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.

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.

Problems and solutions in laboratory testing for hemophilia.

A diagnosis of hemophilia A or hemophilia B begins with clinical assessment of the patient and is facilitated by laboratory testing. The influence of the latter on a diagnosis of hemophilia A or hemophilia B is clear-a diagnosis cannot be made without laboratory confirmation of a deficiency of factor FVIII (FVIII) or factor IX (FIX), respectively. Moreover, the degree of hemophilia severity is specifically characterized by laboratory test results. In turn, patient management, including choice and application of therapies, is influenced by the diagnosis, as well as by identification of respective disease severity. An incorrect diagnosis may lead to inappropriate management and unnecessary therapy, and thus to adverse outcomes. Moreover, identification of factor inhibitors in hemophilia will lead to additional and differential treatments, and incorrect identification of inhibitors or inhibitor levels may also lead to inappropriate management. Problems in hemophilia diagnosis or inhibitor detection can occur at any stage in the clinical diagnosis/laboratory interface, from the "pre-preanalytical" to "preanalytical" to "analytical" to "postanalytical" to "post-postanalytical." This report outlines the various problems in laboratory testing for hemophilia and provides various strategies or solutions to overcome these challenges. Although some outlined solutions are specific to the potential errors related to hemophilia, others are general in nature and can be applied to other areas of laboratory hemostasis. Key to improvement in this area is adoption of best practice by all involved, including clinicians, phlebotomists, and laboratories. Also key is the recognition that such errors may occur, and thus that clinicians should assess laboratory test results in the context of their patient's clinical history and follow-up any potential errors, thus avoid misdiagnoses, by requesting repeat testing on a fresh sample.

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.

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.

The diagnostic dilemma: dual presentations of clinical mucosal bleeding and venous thrombosis associated with the presence of thrombophilia markers and mild reduction in von Willebrand factor.

A prothrombotic and hemorrhagic state can separately manifest in one patient and can potentially cause several diagnostic problems. We report an intriguing case as an example of a potential hemostasis-based diagnostic dilemma. A 29-year-old female patient presented with a personal history of menorrhagia and other mucosal bleeding and renal ovarian thrombosis. Previous investigations had uncovered several diagnostic anomalies, including von Willebrand disease (VWD), factor V Leiden (FVL), antiphospholipid syndrome, and thrombocytopaenia. Previous therapy in this patient included heparin and warfarin for the thrombosis and desmopressin acetate (DDAVP) and antifibrinolytic therapy for surgical management. Subsequent laboratory testing with fresh samples consistently confirmed an equivocal (borderline normal/abnormal) level of von Willebrand factor (VWF) and FVL with activated protein C resistance (APCR). A patient sample, differentially labeled according to the tests being performed, was later distributed for blind testing to participants within several modules of the RCPA Quality Assurance Program (QAP). Most participants reported a low level of VWF consistent with possible mild Type 1 VWD, and most (but not all) reported a positive finding for APCR. All participants correctly reported the sample as heterozygous for the FVL mutation, negative for the Prothrombin gene mutation G20210A, and heterozygous for the methylenetetrahydrofolate reductase (MTHFR) mutation C677T. Interestingly, a significant number of laboratories performing Protein S testing using clot-based procedures also identified a false Protein S deficiency. In conclusion, this exercise showed how, either depending on the clinical review and specific laboratory investigation and tests performed, a pro-bleeding diagnosis (of either VWD or thrombocytopenia) or pro-thrombophilia risk (Antiphospholipid Syndrome or FVL/APCR or false Protein S deficiency) could potentially and differentially arise in the one patient.

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.

Identification of factor inhibitors by diagnostic haemostasis laboratories: a large multi-centre evaluation.

