Anti-Hepatitis C Virus Serology in Immune Thrombocytopenia: A Retrospective Analysis in 101 Patients.

Hepatitis C virus (HCV), an RNA virus, is known to be the major cause of post-transfusion non-A, non-B hepatitis. HCV can induce several expressions of autoimmunity, including both serological abnormalities and clinical disorders. The relationship between the HCV infection and anti-platelet autoimmunity has been occasionally described, but is still far from well-defined. We retrospectively analysed 101 serum specimens, collected between 1988 and 1994, from patients with immune thrombocytopenia (ITP) for the presence of anti-HCV antibodies. Eighty-seven patients were classified as having idiopathic, and 14 secondary ITP (4 systemic lupus erythematosus, 9 non-Hodgkin's lymphoma and 1 Evan's syndrome). Anti-HCV antibodies were determined by second generation tests (ELISA + RIBA). A specimen was considered positive for HCV antibodies in the presence of ELISA reactivity (sample optical density/cut-off > 1.00) accompanied by RIBA reactivity to at least one HCV specific antigen. 20 sera (20%) were positive, with a prevalence higher in secondary than in idiopathic ITP (43% vs. 16%, p < 0.05). No differences were found between anti-HCV positive and negative patients regarding gender, platelet count, platelet associated immunoglobulins, hepatitis B virus serology and liver enzyme profile. On the contrary, mean age was higher in the HCV positive vs HCV negative ones (58±18SD vs. 44±20yrs, p < 0.01), in keeping with the increasing prevalence of HCV infection with ageing. HCV positive patients, showed a poor response to treatment (platelet count lower than 50,000/µl after conventional medical therapy for immune thrombocytopenia) compared to anti-HCV negative ones, (50% versus 7.3%, p < 0.001). When we excluded patients who were exposed to risk factors for HCV infection after ITP diagnosis and before the serum collection, the prevalence of anti-HCV antibodies was not very different (17.6%) from that found in the series as a whole (19.8%). Our results seem to indicate that HCV infection may play a role in triggering several cases ITP, and moreover might constitute a negative prognostic factor for therapy response.

EDTA dependent pseudothrombocytopenia caused by antibodies against the cytoadhesive receptor of platelet gpIIB-IIIA.

AIMS: To clarify the mechanisms involved in the development of EDTA dependent pseudothrombocytopenia, particularly the platelet receptors. METHODS: Platelets were measured in 33 patients with pseudothrombocytopenia, using different anticoagulants to collect blood samples (direct test). The results were compared with the counts obtained by adding patients' serum or immunoglobulins to normal blood samples (indirect test). The role of platelet function was explored using ASA, PGE1, and apyrase as platelet inhibitors. The contribution of platelet receptor/s was investigated using antigens to gpIb-IX and gpIIb-IIIa monoclonal antibodies. Immunoglobulin class was estimated by the ability of IgG, IgA, and IgM antibodies to prevent platelet clumping. RESULTS: Agglutinating antibodies were IgA in 40%, IgG in 30%, and IgM in 10% of patients studied. Both patients' serum and immunoglobulins induced platelet clumping in normal samples anticoagulated with EDTA (indirect test). This was prevented by incubation of blood samples at 37 degrees C and almost completely inhibited by the platelet inhibitors ASA, PGE1, and apyrase. Pseudothrombocytopenia was also entirely prevented by an antigen to gpIIb-IIIa monoclonal antibody that recognises fibrinogen and the von Willebrand factor binding site. Pseudothrombocytopenia was almost completely abolished after the addition of RGD peptide, the recognition sequence of cytoadhesive proteins. CONCLUSIONS: These findings suggest that EDTA dependent pseudothrombocytopenia is caused by agglutinating antibodies that recognise cytoadhesive receptors on platelet gpIIb-IIIa and that an efficient platelet metabolism is required.

A family with an abnormal protein C and a thrombotic tendency.

