Further characterization of the NB 1 antigen as a variably expressed 56-62 kD GPI-linked glycoprotein of plasma membranes and specific granules of neutrophils.

The human neutrophil-specific alloantigen NB1 was identified as a glycosyl-phosphatidylinositol (GPI)-anchored N-glycosylated protein of M(r) 56-62 kD under reducing conditions. Under non-reducing conditions its M(r) was 49-55 kD. This glycoprotein antigen was found to be expressed by only a subpopulation of normal donor neutrophils, and could not be detected on other blood cells. The allotypic epitope recognized by human anti-NB1 IgG was also recognized by the mouse monoclonal antibody 1B5. The percentage of neutrophils stained by these antibodies varied greatly among healthy donors (range 0-100%). When 16 donors were repeatedly tested, the NB1-positive neutrophil fraction appeared to remain remarkably constant over time in most donors, but significant fluctuations were seen in some. NB1 antigen was found to be expressed not only on the plasma membrane, but also intracellularly on the membranes of small vesicles and specific granules. The neutrophils which expressed NB1 antigen on the plasma membrane were the same as those with intracellular expression of this antigen. Crosslinking of NB1 antigen on the plasma membrane with monoclonal antibody 1B5 and goat-anti-mouse Ig resulted in internalization of the complex, while in-vitro stimulation of neutrophils caused an increase of the intensity of plasma membrane staining with anti-NB1, but only of those cells that were positive already prior to stimulation. The NB1 glycoprotein thus appears to identify a distinct subset of neutrophils, the size of which greatly varies among healthy donors. The function of the NB1 glycoprotein remains unclear, but its behaviour upon crosslinking and chemotactic peptide stimulation suggests a possible role as receptor molecule.

Idiopathic thrombocytopenic purpura in pregnancy: a randomized trial on the effect of antenatal low dose corticosteroids on neonatal platelet count.

OBJECTIVE: To determine the efficacy of antenatal low dose oral betamethasone in preventing neonatal thrombocytopenia and/or bleeding in infants of mothers with idiopathic thrombocytopenic purpura (ITP). SETTING: Hospital department of obstetrics and gynaecology, referral centre. PATIENTS: 41 pregnancies in 38 women were randomized. The results of 13 pregnancies were considered non-assessable. The final analysis involved 14 in the betamethasone group and 14 in the non-treatment group. All fulfilled the criteria for ITP. INTERVENTIONS: The treated group received 1.5 mg betamethasone orally per day, from day 259 till day 273 and 1 mg from day 273 till delivery. MAIN OUTCOME MEASURES: Effects of treatment were assessed in terms of maternal platelet counts after the first trimester and neonatal platelet counts at birth and the first week of life and neonatal bleeding episodes. RESULTS: There were no significant differences in neonatal platelet counts at birth. Two infants in the betamethasone group and one in the untreated group had a severe thrombocytopenia either at birth or during the first week of life (less than 50 x 10(9)/l). Seven infants in the betamethasone group and six in the non-treatment group had a mild thrombocytopenia. The overall frequency of neonatal thrombocytopenia was similar: 64% in the betamethasone group and 57% in the untreated group (95% CI of the true difference: -43.5% to +29.5%). There was also no significant difference in neonatal bleeding episodes. CONCLUSIONS: Low-dose betamethasone in pregnant women with ITP does not prevent thrombocytopenia or bleeding in their newborn infants.

Autoimmunity against blood cells in human immunodeficiency-virus (HIV) infection.

In persons with AIDS or at risk from AIDS, autoantibodies against platelets and granulocytes were frequently detected. Platelet-bound immunoglobulins were demonstrated by immunofluorescence in all 16 patients with AIDS, in five out of seven patients with AIDS-related complex/persistent generalized lymphadenopathy (ARC/PGL) and even in seven of 10 healthy sexually active homosexual men. Granulocyte-bound immunoglobulins were found by immunofluorescence in 12 of the 16 AIDS patients, five of the seven patients with ARC/PGL and two of the 10 symptomless men. Red cell bound immunoglobulins were not detected. All patients with AIDS and ARC/PGL and three of the symptomless men were seropositive for human immunodeficiency virus (HIV). The platelet- and granulocyte-bound immunoglobulins could be eluted in 93% and 67% of the cases, respectively. This indicates that specific autoantibodies, rather than circulating immune complexes, which were frequently increased, accounted for the findings. There was no relation between the serological findings and the platelet and granulocyte counts. We conclude that autoantibodies against platelets and granulocytes are common in patients with AIDS and those at risk.

Clinical significance of positive platelet immunofluorescence test in thrombocytopenia.

The sensitivity and specificity of the platelet immunofluorescence test for the diagnosis of idiopathic thrombocytopenia (ITP) was studied in a series of 255 patients. Patients' platelets were tested directly. Diethyl-ether eluates of these platelets and patients' sera were tested indirectly with normal donor platelets. When all three tests were considered, positive results were obtained for 92.0% of the ITP patients with a platelet count of less than 150 X 10(9)/l and for 98.4% of the patients with a count of less than 100 X 10(9)/l. However, for many patients rather weak test results were obtained, with a score of 1/2-1 in 59.8% of the patients. Most patients (94.1%) with a positive direct test had a positive indirect test on the eluate. Thus, platelet-bound antibodies but not platelet-bound immune complexes were present in most, if not all, patients. Positive immunofluorescence tests were obtained for many patients with a diagnosis other than ITP. This resulted in a low specificity of the test for the diagnosis of ITP, evidently because autoimmune thrombocytopenia occurred together with many other diseases and also because antibodies against platelet cryptantigens (expressed by the action of EDTA or by platelet fixation) were present in many patients.

