A multi-laboratory assessment of congenital thrombophilia assays performed on the ACL TOP 50 family for harmonisation of thrombophilia testing in a large laboratory network.

OBJECTIVES: Thrombophilia testing is commonly performed within hemostasis laboratories, and the ACL TOP 50 family of instruments represent a new 'single platform' of hemostasis instrumentation. The study objective was to evaluate these instruments and manufacturer reagents for utility of congenital thrombophilia assays. METHODS: Comparative evaluations of various congenital thrombophilia assays (protein C [PC], protein S [PS], antithrombin [AT], activated protein C resistance [APCR]) using newly installed ACL TOPs 550 and 750 as well as comparative assessments with existing, predominantly STAGO, instrumentation and reagents. Verification of manufacturer assay normal reference ranges (NRRs). RESULTS: HemosIL PC and free PS assays showed good comparability with existing Stago methods (R>0.9) and could be considered as verified as fit for purpose. HemosIL AT showed high relative bias with samples from patients on direct anti-Xa agents, compromising utility. Manufacturer NRRs for PC, PS and AT were verified with minor variance. Given the interference with direct anti-Xa agents, an alternate assay (Hyphen) was evaluated for AT, and the NRR also verified. The HemosIL Factor V Leiden (APC Resistance V) evidenced relatively poor performance compared to existing assays, and could not be adopted for use in our network. CONCLUSIONS: This evaluation of HemosIL reagents on ACL TOP 50 family instruments identified overall acceptable performance of only two (PC, free PS) of four thrombophilia assays, requiring use of third-party reagents on ACL instruments for the other two assays (AT, APCR).

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.

Verification of the ACL Top 50 Family (350, 550, and 750) for Harmonization of Routine Coagulation Assays in a Large Network of 60 Laboratories.

OBJECTIVES: To verify a single platform of hemostasis instrumentation, the ACL TOP 50 Family, comprising 350, 550, and 750 instruments, across a large network of 60 laboratories. METHODS: Comparative evaluations of instrument classes (350 vs 550 and 750) were performed using a large battery of test samples for routine coagulation tests, comprising prothrombin time/international normalized ratio, activated partial thromboplastin time (APTT), thrombin time, fibrinogen and D-dimer, and using HemosIL reagents. Comparisons were also made against existing equipment (Diagnostica Stago Satellite, Compact, and STA-R Evolution) and existing reagents to satisfy national accreditation standards. Verification of manufacturer normal reference ranges (NRRs) and generation of an APTT heparin therapeutic range were undertaken. RESULTS: The three instrument types were verified as a single instrument class, which will permit standardization of methods and NRRs across all instruments (n = 75) to be deployed in 60 laboratories. In particular, ACL TOP 350 test result data were similar to ACL TOP 550 and 750 and showed no to limited bias. All manufacturer NRRs were verified with occasional minor variance. CONCLUSIONS: This ACL TOP 50 Family (350, 550, and 750) verification will enable harmonization of routine coagulation across all laboratories in the largest public pathology network in Australia.

Comparison of the pharmacokinetics of two von Willebrand factor concentrates [Biostate and AHF (High Purity)] in people with von Willebrand disorder. A randomised cross-over, multi-centre study.

Plasma-derived factor concentrates are important in the management of von Willebrand disorder (VWD). In our geographic locality, a single viral inactivation step concentrate (AHF [High Purity]), has been replaced with one using a double viral inactivation step (Biostate). The aim of this study was to compare the pharmacokinetics of von Willebrand factor (VWF) and factor VIII (FVIII) after administration of AHF (High Purity) and Biostate. This study was a single-blind, randomised cross-over, multi-centre investigation in twelve people with VWD, comprising four type 3, two type 2B, one type 2M and five type 1 VWD. The subjects received a single infusion of 60 IU/kg ristocetin cofactor activity (VWF:RCo) of either AHF (High Purity) or Biostate, and after a minimum 15-day wash-out period they received the alternative product. Blood samples were collected for up to 48 hours after each dose for assay of FVIII coagulant activity (FVIII:C) and VWF by VWF:RCo, collagen binding capacity (VWF:CB) and antigen (VWF:Ag). As a measure of delivered VWF 'functionality' we calculated the area-under-the-concentration-time-curve (AUC) ratios of VWF:RCo to VWF:Ag and VWF:CB to VWF:Ag. The effect on platelet adhesiveness by PFA-100 closure times (CTs) was measured prior to and 30 minutes post infusion. VWF multimers were also assessed pre and post infusion. Pharmacokinetic parameters after AHF (High Purity) and Biostate were in close agreement for VWF:RCo (confirming dosing equivalence). Parameters for other study markers were also similar, although Biostate tended to yield relatively lower VWF:Ag and higher VWF:CB levels. Although AHF (High Purity) and Biostate resulted in similar levels of high-molecular-weight (HMW) multimers post-infusion, the relative level of HMW to low-molecular-weight (LMW) multimers were determined to be higher following Biostate. The relative levels of functional VWF (i.e. VWF:CB and VWF:RCo) to VWF:Ag were also higher in Biostate compared to AHF (High Purity). With both study products, PFA-100 CTs 30 minutes post infusion showed minor improvement for only some subjects. In conclusion, the pharmacokinetics of FVIII:C and VWF are not significantly different after administration of AHF (High Purity) and Biostate. Study parameters considered as 'in-vitro' markers of VWF 'functionality' or potential clinical efficacy (i.e. VWF:CB and VWF:RCo relative to VWF:Ag, level of HMW VWF relative to LMW-VWF) were determined to be higher for Biostate than AHF (High Purity). PFA-100 CTs did not adequately reflect changes in these VWF parameters. Based on these results, one would expect Biostate to be at least as effective, if not superior to AHF (High Purity) for the treatment of VWD.

