Soma Mohammed

A laboratory evaluation into the short activated partial thromboplastin time

Although short activated partial thromboplastin times (APTTs) are generally considered to be laboratory artefacts of problematic blood collections, there is mounting evidence that in some cases a short APTT may reflect a hypercoagulable state, potentially associated with increased thrombotic risk and adverse cardiovascular events. We prospectively evaluated the phenomenon of short APTTs in 113 consecutive samples compared with an equal number of age and sex-matched normal APTT samples. We found a significant difference in various test parameters including prothrombin time (PT), Factor (F) V, FVIII, FXI, FXII, von Willebrand factor (VWF) antigen and collagen-binding activity, and in the level of procoagulant phospholipids, as assessed using a novel assay procedure (XACT). Interestingly, there was a significant negative association for fibrinogen, and although elevated, there was no significant association for FIX. On the basis of identified consecutive samples having multiple low APTTs on several sequential days, a proportion of laboratory-defined short APTTs appear to represent in-vivo hypercoagulability. In conclusion, plasma from patients presenting with short APTTs is reflective of a complex hypercoagulant milieu that could feasibly contribute to thrombotic risk, and 20% or more of laboratory definable short APTTs appear to reflect in-vivo phenomenon.

Towards improved diagnosis of von Willebrand disease: Comparative evaluations of several automated von Willebrand factor antigen and activity assays

INTRODUCTION von Willebrand disease (VWD) is reportedly the most common bleeding disorder and arises from deficiency and/or defects of von Willebrand factor (VWF). Laboratory diagnosis and typing has important management implications and requires a wide range of tests, including VWF activity and antigen, and involves differential identification of qualitative vs quantitative defects. METHODS We have assessed several VWF antigen and activity assays (collagen binding [VWF:CB], ristocetin cofactor [VWF:RCo] and the new Siemens INNOVANCE assay [VWF:Ac], employing latex particles and gain of function recombinant glycoprotein Ib to facilitate VWF binding and agglutination without need for ristocetin) using different instrumentation, including the new Sysmex CS-5100, with a large sample test set (n=600). We included retrospective plus prospective study designs, and also evaluated desmopressin responsiveness plus differential sensitivity to high molecular weight VWF. RESULTS VWF:Ag and VWF:RCo results from different methods were respectively largely comparable, although some notable differences were evident, including one high false normal VWF:Ag value (105 U/dL) on a type 3 VWD sample, possibly due to heterophile antibody interference in the latex-based CS-5100 methodology. VWF:Ac was largely comparable to VWF:RCo, but VWF:CB showed discrepant findings to both VWF:RCo and VWF:Ac with some patients, most notably patients with type 2M VWD. CONCLUSIONS (a) VWF:Ag on different platforms are largely interchangeable, as are VWF:RCo on different platforms, except for occasional (some potentially important) differences, and manufacturer recommended methods may otherwise require some assay optimization; (b) VWF:RCo and VWF:Ac are largely interchangeable, except for occasional differences that may also relate to assay design (differing optimizations); (c) VWF:CB provides an additional activity to supplement VWF:RCo or VWF:Ac activity assays, and is not interchangeable with either.

The effect of dabigatran on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples

Summary The new direct oral anticoagulants (DOACS) dabigatran, rivaroxaban, apixaban and edoxaban provide alternatives to warfarin for treatment and prevention of atrial fibrillation and venous thromboembolic disease in various settings. These have been developed as not requiring laboratory monitoring; however, under certain clinical situations, including recent haemorrhage/thrombosis, emergency surgical procedures, testing may be indicated. The aim of this study was to assess findings of haemostasis laboratory tests for one of the DOACs, dabigatran (Pradaxa), tested across a wide range of laboratory assays. Laboratories (n = 72) enrolled in the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) Haematology program were sent set(s) of seven dabigatran spiked plasma samples covering the concentration 0–800 ng/mL. Also, 30 ex vivo patient samples under therapy with dabigatran were assessed. Prothrombin time and activated partial thromboplastin time assays showed some sensitivity to dabigatran; however, a normal result could not inform on drug exclusion. The thrombin time (TT) was very sensitive to dabigatran, and a normal TT could generally be used for drug exclusion. More specialised assays such as the Hemoclot, a direct thrombin inhibition assay, and in-house dilute TT methods, showed good reproducibility and concordance with expected drug levels assessed by mass spectrometry and were effective to quantify drug levels. Dabigatran also affected factor assays, lupus anticoagulant and factor inhibitor measurement, leading to potential misinterpretation of test results. Ex vivo sample testing provided similar and extended information. Dabigatran affects many haemostasis tests. Some can be used to predict the presence, absence or quantity of dabigatran in patient plasma. For others, interference may lead to false conclusions regarding patients’ haemostatic status.

