The effect of the direct factor Xa inhibitors apixaban and rivaroxaban on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples.

The direct oral anticoagulants (DOACs), now including dabigatran, apixaban and rivaroxaban, have given clinicians alternative options to low molecular weight heparins (LMWHs) and vitamin K antagonist therapy, including warfarin, for the treatment of atrial fibrillation and treatment and prevention of venous thromboembolic (VTE) disease. DOACs have been successfully marketed as not requiring monitoring; however, there will be situations where clinicians will request laboratory testing, including emergency department admissions for haemorrhage or thrombosis, or emergency surgical interventions. We report the results of several Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) surveys using apixaban and rivaroxaban spiked samples to either assess the suitability of certain potential screening or drug-quantifying assays, for assessment of drug presence or absence or measurement of levels, as well as assessing potential interference in a wide variety of haemostasis assays. We also include additional evaluations using ex vivo samples from patients given apixaban and rivaroxaban for various therapeutic reasons. The prothrombin time (PT) and activated partial thromboplastin time (APTT) show better sensitivity with rivaroxaban than apixaban. Anti-Xa assays show good concordance and reproducibility with expected drug levels; however, availability of these assays may be limited to larger institutions. Interference of apixaban and rivaroxaban on haemostasis testing extends beyond routine coagulation assays to encompass a plethora of specialised assays, including factor assays, lupus inhibitor, and FVIII inhibitor estimation. In conclusion, this report highlights the influence of these drugs on most tests performed in haemostasis laboratories, and the potential for some tests to predict the presence, absence or level of these drugs in plasma.

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

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.

A retrospective examination of mean relative telomere length in the Tasmanian Familial Hematological Malignancies Study.

Telomere length has a biological link to cancer, with excessive telomere shortening leading to genetic instability and resultant malignant transformation. Telomere length is heritable and genetic variants determining telomere length have been identified. Telomere biology has been implicated in the development of hematological malignancies (HMs), therefore, closer examination of telomere length in HMs may provide further insight into genetic etiology of disease development and support for telomere length as a prognostic factor in HMs. We retrospectively examined mean relative telomere length in the Tasmanian Familial Hematological Malignancies Study using a quantitative PCR method on genomic DNA from peripheral blood samples. Fifty-five familial HM cases, 191 unaffected relatives of familial HM cases and 75 non-familial HM cases were compared with 758 population controls. Variance components modeling was employed to identify factors influencing variation in telomere length. Overall, HM cases had shorter mean relative telomere length (p=2.9×10-6) and this was observed across both familial and non-familial HM cases (p=2.2x10-4 and 2.2x10-5, respectively) as well as additional subgroupings of HM cases according to broad subtypes. Mean relative telomere length was also significantly heritable (62.6%; p=4.7x10-5) in the HM families in the present study. We present new evidence of significantly shorter mean relative telomere length in both familial and non-familial HM cases from the same population adding further support to the potential use of telomere length as a prognostic factor in HMs. Whether telomere shortening is the cause of or the result of HMs is yet to be determined, but as telomere length was found to be highly heritable in our HM families this suggests that genetics driving the variation in telomere length is related to HM disease risk.

Evaluating errors in the laboratory identification of von Willebrand disease in the real world.

INTRODUCTION: von Willebrand disease (VWD), reportedly the most common bleeding disorder, arises from deficiency and/or defects of von Willebrand factor (VWF). Assessment requires a wide range of tests, including VWF activity and antigen. Appropriate diagnosis including differential identification of qualitative vs quantitative defects has important management implications, but remains problematic for many laboratories and clinicians. METHODS: Data using a large set (n=29) of varied plasma samples comprising both 'quantitative' VWF deficiency ('Type 1 and 3' VWD) vs 'qualitative' defects ('Type 2 VWD') tested in a cross-laboratory setting has been evaluated to assess the ability of real world laboratories to differentially identify these sample types. RESULTS: Different VWF assays and activity/antigen ratios show different utility in VWD and type identification. VWD identification errors were often linked to high inter-laboratory test variation and result misinterpretation (i.e., laboratories failed to correctly interpret their own test panel findings). Thus, moderate quantitative VWF deficient samples were misinterpreted as qualitative defects on 30/334 occasions (9% error rate); 17% of these errors were due to laboratories misinterpreting their own data, which was instead consistent with quantitative deficiencies. Conversely, whilst qualitative VWF defects were misinterpreted as quantitative deficiencies at a similar error rate (~9%), this was more often due to laboratories misinterpreting their data (~50% of errors). For test-associated errors, ristocetin cofactor was associated with the highest variability and error rate, which was at least twice that using collagen binding. CONCLUSION: These findings in part explain the high rate of errors associated with VWD diagnosis.