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.

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.

Time to think outside the box? Proposals for a new approach to future pharmacokinetic studies of von Willebrand factor concentrates in people with von Willebrand disease.

Plasma-derived factor concentrates are important in the management of von Willebrand disease (VWD). We review the current literature regarding pharmacokinetic studies of von Willebrand factor (VWF) concentrates used to treat VWD. Using additional local experience of a crossover pharmacokinetic (PK) study comparing a currently licensed double virally inactivated concentrate, Biostate, with that of its single virally inactivated predecessor, AHF (High Purity), we propose that consideration now be given to the use of new and novel test parameters for future PK studies. In particular, we propose that an evaluation of VWF collagen binding (VWF:CB) using an assay confirmed to be sensitive to high-molecular-weight forms of VWF be used in all future studies in addition to standard parameters such as factor VIII coagulant (FVIII:C), VWF antigen (VWF:Ag), and ristocetin cofactor (VWF:RCo). We further propose the use of calculated ratios of VWF:RCo to VWF:Ag and VWF:CB capacity to VWF:Ag as a measure of delivered VWF "functionality." We also report some new data regarding assessment of VWF:multimers using standard procedures together with densitometry and a novel scoring system to support our proposals. We suggest that these test parameters may be useful in future PK studies to better assess and compare the potential utility and clinical efficacy of VWF factor concentrates for use as therapy for VWD.

Reducing errors in identification of von Willebrand disease: the experience of the royal college of pathologists of australasia quality assurance program.

Regular multilaboratory surveys of laboratories derived primarily from Australia, New Zealand, and Southeast Asia have been conducted during the last 8 years to evaluate testing proficiency in the diagnosis of von Willebrand disease (vWD). We summarize and update the findings of these surveys with a particular emphasis on diagnostic errors and error rates associated with particular tests or test panel limitations. A total of 43 plasma samples have been dispatched to survey participants. These have included 13 normal samples, five type 1 vWD samples, eight type 2 vWD samples (three 2A, three 2B, one 2M, and one 2N), and four type 3 vWD samples. In addition to numerical test results, participant laboratories (currently, n = 49) were asked to provide diagnostic interpretations regarding results, and whether or not vWD was suggested, and if so, a 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 in 13.3% of cases, and laboratories performing von Willebrand factor (vWF):ristocetin cofactor activity (RCo) without vWF:collagen-binding activity (CB) were seven times more likely to make such an error compared with those performing vWF:CB. Similarly, type 2 vWD plasma was misidentified as type 1 or type 3 vWD in an average of 20.1% of cases, and laboratories performing vWF:RCo without vWF:CB were three times more likely to make such an error compared with those performing vWF:CB. Finally, normal plasma was misidentified as vWD in an average of 6.7% of cases, and laboratories performing vWF:RCo without vWF:CB were four times more likely to make such an error compared with those performing vWF:CB. We conclude that although laboratories are generally proficient in tests for vWD, diagnostic errors do occur and error rates are substantially reduced when test panels are more comprehensive and include the vWF:CB.

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.

Role of thrombospondin-1 in control of von Willebrand factor multimer size in mice.

Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF can be controlled by proteolysis at the Tyr(842)-Met(843) peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold VWF multimers together by thrombospondin-1 (TSP-1). The average multimer size of plasma VWF in TSP-1 null mice was significantly smaller than in wild type mice. In addition, the multimer size of VWF released from endothelium in vivo was reduced more rapidly in TSP-1 null mice than in wild type mice. TSP-1, like ADAMTS13, bound to the VWF A3 domain. TSP-1 in the wild type mice, therefore, may compete with ADAMTS13 for interaction with the A3 domain and slow the rate of VWF proteolysis. TSP-1 is stored in platelet alpha-granules and is released upon platelet activation. Significantly, platelet VWF multimer size was reduced upon lysis or activation of wild type murine platelets but not TSP-1 null platelets. This difference had functional consequences in that there was an increase in collagen- and VWF-mediated aggregation of the TSP-1 null platelets under both static and shear conditions. These findings indicate that TSP-1 influences plasma and platelet VWF multimeric size differently and may be more relevant for control of the VWF released from platelets.