published_or_final_version / Pathology / Master / Master of Medical Sciences

Thrombotic microangiopathy is featured by microangiopathic haemolytic anaemia, thrombocytopenia and the presence of peripheral fragmented red cells. Thrombotic thrombocytopenic purpura (TTP) is the major disease entity of concern, which is caused by a congenital or acquired deficiency of a von Willebrand factor (vWF) cleaving protease known as ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type 1 motif, member 13). Deficiency of this protease, leads to accumulation of uncleaved ultra-large hyperactive vWF multimers in peripheral circulation causing the extensive microvascular platelet aggregation in a TTP event. However, the differential diagnosis is sometimes difficult because symptoms and signs can be non-specific and the condition may resemble a number of disorders. Early recognition and definite diagnosis of TTP is critical to enable prompt plasma exchange therapy. Specific and sensitive ADAMTS13 assays will be potentially helpful. In this review, archive frozen plasma samples from six patients presented with prominent thrombotic microangiopathy were retrospectively analysed for ADAMTS13 by immunoassays. The relationship between ADAMTS13 antigen, activity and its autoantibodies and TTP was studied. Local reference ranges of these assays were also determined. The assay results were validated by identifying the clinically-confirmed cases of TTP, with also prospective serial measurements of ADAMTS13 in a few cases. Patients presented with acute TTP were characterized by a severely deficient ADAMTS13 antigen and activity level, as well as a positive autoantibody titre detected for its acquired immune aetiology ; while patients with non-TTP conditions only had mildly reduced ADAMTS13 antigen but variably decreased activity level and a negative autoantibody titre . A pooled analysis of patients and normal subjects also demonstrated a positive correlation between ADAMTS13 antigen and activity. / published_or_final_version / Pathology / Master / Master of Medical Sciences

We report on 2 cases of chronic relapsing thrombotic thrombocytopenic purpura, in which anti-phospholipid antibodies were also found. The first patient was felt to have the antiphospholipid antibody syndrome, while the second patient had anti-phospholipid antibodies without clinical manifestations of the anti-phospholipid antibody syndrome. We discuss chronic relapsing thrombotic thrombocytopenic purpura and the anti-phospholipid antibody syndrome. Furthermore, we introduce the possibility of an association between chronic relapsing thrombotic thrombocytopenic purpura and the presence of anti-phospholipid antibodies.

Abstract Background: Deficiency of Von Willebrand Factor Cleaving Protease (VWFCP) has been implicated as the cause of Thrombotic Thrombocytopenic Purpura (TTP). TTP is a lifethreatening disease characterised by microangiopathic thrombosis due to accumulation of Ultralarge Von Willebrand Factor (ULVWF) multimers. The clinical features of TTP include microangiopathic haemolysis and thrombocytopenia. TTP is being seen with increased frequency in the context of HIV. However, in the context of HIV infection, cytopenias are often multifactorial in nature and levels of VWFCP in HIV-related thrombocytopenia have not specifically been assessed. This study assessed VWFCP activity in the setting of patients with HIV and thrombocytopenia in the absence of TTP, in order to determine the utility of a VWFCP assay in the diagnosis of HIV-related TTP. Acquired VWFCP deficiency is generally assumed to be due to the presence of autoantibody inhibitors to the enzyme, but limited data are available regarding VWFCP activity in HIV positive TTP patients. There is also currently no assay available for measuring VWFCP activity in our laboratory. Aim of Study: To establish a practical assay for VWFCP activity for routine use in our laboratory. The rapid collagen binding assay, based on the ELISA method of Rick, et al., 2002, was chosen. This was initially used to measure VWFCP activity in patients with HIV with and without thrombocytopenia (of any cause except TTP), in order to ascertain whether assessment of VWFCP activity is likely to be of value in facilitating early diagnosis of HIV related TTP. The ELISA assay was performed to establish cut-off values for VWFCP in HIV negative controls and two HIV positive groups (HIV thrombocytopenia / low platelets and HIV normal platelets). Depending on the outcome of this, the assay could then be performed to assess VWFCP activity in HIV positive patients with TTP. Methods: The rapid collagen binding assay for VWFCP activity was established and optimised for routine use in our laboratory. The cut-off values for percentage Residual Collagen Binding Activity (RCBA) in both HIV negative and HIV positive groups were identified. The assay could then be used to assess VWFCP activity in 20 HIV positive patients with TTP at the time of presentation. In patients with reduced VWFCP activity, patient plasma was mixed with normal pool plasma in a 50:50 mix, to assess for the presence of inhibitors. Correlation of VWFCP activity, inhibitors and other laboratory and clinical parameters were performed. Results: The cut-off values for percentage RCBA in both HIV negative (<37.12%) and HIV positive (<51.51%) patients were established. The % RCBA for the HIV negative control group was statistically significantly different from the HIV positive group with normal platelets (p=0.0001) and from the HIV positive group with low platelets (p=0.0006). The cut-off value in the two HIV positive patient groups was higher than for HIV negative control patients, indicating mildly reduced VWFCP enzyme activity in HIV positive patients (regardless of the platelet count), in the absence of TTP. However, no significant difference in the cut-off value was noted between HIV positive patients with low platelet counts versus HIV positive patients with normal platelet counts (p=0.7783). The assay could therefore be used in HIV positive patients with TTP. VWFCP activity was assessed in twenty HIV positive patients with TTP. Two groups of HIV positive patients with TTP were identified based on VWFCP activity. Six patients (30%) had normal (one borderline) VWFCP activity (RCBA <51.51%), while the remaining 14 patients had severely reduced VWFCP levels (RCBA >90%). Of the patients with reduced VWFCP activity, only 5 patients had a detectable inhibitor, while an inhibitor was not detected in the remaining 8 patients. Conclusion: The rapid collagen binding ELISA assay is a cost effective semi-quantitative assay for the assessment of VWFCP activity. VWFCP activity in HIV positive patients appears to be slightly lower, however is not related to the platelet count. This suggests a slight baseline deficiency of VWFCP in the setting of HIV. The baseline VWFCP cut-off value in HIV allowed assessment of HIV positive patients with TTP. The results suggest heterogeneity of VWFCP activity in HIV-related TTP. A negative result (normal VWFCP activity) does not exclude TTP in patients with HIV-related TTP and other pathogenic factors may therefore be involved.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted. Includes bibliographical references.