External quality assessment of factor VIII inhibitor assays.

Inhibitors to coagulation factors cause prolongation of routine hemostasis laboratory test results and have clinical relevance in the management of congenital and acquired hemophilia patients. Factor VIII (FVIII) inhibitors can be either allo-antibodies (in hemophilia A) or auto-antibodies (in acquired hemophilia) directed against FVIII. The most commonly used assays for detecting these inhibitors are the classical Bethesda assay or a modified (Nijmegen) method. Previous laboratory assessments from the Royal College of Pathologists of Australia Quality Assurance Program (RCPAQAP) Haematology and other external quality assessment programs have shown wide variability in FVIII inhibitor results and method performance, as well as a significant degree of false-positive and false-negative interpretations. Despite its limitations, the Bethesda assay is still the primary assay used in laboratories for detecting the presence and strength of a FVIII inhibitor. Therefore, it is of utmost importance that this assay is performed well. The current report reviews the most recent findings from the RCPAQAP Haematology, which show there is still a need for better standardization and improvement in the detection of low-level FVIII inhibitors to ultimately provide better clinical management of affected patients.

External quality assurance for heparin monitoring.

Although there is considerable debate regarding the usefulness of laboratory heparin monitoring, these test processes reflect a substantial portion of hemostasis laboratory activity. Accordingly, external quality assurance (EQA) remains an essential component of such testing, and ensures that laboratories provide the best available service for patient management. This report provides an overview of recent and past EQA related to heparin monitoring using data from the Royal College of Pathologists of Australasia Haematology Quality Assurance Program, and heparin-containing plasma samples with concentrations ranging from 0 to 1.4 U/mL. Laboratory tests evaluated comprised activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen, and anti-Xa assays. Results for APTT and TT testing were largely as expected, showing prolongation with increasing concentrations of heparin. Fibrinogen assays were generally unaffected by the presence of therapeutic heparin levels. Although cross-laboratory median values for the anti-Xa assay were close to target values, substantial interlaboratory variation in results, expressed as coefficient of variation (CV), was observed in all exercises conducted over an 8-year period (5 to 28% for low-molecular weight heparin [LMWH] and 19 to 37% for unfractionated heparin). Duplicate samples sent in consecutive surveys resulted in similar median values. The use of a survey-provided standard as assay calibrant improved CVs in earlier surveys, but not in the most recent survey.

Internal quality control and external quality assurance in testing for antiphospholipid antibodies: Part II--Lupus anticoagulant.

In addition to the presence of appropriate clinical features, the diagnosis of the antiphospholipid antibody syndrome (APS) fundamentally requires the finding of positive antiphospholipid antibody (aPL) test result(s), with these comprising clot-based assays for the identification of lupus anticoagulant (LA) and immunologic ("solid-phase") assays such as anticardiolipin antibodies (aCL) and anti-ß2-glycoprotein I antibodies (aß2GPI). This article is the second of two that review the process for, and provide recommendations to improve, internal quality control (IQC) and external quality assurance (EQA; or proficiency testing) for aPL assays. These processes are critical for ensuring the quality of laboratory test results, and thence the appropriate clinical diagnosis and management of APS. This article covers LA testing. We provide some updated findings from the Royal College of Pathologists of Australasia Haematology Quality Assurance Program, and cover testing results for the past 3 years (2009 to 2011 inclusive). In brief: (1) essentially all laboratories currently perform LA testing using activated partial thromboplastin time (APTT) and dilute Russell viper venom time (dRVVT) methods, and about one-third also employ the kaolin clotting time (KCT); (2) KCT usage has dropped slightly, from around 50% of laboratories in 2009, to around 35% in 2011, presumably reflecting take up of the latest consensus recommendations; (3) other methodologies such as silica clotting time (SCT) and the platelet neutralization procedure (PNP) are only used by <5% of laboratories; (4) interlaboratory coefficients of variation (CVs) are in general moderate, and substantially better than those reported for solid-phase assays such as aCL and aß2GPI, with median (range) values being 11.6% (9.2 to 25.5%) for APTT ratios, 16.7% (10.1 to 19.2%) for KCT ratios, and 11.7% (5.7 to 17.4%) for dRVVT ratios; (5) CVs increase slightly with increasing LA positivity; (6) most laboratories correctly interpreted test findings for LA, reporting normal samples as normal, and LA-positive samples as positive (albeit with varying gradings of positivity); and (7) however, some laboratories found interpretation to be challenging for some samples, namely a weak LA sample (which was reported as normal by around 50% of laboratories) and a very strong LA sample (which was reported as normal by around 10% of laboratories, primarily those that did not perform mixing studies).