An Italian family with a dysfunctional protein C (type II deficiency) tentatively designated Protein C Padua has been investigated. Five family members had this defect. Thrombotic manifestations occurred in the propositus, his sister, and his 87-year-old mother, who suffered from a transitory ischemic attack (TIA). All other deficient patients were asymptomatic. The abnormality was consistent with normal protein C (PC) antigen level but reduced both anticoagulant and amidolytic activities after PC activation by Protac. When thrombin was used as activator, PC activity level was also reduced. The abnormal PC molecule seemed to have a defect involving the activation region or the active site mechanism (reduced activity level regardless of the PC activators and the methods used in the functional assays). The electrophoretic mobility of the dysfunctional protein, assayed by crossed-immunoelectrophoresis (CIE), was normal in presence of both Na citrate and calcium lactate. The kinetics of antigen-antibody complex formation between the abnormal PC and anti-PC polyclonal antibody was investigated by means of a laser nephelometer and a peculiar pattern was found in the affected patients suggesting a possible anomalous interaction between the antibody and the abnormal protein.

Heterozygous protein C deficiency associated with multiple congenital hemangiomas--a case report.

Congenital protein C deficiency is described as associated with recurrent thrombotic manifestations. The proband, a fourteen-year-old female, has a history of severe and frequent thrombotic disease, moreover, she presents congenital multiple hemangiomas. Family history was positive for protein C deficiency since the mother of the proposita is also affected and has manifested deep venous thrombophlebitis. Two additional relatives on the maternal side were not available for study but were reported to be symptomatic. None of the family members presented angiomatosis. This case report represents the first description of the association of protein C deficiency with multiple hemangiomas.

Hereditary heparin cofactor II deficiency and thrombosis: report of six patients belonging to two separate kindreds.

We describe six patients belonging to two families with congenital heparin cofactor II deficiency (HC II). The affected members had low levels of HC II antigen and activity, and no abnormalities in HC II electrophoretic mobility were detected in the presence of heparin or dermatan sulphate during the first migration of crossed immunoelectrophoresis. These data suggested that patients had a type I (true) HC II deficiency. The association of thrombotic manifestations with congenital HC II deficiency has not been completely clarified. In these two families, thrombotic events occurred in two out of six affected members. In addition, there was a high incidence of spontaneous abortion in the affected females. Finally, the association of congenital HC II deficiency with angiomatosis was also observed in one patient.

Laser nephelometer evaluation of factor IX.

Laser Nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetic of the reaction between antigen and antibody. In a few instances the method was also applied in the characterization of abnormal molecules. We developed assays for the measurement of Factor IX antigen and the results were compared with those obtained by conventional immunological methods such as rocket immunoelectrophoresis. Plasmas from patients with haemophilia B, on coumarin treatment, with liver cirrhosis were studied. A standard reference curve was obtained using pooled normal plasma. The factor IX levels obtained by laser nephelometer correlated fairly well with those obtained by electroimmunoassay.

Heterozygous protein-S deficiency: a study of a large kindred.

Another family with protein-S deficiency is described here. The defect is characterized by a reduced level of total protein-S antigen; in addition, a markedly reduced level of free protein-S antigen was found. We have studied 20 family members. Ten of them showed reduced levels of protein-S antigen. Five of the affected patients manifested 'thrombophilia'. All the symptomatic patients developed the first thrombotic event at a young age. Heparin and oral anticoagulants were useful for the treatment of the acute phase of the thrombotic events, and in 1 symptomatic patient, the life-long oral anticoagulation treatment was effective in preventing relapses. On the other hand, all the symptomatic but untreated family members developed several recurrent thrombotic episodes.

Severe arterial cerebral thrombosis in a patient with protein S deficiency (moderately reduced total and markedly reduced free protein S): a family study.

Deficiency of protein S has been associated with an increased risk of thrombotic disease as already shown for protein C deficiency. Deficiencies of any of these two proteins predispose to venous thrombosis but have been only rarely associated with arterial thrombosis. In this study we describe a case of severe cerebral arterial thrombosis in a 44-year old woman with protein S deficiency. The defect was characterized by moderately reduced levels of total and markedly reduced levels of free protein S. C4b-bp level was normal. Protein C, AT III and routine coagulation tests were within the normal limits. In her family two other members showed the same defect. All the affected members had venous thrombotic manifestations, two of them at a relatively young age. No other risk factors for thrombotic episodes were present in the family members. The patient reported was treated with ASA and dipyridamole and so far there were no relapses.

Unsuitability of laser nephelometer in the evaluation of factor VIII antigen.

We developed assays for the measurement of Factor VIII in a Laser Nephelometer and the results were compared with those obtained by conventional immunological methods. Marked discrepancies were obtained using different antisera and only the Dako antiserum lot 42 B yielded satisfactory results. Other antisera used (other lots of Dako antiserum, Behringwerke antiserum and Immunoscientific antiserum) failed to yield satisfactory results. From our data it seems that Laser Nephelometer has no role in the evaluation of Factor VIII complex. Statements to the contrary have to be accepted with great caution.