Alloimmunization against the platelet-specific Zwa antigen, resulting in neonatal alloimmune thrombocytopenia or posttransfusion purpura, is associated with the supertypic DRw52 antigen including DR3 and DRw6.

The strong association between HLA-DR3 and Zwa alloimmunization in Zwa-negative mothers, resulting in the production of anti-Zwa antibodies and ultimately in neonatal alloimmune thrombocytopenic purpura (NAITP) in the newborn, could be confirmed. In addition, we have shown an equally strong association between HLA-DR3 and Zwa alloimmunization in Zwa-negative recipients of blood transfusions, resulting in posttransfusion purpura (PTP). However, the strongest association both in mothers of NAITP patients and in PTP patients was observed with the supertypic DRw52 antigen. In fact, all individuals carried this antigen. Normally, DRw52 includes DR3, DR5, DRw6, and DRw8 almost completely. However, in our material, DRw52 included only DR3 and DRw6 with one exception. Another interesting observation is that all PTP patients were women with previous pregnancies without any clinical signs of a bleeding disorder in their children. This indicates that alloimmunization against Zwa during pregnancy is DR3- and/or DRw52-positive women does not always lead to the development of NAITP, but these women may be at risk for the development of PTP after blood transfusion.

Thrombocytopenia associated with gold therapy: a drug-induced autoimmune disease?

We studied 13 patients with rheumatoid arthritis (RA) and gold-induced thrombocytopenia. Platelet-specific autoantibodies of the IgG and often also of the IgM class were detected by immunofluorescence on the patient's platelets and in ether eluates from these platelets. In nine patients we also detected autoantibodies in the serum. The antibodies were unreactive with platelets from patients with Glanzmann's disease in most cases, and were not EDTA dependent. Thus, they had the serological characteristics of true autoantibodies. The reaction of the antibodies with platelets was not influenced by the addition of extra gold. By atomic absorption spectrophotometry we found that the ether eluates, which were often strongly reactive with donor platelets, were free of gold. This indicated that the autoantibodies in the thrombocytopenic patients were not dependent on gold for their reaction. The possibility is raised that gold treatment of rheumatoid arthritis patients induces or enhances the formation of platelet-specific autoantibodies, and that gold-induced thrombocytopenia is a drug-induced autoimmune disease.

The serology of febrile transfusion reactions.

Sera from 40 patients with febrile, nonhemolytic transfusion reactions were tested for the presence of alloantibodies using a number of techniques, including immuno-fluorescence tests on granulocytes, lymphocytes and platelets, a modified NIH lymphocytotoxicity test and the leukocyte agglutination test. Cells of at least 9 donors were used as target cells. Alloantibodies were detected in all sera. The frequency of the occurrence of antibodies was not much higher in sera obtained about 1 month after the transfusion reaction as in sera obtained within 4 days. Most of these antibodies were anti-HLA, but quite frequently platelet-specific antibodies were found, and sometimes these were the only antibodies detected. Granulocyte-specific antibodies were the least frequent. The nature of the antibodies was specified by their difference in reactivity with the cells of multiple donors, by applying panels of cells from typed donors and by absorption and elution experiments. It appeared that not only granulocyte-specific but also HLA- and perhaps platelet-specific antibodies may be responsible for a febrile transfusion reaction. We did not find that the occurrence of rigors, together with fever, was associated with particular serologic results.

Platelet autoantibodies in septicaemia.

Thirty-three patients, 16 of whom had a platelet count below 100 X 10(9)/l, were studied for the presence of platelet antibodies using the platelet suspension immunofluorescence test (PSIFT). In the acute phase platelet-bound immunoglobulins (PBIg) were found in 62% of the thrombocytopenic patients, but also in 65% of the patients with a normal platelet count. Fourteen patients were re-examined after successful treatment of their infection. At that time all had a normal platelet count, yet PBIg were found in 71%. In all but four patients the PBIg could be eluted with ether and were shown to bind to normal platelets but not to Glanzmann type I platelets in 11 of 12 patients tested, indicating the platelet-specific autoantibody nature of the PBIg. Platelet autoantibodies were also demonstrated in the sera: in 38% in the acute phase and in 43% after treatment. In those patients in whom this could be tested the antibodies appeared to be strictly or partially EDTA-dependent. It is postulated that the antibodies are mainly directed against cryptantigens, exposed in vivo only on platelets damaged by the septicaemia and in vitro in the presence of EDTA. This would also explain the lack of relationship between the platelet count and the serological findings.

Platelet freezing for serological purposes with and without a cryopreservative.

We compared the reactivity of fresh platelets with platelets frozen in dimethylsulfoxide (DMSO) or without a cryopreservative by testing agglutinating and non-agglutinating platelet-specific alloantibodies in titration. Frozen platelets, irrespective of the method of preservation, showed no loss of antigenicity as compared with fresh platelets. The yield of platelets frozen without a cryopreservative proved to be higher (80%) than platelets frozen in the presence of DMSO (50%). Platelets sensitized with autoantibodies from patients with autoimmune thrombocytopenia could also be stored by simple freezing in platelet-rich plasma and used for (re)testing.