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.

Anticipation in familial hematologic malignancies.

We describe a collection of 11 families with ≥ 2 generations of family members whose condition has been diagnosed as a hematologic malignancy. In 9 of these families there was a significant decrease in age at diagnosis in each subsequent generation (anticipation). The mean age at diagnosis in the first generation was 67.8 years, 57.1 years in the second, and 41.8 years in the third (P < .0002). This was confirmed in both direct parent-offspring pairs with a mean reduction of 19 years in the age at diagnosis (P = .0087) and when the analysis was repeated only including cases of mature B-cell neoplasm (P = .0007). We believe that these families provide further insight into the nature of the underlying genetic mechanism of predisposition in these families.

Evidence for a common genetic aetiology in high-risk families with multiple haematological malignancy subtypes.

A family history of a haematological malignancy (HM) is known to be a risk factor for HMs. However, collections of large families with multiple cases of varied disease types are relatively rare. We describe a collection of 12 families with dense aggregations of multiple HM subtypes. Cases were ascertained from a population based study conducted between 1972 and 1980 in Tasmania, Australia. Diagnoses were confirmed through review and re-examination of stored tissue, pathology reports, Tasmanian Cancer Registry and flow cytometry records. Family trees were generated and kinship coefficients were calculated for all pairs of affected individuals. 120 cases were found in these families. Cases diagnosed with chronic lymphocytic leukaemia (CLL) demonstrated the most significantly increased aggregation (P < 0.0001). There was also significant evidence that those individuals diagnosed at an older age (>53 years), did not aggregate together in families with disease that presented at an earlier age (<20 years) (P = 0.009).

Cytogenetic investigations of chronic lymphocytic leukemia.

This study aimed to determine which culture method would yield the highest culture success rate, mitotic index, banding resolution, and abnormality rate in investigation of patients with chronic lymphocytic leukemia (CLL). A range of culture techniques for conventional cytogenetic (CC) analyses was compared: 24-hour unstimulated, 72 hours incubation with additional fetal calf serum, 72 hours stimulation with interleukin 4, 72 hours stimulation with lipopolysaccharide (LPS), 72 hours stimulation with TPA (12-O-tetradecanoylphorbol 13-acetate), and 72 hours stimulation with CpG-oligonucleotide DSP30 + Interleukin-2 (IL-2). CC abnormality rates were also compared to fluorescence in situ hybridization (FISH) results using probes for CLL (LSI D13S319/13q34/CEP 12: LSI ATM/p53). Forty-five samples from 24 patients (consisting of 11 newly diagnosed and 13 previously diagnosed patients) were included. For CC, a 100.0% culture success rate was achieved (n = 45) by means of an EDTA (ethylenediaminetetraacetic acid) peripheral blood sample with an associated 62.5% CC abnormality rate (n = 24). FISH detected an abnormality rate of 75.0% (n = 24). The combined CC and FISH abnormality rate was 87.5% (n = 24). This study demonstrates that CC that uses TPA and DSP30 + IL-2 on EDTA peripheral blood is effective in the investigation of CLL and may be used as a supplement to FISH studies.

Investigations from external quality assurance programs reveal a high degree of variation in the laboratory identification of coagulation factor inhibitors.

