Geoff Kershaw

Factor V inhibitors: rare or not so uncommon? A multi-laboratory investigation

Acquired deficiencies of, or inhibitors to, factor V are considered rare events. We report a series of 14 acquired factor V deficiencies, 10 of which were confirmed to have inhibitors to factor V, as identified within Australia in the past 5 years following a multi-laboratory investigation. The initial index case seen by one laboratory was followed within 4 months by a separate similar case. This prompted local contact with colleagues (n = 20) working in other haemostasis referral laboratories to identify the current case series. In total, nearly one-half of all haemostasis referral laboratories contacted had seen a case within the past 5 years. Clinical features and the apparent associated risk of bleeding complications generally varied, as did laboratory findings and the likely causal event. There were three females and 11 males. Age ranged from 44 to 95 years (median, 81 years). The level of inhibitor ranged from undetectable to over 250 Bethesda units. The probable cause leading to development of the inhibitors ranged from exposure to bovine thrombin, exposure to antibiotics, surgery and malignancy. Of additional interest was the apparent association of anti-phospholipid antibodies in many of the cases. For example, in the two similar index cases, with factor V inhibitor titres > 200 Bethesda units, high levels of anti-cardiolipin antibodies (> 70 GPL units) were also detected. Although less clear because of inhibitor interference, many of the cases also showed evident co-associated lupus anticoagulant activity. In conclusion, we report a series of factor V inhibitors recently identified within our geographic region that would represent an annual incidence of around 0.29 cases per million Australians. Although considered a rare finding, there is a high likelihood that most haemostasis referral laboratories will see a case every five or so years.

Laboratory diagnosis of von Willebrand disorder (vWD) and monitoring of DDAVP therapy: efficacy of the PFA-100® and vWF:CBA as combined diagnostic strategies

We have coevaluated a combination of test processes for diagnosing von Willebrand disease (vWD) and monitoring deamino‐δ‐D‐arginine vasopressin (DDAVP) therapy. Using normal controls (n=23), closure time (CT) ranges measured by PFA‐100® were (mean ± 2SD): (i) collagen/ADP cartridge (C/ADP): 67–127 s (ii) collagen/epinephrine (C/Epi): 94–162 s. From a panel of 125 patients undergoing evaluation for clinical haemostatic defects, 29/30 samples from patients with vWD [17/18 type 1, 1/1 type 3, 3/3 type 2A, 7/7 type 2B and 1/1 pseudo‐vWD] gave prolonged CTs using C/Epi. The C/ADP was less sensitive, being normal in 7/18 of the type 1 vWD individuals, with higher sensitivity to more severe vWD. Individuals with haemophilia (six factor VIII‐deficient, one factor XI‐deficient) gave normal CTs, while those with clinical thrombocytopenia (n=13) gave normal or prolonged CTs, somewhat dependent on platelet count. The PFA‐100® was also evaluated as a part of the laboratory monitoring procedure in patients with either vWD or haemophilia undergoing a DDAVP trial as a therapeutic management process. For vWD, correction of an initially prolonged CT by DDAVP, accompanied by normalization of von Willebrand factor (vWF) measurable by von Willebrand factor antigen, vWF collagen binding activity and vWF ristocetin cofactor assays (vWF:Ag, vWF:CBA and vWF:RCof), was achieved in type 1 vWD (n=5). In an individual with type 2A vWD, DDAVP normalized vWF:Ag and vWF:RCof, but had no apparent effect on the baseline maximally prolonged CT. In an individual with type 2B vWD, factor VIII/vWF concentrate also normalized vWF:Ag and vWF:RCof, but similarly had no apparent effect on the baseline maximally prolonged CT. vWF:CBA did not normalize for either of these individuals, potentially suggesting that normalization of vWF:CBA might be required for normalization of CT. This concept is supported by correlation analysis undertaken between CT and various vWF parameters. Among these, vWF:CBA held the strongest relationship in our data set, which showed an inverse progressive rise in CT for falling vWF:CBA. Based on these results, we would conclude that the PFA‐100® is highly sensitive to the presence of vWD, and may thus provide a valuable screening test for vWD. Furthermore, the combined utility of the PFA‐100® and vWF:CBA as markers of DDAVP responsiveness may prove to be simple, quick but powerful, predictors for its clinical efficacy.

Rituximab treatment of mild haemophilia A with inhibitors: a proposed treatment protocol

Summary.  Inhibitors are an uncommon complication of mild haemophilia A but represent a severe disease, typically with high titre inhibitors and an associated high rate of bleeding. We present data from three patients with MHAI who were successfully treated with Rituximab alone and unequivocally prove that such inhibitors respond to this agent. A treatment protocol is suggested.

Laboratory diagnosis of von Willebrand disorder: use of multiple functional assays reduces diagnostic error rates.