Identification and prevalence of von Willebrand disease type 2N (Normandy) in Australia

We report an investigation of type 2N von Willebrand disease (VWD), covering the past 7 years and evaluating 1031 plasma samples from over 500 patients. Samples included specific requests for investigation of possible type 2N VWD (including family studies) and samples from ‘hemophilia’ or nonspecified VWD investigations that could unknowingly be type 2N VWD. In total, 13 new patients with type 2N VWD were identified, four of whom initially presented with normal levels of factor VIII and only three of whom (i.e. 23%) derived from specific clinical requests for investigation of type 2N VWD. Furthermore, type 2N VWD was excluded in 91% of specific clinical requests for type 2N VWD investigations. Poststudy evaluation indicates that type 2N VWD in this geographic region has an incidence rate similar to that in other westernized regions, accounting for around 1–2% of all identified VWD cases and about 13% of all type 2 VWD cases. In conclusion, this study highlights that clinicians requesting laboratory investigations related to a bleeding tendency often fail to appropriately recognize the possibility of type 2N VWD, that a normal plasma factor VIII will not exclude type 2N VWD, and although a relatively uncommon form of VWD overall, type 2N VWD represents a significant qualitative disorder.

Evaluation of a von Willebrand factor three test panel and chemiluminescent-based assay system for identification of, and therapy monitoring in, von Willebrand disease.

von Willebrand disease (VWD) is reportedly the most common bleeding disorder and arises from deficiency and/or defects of von Willebrand factor (VWF). Laboratory diagnosis and typing of VWD has important management implications and requires a wide range of tests, including VWF antigen (VWF:Ag) and various activities, involving differential identification of qualitative vs quantitative VWF defects. We have assessed a new hemostasis instrument, the chemiluminescent assay based ACL AcuStar™, and an associated HemosIL AcuStar three test panel comprising VWF:Ag, VWF ristocetin cofactor (VWF:RCo) and VWF collagen binding (VWF:CB) (Instrumentation Laboratory, Bedford, Ma. USA) for ability to identify VWD, to help provisionally type VWD, and for potential use in therapy monitoring. This test system was compared to previously evaluated and validated test systems including VWF:RCo on CS-5100 and BCS analyzers, the new Siemens INNOVANCE assay (VWF Ac) on CS-5100, and VWF:Ag and VWF:CB assays performed by automated ELISA. We employed a large total sample test set (n=535) comprising plasma and platelet-lysate samples from individuals with and without VWD, some on treatment, normal plasmas, and normal and pathological controls. We also evaluated desmopressin (DDAVP) responsiveness, plus differential sensitivity to reduction in high molecular weight (HMW) VWF. The chemiluminescent test panel (VWF:Ag, VWF:RCo, VWF:CB) showed good comparability to similar assays performed by alternate methods, and broadly similar data for identification of VWD, provisional VWD type identification, DDAVP and VWD therapy, and HMW VWF sensitivity, although some notable differences were evident. The chemiluminescent system showed best low level VWF sensitivity, and lowest inter-assay variability, compared to all other systems. In conclusion, we have validated theACL AcuStar and the chemiluminescent HemosIL AcuStar VWF test panel for use in VWD diagnostics, and have identified some favorable characteristics that may improve the future diagnosis of VWD.

Type 2M von Willebrand disease - more often misidentified than correctly identified.

Appropriate diagnosis of von Willebrand disease (VWD), including differential identification of qualitative vs. quantitative von Willebrand factor (VWF) defects has important management implications, but remains problematic.