The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titers during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either with the original or the Nijmegen-modified Bethesda assay. In addition, several circumstances can arise in which the laboratory may test samples that potentially reflect false identification of factor inhibitors. These include lupus anticoagulants and other events generally related to preanalytical variables, including incorrect sample presentations. This article reviews each of these elements, largely from the perspective of cross-laboratory studies undertaken within the framework of external quality assurance (EQA), a peer-laboratory process that aims to assess the ongoing performance of groups of similar laboratories. This review details the experience of the Royal College of Pathologists of Australasia Haematology Quality Assurance Program, and it also reflects on the experience of other EQA organizations. Our analysis reveals a wide variety of test practice among inhibitor testing laboratories, a wide variation in detected inhibitor levels in cross-tested samples, and substantial evidence of false-positive and false-negative detection of factor inhibitors. These findings hold some significance for the clinical management of patients affected by these inhibitors. There is still much need for standardization and improvement in factor inhibitor detection, and we hope that this report provides a basis for future improvements in this area.

Lower limit of assay sensitivity: an under-recognised and significant problem in von Willebrand disease identification and classification.

UNLABELLED: von Willebrand disease (VWD) is the most common inherited bleeding ailment, and is characterised by low levels of, or abnormal function in, the plasma protein von Willebrand factor (VWF). However, the laboratory testing process is problematic because of both the heterogeneity of VWD and the limitations in the tests used to identify reduced or abnormal VWF. OBJECTIVE: This study reports on the lower levels of sensitivity for the different assays used in the diagnostic process for VWD and their significance in the diagnostic identification and classification ofVWD. METHODS: The RCPA Haematology QAP is an international external quality assurance (EQA) program that includes VWF/VWD testing within one of its special haemostasis modules. Over the past 10 years, over 50 samples have been distributed to participants, including five samples devoid of VWF and derived from either true Type 3 VWD patients or else from commerciallypurchased VWF deficient plasma. Samples were tested blind by study participants, who report back both numerical values (for VWF and Factor VIII:C) and an interpretation regarding whether or not VWD is suggested by laboratory findings, and if so, the probable VWD subtype. RESULTS: Returned data indicates that the lower level of sensitivity (LLS) tends to be around 5-10U/dL for Factor VIII:C, VWF antigen(VWF:Ag), VWFcollagen binding (VWF:CB), and VWF 'activity' (VWF:Act), but canreach 20U/dL or more for VWF ristocetin cofactor (VWF:RCo). There does not appear to be any improvement over the past decade despite ongoing automation of methodology, and indeed, automation does not seem to provide better LLS performance. CONCLUSIONS: Limitations in the LLS of VWD testing have significant implications in terms of the identification and classification of an individual's VWD, given that these laboratory assays are used to identify VWD andhelp characterise functional VWF discordance, and that the majority of severe VWD subtypes have levels of VWF below 20U/dL. Thus, laboratories will sometimes be unable to distinguish whether VWF deficient samples derive from Type 3 VWD or severe Type 1 VWD or even Type 2 VWD.

Mis-identification of factor inhibitors by diagnostic haemostasis laboratories: recognition of pitfalls and elucidation of strategies. A follow up to a large multicentre evaluation.

AIMS: We previously reported the ability of diagnostic haemostasis facilities to identify coagulation factor abnormalities and inhibitors, through a large multi-centre study conducted on behalf of the Royal College of Pathologists of Australasia (RCPA) Quality Assurance Program (QAP). In the current report, additional data evaluation aims to (1) help identify the reasons behind the failures in inhibitor identification, (2) highlight the pitfalls in inhibitor testing, and (3) help elucidate some strategies for overcoming these problems and to assist in better identification and characterisation of inhibitors. METHODS: Forty-two laboratories blind tested a set of eight samples for the presence or absence of inhibitors. These included true factor inhibitors (FVIII and FV), and other samples that reflected potential pre-analytical variables (e.g., heparin contamination, serum, EDTA plasma, aged plasma) that might arise and complicate inhibitor detection or lead to false inhibitor identification. RESULTS: There was a wide scatter of inhibitor results, with false positive and false negative inhibitor identification, and mis-identification of inhibitors (e.g., FVIII inhibitor identified where FV inhibitor present). Further analysis of the pattern of reported laboratory results, including routine coagulation testing and factor profiles, allowed some additional interpretative power to provide strategies that will assist laboratories to improve the accuracy of inhibitor identification in the future. CONCLUSIONS: There are currently occasional lapses in factor inhibitor identification, which this report will hopefully help address.