We have assessed the proficiency of diagnostic haemostasis facilities to correctly identify coagulation factor abnormalities and inhibitors. Forty-two laboratories participating in the external Quality Assurance Program (QAP) conducted by the RCPA agreed to participate and were each sent a set of eight samples (each 3 x 1 ml) for evaluation. They were asked to blind test these samples for the presence or absence of inhibitors, and where identified, to perform further analysis (including specific inhibitor analysis). In order to make the exercise more challenging, in addition to true factor inhibitors, samples were provided that reflected potential pre-analytical variables that might arise and complicate inhibitor detection or lead to false inhibitor identification. In brief, the sample set comprised a true high level factor (F) V inhibitor, a true moderate level FVIII inhibitor (but sample was defibrinogenated), a true lupus anticoagulant (LA), a normal (but slightly aged) plasma sample, a normal serum sample, a normal EDTA sample, an oral anticoagulant/vitamin K deficiency sample, and a gross heparin ( approximately 10 U/ml) contaminated sample. Sixty-three percent of participants correctly identified the true FV inhibitor as such, although the reported range varied greatly [10 to >250 Bethesda units (BU/ml)] and 46% correctly identified the true FVIII inhibitor, despite the complication of the sample presentation, although the reported range also varied (7 to 64 BU/ml). Some laboratories either failed to identify the inhibitor present, or misidentified the inhibitor type. The LA, the oral anticoagulant/vitamin K deficiency, the normal serum sample, and the normal (aged) sample were also correctly identified by most laboratories, as was the absence of specific factor inhibitors in these samples. However, a small subset of laboratories incorrectly identified the presence of specific factor inhibitors in some of these samples. The heparin sample was also correctly identified by most (68%) laboratories. In contrast, the normal EDTA sample was misidentified as a FV and/or FVIII inhibitor by most (68%) laboratories, and only one laboratory correctly identified this as an EDTA sample. Thus, we conclude that although laboratories are able, in most cases, to identify the presence of true factor inhibitors, there is a large variation in identified inhibitor levels and there are also some significant errors in identification (i.e. false negatives and misidentifications). In addition, there is a significant false positive error rate where some laboratories will identify the presence of specific factor inhibitors where no such inhibitor exists (i.e. false positives).

An international survey of current practice in the laboratory assessment of anticoagulant therapy with heparin.

AIMS: We conducted a survey of laboratory practice for assessment of heparin anticoagulant therapy by participants of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP). METHODS: A questionnaire was sent to 646 laboratories enrolled in the Haematology component of the QAP, requesting details of tests used for monitoring heparin therapy. RESULTS: Seventy laboratories (10.8%) returned results that indicated that they performed laboratory monitoring of heparin therapy. Most laboratories (69/70 = 98.6%) use the activated partial thromboplastin time (APTT) to monitor unfractionated heparin, with eight (11.4%) also using the APTT for monitoring low molecular weight (LMW) heparin. Five (7.1%) laboratories use the thrombin time (TT) test to help monitor heparin therapy and 37 (52.9%) laboratories use an anti-Xa assay to monitor heparin (either LMW or unfractionated). Normal reference ranges (NRR) for APTT differed considerably between laboratories, even those using the same reagent. Therapeutic ranges (TR) also differed considerably between laboratories, for both APTT and the anti-Xa assay. Laboratory differences in NRR and TR using the same reagents could only be partly explained by the use of different instrumentation. CONCLUSIONS: There is a large variation in current laboratory practice relating to monitoring of heparin anticoagulant therapy. This finding is similar to that of a similar survey conducted by the RCPA QAP almost a decade ago. This study suggests that better standardisation is still required for laboratory monitoring of heparin therapy.

How useful is the monitoring of (low molecular weight) heparin therapy by anti-Xa assay? A laboratory perspective.

We have conducted a series of laboratory-based surveys to assess variability in assay results utilized to monitor heparin anticoagulant therapy. These surveys involved laboratories participating in the Haematology component of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP). Thirty five of 646 laboratories that were sent a preliminary questionnaire indicated that they performed anti-Xa assays and these laboratories were sent a panel of four plasma samples. These plasma samples contained respectively: (i) no added heparin, (ii) low molecular weight heparin (LMWH), enoxaparin, added to a level of approximately .5 U/mL, (iii) unfractionated heparin added to a level of approximately .5 U/mL, and (iv) LMWH added to a level of approximately 1.0 U/mL. Tests to be performed were the activated partial thromboplastin time (APTT), the thrombin time (TT), fibrinogen, and anti-Xa. As expected, returned results for APTT and TT showed some elevation in heparinized samples while fibrinogen assays were not affected. Anti-Xa assays yielded the following results (median [range]): (i) .01 [0-.11], (ii) .43 [.33-.80], (iii) .23 [.10-.49], and (iv) .90 [.60-1.30]. Thus, although median values were close to those anticipated, there was a wide variation in returned results. In a repeat exercise a few months later laboratories were also asked about their therapeutic ranges (TRs) and provided with an additional vial of LMWH-spiked (1.0 U/mL) plasma labeled as 'heparin-standard' to be used as an assay calibrant. TRs varied substantially between laboratories, from low ranges of .2-.4 to high ranges of .8-1.2. Anti-Xa assay results were similar to those of the first survey: (median [range]): (a) repeat testing: (i) .02 [0-.28], (ii) .47 [.34-.80], (iii) .25 [.14-.58], (iv) .95 [.65-1.31]; (b) repeat testing using survey provided 'heparin-standard': (i) .02 [0-.24], (ii) .55 [.4-.83], (iii) .28 [.10-.63], (iv) 1.00 [.9-1.16]. Thus using the provided 'heparin-standard' yielded lower variability in results for LMWH. In conclusion, the high variability of anti-Xa assay results coupled with the widely variable TRs suggests that therapeutic heparin monitoring is poorly standardized, and this raises some concerns over the clinical value of such monitoring.