A clinical audit of congenital thrombophilia investigation in tertiary practice

Background: The presumed cause of congenital thrombophilia can now be explained in ∼50% of familial thrombosis cases following evaluation of a range of markers, primarily comprising factor V Leiden (FVL), activated protein C resistance (APCR), protein C (PC), protein S (PS) and antithrombin (AT). However, the effectiveness of such evaluations is largely determined by limiting improper investigations, either in inappropriate patients or at unsuitable timepoints. Aim: To evaluate clinical ordering patterns for a range of thrombophilia associated tests at a tertiary level public facility. Methods: Several independent audits into clinical requests for FVL, APCR, PC, PS, and AT testing were performed at our institution. Results: We identified a wide variety of clinical ordering background, although most requests related to ‘thrombosis’ or ‘obstetric’ indications. For FVL, the detection rate of heterozygotes continues to decline and is currently ∼10% of investigations. For APCR, review of clinical requests and clinical notes indicated that around 36% of investigations occurred whilst patients were on anticoagulant therapy. For PC, PS and AT investigations, additional testing of samples that yielded low test results for PC, PS and/or AT indicated that an alarming 80% of these cases likely derived from patients on anticoagulant therapy. Conclusion: These results continue to reflect on poor patient or timing selection for congenital thrombophilia investigations that compromises the utility of these tests. In total, this would yield a very high rate of false positive identification for disorders that patients do not have, raising the question: are broadly based congenital thrombophilia investigations doing more harm than good?

International normalized ratio monitoring of vitamin K antagonist therapy: comparative performance of point-of-care and laboratory-derived testing.

The monitoring of warfarin therapy using the international normalized ratio (INR) has now moved outside the laboratorys control by use of point-of-care (POC) devices. Although this provides patients with the convenience of immediate results and clinical assessment, POC-INRs are often performed by nonlaboratory staff with little experience in quality control. The Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) Haematology has devised a POC-INR external quality assessment (EQA) program that is suitable for both laboratory and nonlaboratory operators (e.g., nurses) to perform INR testing with good accuracy and precision. A comparison of the performance of the POC versus the laboratory-derived INR testing over the past 8 years has shown that the variation in test results (expressed as coefficient of variation; CV) for laboratory INRs increases with more prolonged INR values, whereas CVs for the POC-INR testing were generally lower, with a reduced dependency on INR values. In our program, the CoaguChek XS (Roche, Basel, Switzerland) showed the best performance among the POC devices. A comparative assessment with other EQA providers showed agreement and disparity with our data in terms of comparative CVs obtained between the laboratory and POC-INRs. The growth of the RCPAQAP POC-INR program from 29 to 360 in the past 12 years highlights the importance of providing suitable EQA for POC-INR staff who are unfamiliar with laboratory practice. This helps maintaining consistent results, which have important implications for the therapeutic management of patients on vitamin K antagonist therapy.

Laboratory identification of factor VIII inhibitors in the real world: the experience from Australasia

Summary.  The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titres during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either using the original or Nijmegen‐modified Bethesda assay. In view of previously demonstrated high variability in laboratory results for inhibitor assays, we have more extensively examined laboratory performance in the identification of FVIII inhibitors. Over the past 3 years, we conducted two questionnaire‐based surveys and two wet‐challenge surveys utilizing eight samples comprising no FVIII inhibitor (n = 1), or low‐titre (n = 2), medium‐titre (n = 3) or high‐titre (n = 2) FVIII inhibitor. Four samples were tested by 42 laboratories in 2007, and four by 52 laboratories in 2009. High inter‐laboratory variation was evident, with CVs around 50% not uncommon, and some 10% of all laboratories (or around 15% of laboratories using Bethesda method) failed to detect low‐level inhibitors of around 1 BU mL−1. Laboratories using the Nijmegen method appeared to perform better than those using a standard Bethesda assay, with lower evident assay variation and no false negatives. There was a wide variety of laboratory practice, with no two laboratories using exactly the same process for testing and interpretation of factor inhibitor findings. In conclusion, our study indicates that there is still much need for standardization and improvement in factor inhibitor detection, and we hope that our findings provide a basis for future improvements in this area.

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.