Regular multilaboratory surveys of laboratories primarily in Australia, New Zealand, and Southeast Asia have been conducted over the past 8 years to evaluate testing proficiency in the diagnosis of von Willebrand disorder (VWD). We have reassessed the findings of these surveys with a particular emphasis on the diagnostic errors and error rates associated with particular tests or test panel limitations. The 37 plasma samples dispatched to survey participants include 9 normal samples, 4 type 1 VWD samples, 8 type 2 VWD samples (2A x 3, 2B x 3, 2M x 1, and 2N x 1), and 4 type 3 VWD samples. In addition to providing numerical test results, participant laboratories (average, n = 35) were asked to provide diagnostic interpretations of their test results regarding whether VWD was evident and, if so, the probable subtype. Although laboratories usually provided correct interpretative responses, diagnostic errors occurred in a substantial number of cases. On average, type 1 VWD plasma was misidentified as type 2 VWD plasma in 11% of cases, and laboratories that performed the ristocetin cofactor assay for von Willebrand factor (VWF:RCo) without performing the collagen-binding activity assay for VWF (VWF:CB) were 6 times more likely to make such an error than those that did perform the VWF:CB. Similarly, type 2 VWD plasma samples were misidentified as type 1 or type 3 VWD in an average of 20% of cases, and laboratories that performed the VWF:RCo without the VWF:CB were 3 times more likely to make such an error than those that performed the VWF:CB. Finally, normal plasma was misidentified as VWD plasma in an average of 5% of cases, and laboratories that performed the VWF:RCo without the VWF:CB were 10 times more likely to make such an error than those that performed the VWF:CB. We conclude that laboratories are generally proficient in their testing for VWD and that diagnostic error rates are substantially reduced when test panels are more comprehensive and include the VWF:CB.

Evaluating laboratory approaches to the identification of lupus anticoagulants: A diagnostic challenge from the RCPA Haematology QAP

Background: Laboratory identification of lupus anticoagulants (LA), an important component of the clinical diagnosis of the autoimmune disorder antiphospholipid syndrome (APS), is challenged by the heterogeneity of tests available, the diagnostic and laboratory approach undertaken, and the heterogeneity of the autoantibodies present. Aim: To assess the laboratory approach for investigation of LA, as well as the utility of various tests and test approaches, given a difficult clinical scenario in which LA might or might not be present. Methods: Ninety-three participants in the Royal College of Pathologists of Australasia (RCPA) Haematology Quality Assurance Program (QAP) were sent 4 mL of a complex but strongly positive LA sample blinded to the nature of the abnormality. Results: Seventy-three (79%) participants returned results and in most cases diagnostic interpretations. The laboratory approach to LA investigation of this sample was quite varied: 34.7% of participants concluded the sample was LA negative, with 91.7% of these performing dilute Russell viper venom time (dRVVT) testing without mixing, whereas 43.5% of participants identified a strong LA, with 96.7% of these having performed mixing studies. Most laboratories reporting negative LA instead identified the false presence of specific factor inhibitors against a variety of factors, including II, V and VIII. Conclusions: For this difficult challenge, performance of non-mixing dRVVT was associated with a high false negative LA rate.

EVALUATION OF THE TECHNICON H-1 HEMATOLOGY ANALYSER

&NA; The Technicon H‐1 is a new, random‐access hematology instrument performing a full blood count with leukocyte differential including eosinophils and basophils. Technical assessment showed good linearity and precision. Comparison with a Coulter S‐PLUS (II) showed close correlation for all full blood count parameters except MPV and MCHC on 149 unselected hospital inpatients over a wide clinical range. No significant carry‐over was detected in hemoglobin, red cell, white cell and platelet estimations. Differentials agreed closely with Technicon H6000 results for neutrophils, lymphocytes, monocytes and eosinophils. Poorer correlation with 100‐cell manual differentials for all cell types except neutrophils probably reflects the relative lack of precision in manual methods. Technological innovations on the H‐1 include a laser‐based optical system from which several new hematological parameters are derived. The contribution they make towards improved patient care awaits assessment.

Effects of FEIBA on platelet and leucocyte activation in severe haemophilia patients with inhibitors

Factor eight inhibitory bypassing agent (FEIBA) is used as a therapeutic option in haemophilia patients who have developed inhibitors. The measurement of thrombin generation has been applied to monitor the efficacy of FEIBA. However, a major concern about the clinical use of FEIBA is whether or not an increase in thrombin activity causes subsequent platelet activation and risk of thrombosis. Our aim is to evaluate whether FEIBA causes platelet and leucocyte activation in haemophilia patients with inhibitors. We evaluated the effects of FEIBA on platelet and leucocyte activity in correlation with thrombin generation. Initially, an in vitro study was conducted to evaluate the effects of FEIBA on platelet and leucocyte activity (using flow cytometry) using peripheral blood from normal volunteers. We then performed an ex vivo study looking at the effect of FEIBA on the above parameters in two haemophiliacs with high-titre inhibitors. A parallel study was also carried out ex vivo to evaluate thrombin generation using a thrombinoscope. FEIBA did not cause platelet or leucocyte activation in either the in vitro or ex vivo studies but showed a predictable increase in thrombin generation. Our study is the first one to address the effect of FEIBA on platelet and leucocyte function. We found no evidence of ‘systemic’ platelet activation. The findings suggest that whilst FEIBA improves global haemostasis, platelet activation is likely to be contained to the site of injury and systemic platelet activation, a previously feared consequence of FEIBA infusion that that may have contributed to thrombotic risk is absent.