. Platelet associated IgG (PAIgG) was measured in all patients by a quantitative enzyme linked assay. Platelet antibodies were found in 11 of 24 (46%) thrombocytopenic patients with LPD, 10 of 16 (62%) patients with SLE and thrombocytopenia, and in all patients with RA and PA who had low platelet counts at the time of study. In addition, elevated PAIgG levels were found in the following non-thrombocytopenic patients: 9 of 43 (21%) patients with LPD, 2 of 12 (17%) with SLE, 2 of 12 (17%) with AIHA, 2 of 39 (5%) with PA and 5 of 61 (8%) patients with RA. The nature and the role of raised PAIgG levels in diseases other than autoimmune thrombocytopenia is controversial. Our reasons for interpreting these as true platelet autoantibodies in this selected group of disorders and the clinical implications of our results are discussed.

Thalassaemia, iron, and pregnancy.

Haematological values of 35 pregnant women with beta-thalassaemia trait were followed during pregnancy. The discriminant function, calculated from haematological indices, was of no value in diagnosing beta-thalassaemia trait during pregnancy. Initially patients were given iron supplements only if the serum iron and total iron binding capacity levels indicated iron deficiency, but bone marrow biopsies performed in the first 22 patients at 32 weeks indicated deficient iron stores. These patients were therefore given iron irrespective of their serum iron level. All subsequent patients with beta-thalassaemia were also put on iron routinely at booking. Retrospectively the patients were divided into two groups. Patients in group 1 (18 patients) had received iron for less than 12 weeks, and their haemoglobin levels fell significantly during pregnancy (P less than 0-001). Haemoglobin levels in 16 patients who had received iron for more than 12 weeks (group 2), however, did not fall significantly during pregnancy (P less than 0-6). It is suggested (contrary to common practice) that patients with beta-thalassaemia trait should be given iron supplements during pregnancy. Serum folate and vitamin B12 levels did not change significantly in these patients and there was no increase in the incidence of maternal or fetal complications.

Multiple myeloma and immune thrombocytopenia.

Immune thrombocytopenia (ITP) is frequently encountered in patients with lymphoproliferative disorders. However this is only rarely reported in patients with multiple myeloma. We describe three cases who presented initially with the clinical manifestations of ITP but were subsequently found to have multiple myeloma. Platelet count increments to standard treatment modalities for ITP were observed in all three patients with transient or partial response. The importance of recognizing the immune mediated thrombocytopenia in patients with myeloma and the implications of this combination are discussed.

Factor VIII inhibitor and raised platelet IgG levels associated with methyldopa therapy.

An inhibitor to the plasma coagulation factor VIII and abnormally high levels of platelet associated IgG (PAIgG) were found in a patient whilst on methyldopa. Both these parameters fell on initial withdrawal, but on rechallenge with the drug only the PAIgG rose to the high presenting levels. No inhibitory activity to factor VIIIc could be detected in a concentrated eluate prepared from the patient's platelets. These results may imply that two distinct antibodies were provoked by the administration of methyldopa, but could also be explained by the action of anti VIIIc alone on the patient's platelets.

Reticulocytopenia and "absence" of red cell autoantibodies in immune haemolytic anaemia.

A raised reticulocyte count is common in patients with immune or autoimmune haemolytic anaemia, and the result of the direct antiglobulin test (DAGT) is usually positive because of IgG or IgG and complement components on the red cells. We report on three patients who had low reticulocyte counts when they were most anaemic, and in whom no red cell autoantibodies could be detected by the DAGT. We postulate that reticulocytes may be selectively destroyed if antibodies are directed against antigenic sites on these young red cells, thus giving rise to a population of cells whose antigenic sites are poorly expressed. This theory might explain the low reticulocyte counts and the "absence" of antibodies (as tested by the DAGT) in such patients. Radioisotopic studies with 51Cr and 59Fe may provide useful information on the rate and sites of red cell destruction.