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.

Multilaboratory testing of thrombophilia: current and past practice in Australasia as assessed through the Royal College of Pathologists of Australasia Quality Assurance Program for Hematology.

We have conducted a series of multilaboratory surveys during the last 6 years to evaluate testing proficiency in the detection of congenital and acquired thrombophilia. For lupus anticoagulant (LA) testing, participant laboratories used a panel of tests, including activated partial thromboplastin time (aPTT; 100% of laboratories), kaolin clotting time (26 to 70%), and Russell's viper venom time (RVVT; 75 to 100%). Coefficients of variation (CVs) for assays ranged from 5 to 40%. RVVT assays appeared to be most sensitive and specific for detection of LA (fewer false-negatives or -positives), although laboratories performed best when they used a panel of tests. For congenital thrombophilia, tests evaluated comprised protein C (PC), protein S (PS), antithrombin (AT), and activated protein C resistance (APCR). Most participant laboratories performed PC using chromogenic (approximately 75%), or clot based (approximately 15%) assays, with few (< 10%) performing antigenic assessments. PS was most often assessed (approximately 60%) by immunological or antigenic assays, usually of free PS, or by functional or clot-based assays (approximately 40%). AT is usually assessed by functional chromogenic assays (approximately 95%). APCR was assessed using aPTT (approximately 50%) or RVVT (approximately 50%) clot-based assays, with the aPTT APCR typically performed using factor V-deficient plasma predilution, but the RVVT APCR typically performed without. Laboratories using the RVVT APCR generally performed better in detection of factor V Leiden-associated APCR, with the aPTT method group yielding higher false-negative and/or false-positive findings (approximately 5% of occasions). Some clot-based PC and PS assays appeared to be influenced by APCR status, and yielded lower apparent PC and PS levels with positive APC resistance. The overall error rate for PC, PS, and AT was approximately 2 to 8% (i.e., false-normal interpretations for deficient plasma or false-abnormal interpretations for normal plasma). The CVs for these assays ranged from 5 to 40%, with highest CVs typically obtained with PS assays.

Laboratory diagnosis of von Willebrand disorder. Current practice in the southern hemisphere.

A survey of 44 laboratories was conducted to evaluate current testing proficiency in the diagnosis of von Willebrand disorder (vWD) and to assess recent changes in test practices. Laboratories performed their usual panel of tests for vWD and interpreted results for the likelihood of vWD and potential subtype. Samples were as follows: normal plasma; borderline normal or abnormal levels of von Willebrand factor (vWF) and factor VIII; type 3 vWD; type 2A vWD; and 2 samples from a healthy person, processed after handling at 22 degrees C and 4 degrees C, respectively. Interassay and within-method coefficients of variation were similar for all assays (approximately 15%-25%). Most laboratories reported test values consistent with expected findings and made correct interpretations, although discrepant results for 5% to 10% of responses are of concern. For the sample stored at 4 degrees C, all laboratories detected low or borderline levels of vWF and factor VIII coagulant, and no laboratory identified this sample as from a healthy person. In contrast, for the sample stored at 22 degrees C, most laboratories reported normal results. Compared with previous results, performance of some assays has declined while that of others has increased. Laboratories generally are proficient in tests for vWD, and transport of samples at 4 degrees C before processing may lead to false identification of vWD, suggesting that NCCLS guidelines should be reviewed.