The prevalence of AIDS, AIDS related complex and HIV seropositivity in a large population of patients with congenital clotting disorders.

We have investigated up to the beginning of 1987 114 patients with congenital clotting disorders. 84 had received plasma and/or clotting factors concentrates. 18 out of 84 (21%) had leukopenia, thrombocytopenia, or both. 64 out of 84 (76%) had been infected by hepatitis B virus. The great majority of them (62 out 64) developed adequate immunity (anti Hbs antibodies). Despite this, 47 out 84 (57%) showed persistently elevated transaminases. 17 out of 84 (20%) had HIV-seropositivity. Among them, 7 are free of symptoms related to such a virus up to present time, 8 developed AIDS-related complex and 2 had the full-blown AIDS and died. Non significant difference in HIV seroconversion or its clinical manifestations was noted depending on the administration of factor VIII concentrates versus prothrombin complex concentrates. In contrast, plasma administration appeared to be associated with a lower risk of viral transmission. No abnormality was observed in patients who had never received haemoderivatives, except the presence of anti Hbs antibodies in 1 of them.

The report of an Italian family with heterozygous protein C deficiency.

A heterozygote protein C deficit was found in 4 members of the same family. The propositus is a 40 year old male with a clear thrombotic tendency. This included repeated thrombophlebitis of the right leg, and one episode of pulmonary embolism. Arterial thrombosis was not noted. The anticoagulant therapy undertaken by the patient appears to be of some benefit in the sense that no recurrence of thrombotic manifestations occurred. One brother and two nephews of the propositus, even though asymptomatic showed reduced levels of Protein C both as activity and antigen. The parallel reduction of Protein C activity and antigen points towards a "true" deficit of Protein C. The normal, although reduced, pattern in the crossed immunoelectrophoresis supplies further confirmation to this interpretation.

Factor VIII/von Willebrand factor abnormalities during L-asparaginase treatment in patients with acute lymphoblastic leukemia.

Factor VIII/von Willebrand factor (VIII/vWf) related properties were studied in 10 patients affected by acute lymphoblastic leukemia during L-asparaginase-vincristine-prednisone treatment. These properties remained within the normal range during the period of observation without any difference from the basal values. On the contrary, VIII:C activity was already increased before medication and showed gradual additional elevation during the observation period, reaching a peak 1 week after discontinuation of L-asparaginase administration. Crossed immunoelectrophoresis of vWf, performed weekly in 2 patients during the period of medication, demonstrated a normal pattern before the beginning of treatment, but an apparently faster migrating peak during L-asparaginase therapy, suggesting a qualitative abnormality of vWf. No abnormal bleeding tendency was found in any of the patients.

A new family with congenital factor XIII deficiency showing a deficit of both subunit A and B. Type I factor XIII deficiency.

In this study we present a new case of Factor XIII deficiency. The proposita, a 34 year old woman, showed a deficiency of both subunit a and subunit b, and a moderate bleeding tendency. Because of the concomitant decrease of subunits a and b the proposita is considered to be an example of Type I disease. Factor XIII levels were less than 10% both as activity and antigen. Several family members showed intermediate levels of both subunit a and b and were asymptomatic. They were considered to be heterozygotes. The hereditary pattern is autosomal incompletely recessive. Type I disease appears much less frequent than Type II.

Factor X antigen--factor X activity discrepancy in coumarin treated patients. No relationship with dilution curve.

The plasma of 12 patients on coumarin medication was investigated with regard to factor X antigen, factor X activity, factor X antigen--factor X activity difference (delta) and dilution curve. The plasmas were divided into 3 groups according to the level of anticoagulation (under anticoagulated, normally anticoagulated, over anticoagulated). The average factor X antigen and the average factor X activity were 56.7, 53.0, 34.6 and 25.5, 20.1, 11.1%, respectively for the three groups of patients. The antigen-activity differences (delta) were 21.2, 22.5 and 23.5%, respectively. The degree of inhibition in conventional units was 2.6, 1.7 and 0.4 units for the three groups of patients, respectively. The decrease of "inhibition" with the increase of anticoagulation is not in agreement with the presence of similar levels of pre-factors or delta antigen-activity in the plasma of the three groups of patients. These data are against the presence of inhibitors in coumarin treated patients.