| Summary: | 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.
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