2B or not 2B? Disparate discrimination of functional VWF discordance using different assay panels or methodologies may lead to success or failure in the early identification of type 2B VWD.

Laboratory proficiency in the identification of functional von Willebrand factor (VWF) discordance in type 2B von Willebrand disease (VWD) was assessed by external quality assurance surveys conducted by the RCPA Haematology QAP, and using six different type 2B VWD plasma samples (three historical and three previously unpublished) tested by up to 52 laboratories. For the three most recent samples, functional VWF discordance was either not identified in testing or by interpretation with misidentification as 'normal' or 'type 1 VWD', on average for 25.7% of test occasions when laboratories performed VWF:Ag and VWF:RCo as their primary VWF test panel, but somewhat fewer occasions (10.9%) for laboratories that incorporated VWF:CB as an additional functional VWF assay. VWF assay sub-methodologies also influenced the appropriate identification of samples as potentially type 2 VWD, and VWF functional discordance was more consistently identified when laboratories used (i) automated platelet agglutination for VWF:RCo compared to classical platelet aggregometry, (ii) inhouse VWF:CB assays compared to commercial kit methods, and (iii) automated LIA-based 'VWF:Activity' assays compared to ELISA based assays. We conclude that:(i) laboratories are generally proficient in tests for VWD but interpretative diagnostic errors do occur; (ii) correct diagnosis is more likely when test panels are more comprehensive and include the VWF:CB; (iii) sub-methodology influences the appropriate identification of VWF functional discordance. On the basis of these findings, we provide a series of recommendations to enable the appropriate laboratory identification of VWD, in particular type 2B VWD.

Utility of the PFA-100 as a screening test of platelet function: an audit of haemostasis laboratories in Australia and New Zealand.

The PFA-100 is a relatively new laboratory instrument, first described in 1995. There have since been numerous studies assessing its utility as a screening tool for platelet dysfunction and/or von Willebrand's disease (VWD). The PFA-100 displays variable sensitivity to different types of platelet disorders, as well as to antiplatelet medication (e.g. aspirin), with similar caveats for monitoring of primary haemostasis-promoting therapies in platelet dysfunction. There is therefore considerable uncertainty regarding its utility within this context, and we have accordingly performed an audit of usage among participants of the Royal College of Pathologists of Australasia Quality Assurance Program. Of 105 laboratories surveyed, 40 responded that they performed platelet function testing, with 26 (65%) further indicating they utilized the PFA-100. We report a wide variety of laboratory usage among these users, including numbers of tests performed [annual median (range) = 270 (15-6000)], sources of requests (clinical sources and localities), testing criteria and follow-up action. Most tests were completed within 4 h of collection, as recommended by the manufacturer, and most tests were performed as a replacement, or as a preliminary screen of platelet function (i.e. classical aggregation). Most abnormal findings, however, were attributed to antiplatelet medication such as aspirin.

Accuracy and clinical utility of the CoaguChek XS portable international normalised ratio monitor in a pilot study of warfarin home-monitoring.

AIM: To evaluate the accuracy of the CoaguChek XS international normalised ratio (INR) monitor compared with the laboratory method. METHODS: The accuracy and ease of use of the recently marketed CoaguChek XS portable INR monitor was evaluated in 17 patients involved in a trial of warfarin home monitoring. INR results from the monitor were compared with those from the laboratory method. Clinical applicability was measured by discrepant INR values, defined in the literature by expanded and narrow agreement criteria, and by the proportion of INR values differing by >15% and by >20% from those derived by the laboratory method. RESULTS: Participants provided 59 comparison INR measurements for analysis. The paired results were highly correlated (r = 0.91). Expanded and narrow agreement between paired INR values occurred 100% of the time. Only three CoaguChek XS (5.1%) results differed by >15% compared with the laboratory method; no results differed by >20% or were discrepant by >0.5 INR units. CONCLUSIONS: In the hands of patients the CoaguChek XS showed good correlation with laboratory determination of INR and compared well with expanded and narrow clinical agreement criteria. Both patients and doctors were highly satisfied with the accuracy and ease of use of the CoaguChek XS.