Laboratory testing for activated protein C resistance: rivaroxaban induced interference and a comparative evaluation of andexanet alfa and DOAC Stop to neutralise interference.

Background Investigation of hemostasis is problematic when patients are on anticoagulant therapy. Rivaroxaban especially causes substantial interference, extending many clot-based tests, thereby leading to false positive or negative events. In particular, rivaroxaban affects some assays for activated protein C resistance (APCR). Methods We assessed, in an international setting, cross laboratory (n = 31) testing using four samples to evaluate rivaroxaban induced interference in APCR testing, and whether this interference could be neutralised. The samples comprised: (A) pool of normal plasma (APCR-negative control); (B) this normal pool spiked with rivaroxaban (200 ng/mL) to create rivaroxaban-induced interference (potential 'false' positive APCR event sample); (C) the rivaroxaban sample subsequently treated with a commercial direct oral anticoagulant 'DOAC-neutraliser' (DOAC Stop), or (D) treated with andexanet alfa (200 µg/mL). Testing was performed blind to sample type. Results The rivaroxaban-spiked sample generated false positive APCR results for some, but unexpectedly not most APCR-tests. The sample treated with DOAC Stop evidenced a correction in the rivaroxaban-affected APCR assays, and did not otherwise adversely affect the rivaroxaban 'unaffected' APCR assays. The andexanet alfa-treated sample did not evidence correction of the false positive APCR, and instead unexpectedly exacerbated false positive APCR status with many tests. Conclusions DOAC Stop was able to neutralise any APCR interference induced by rivaroxaban. In contrast, andexanet alfa did not negate such interference, and instead unexpectedly created more false-positive APCR events.

Reducing the effect of DOAC interference in laboratory testing for factor VIII and factor IX: A comparative study using DOAC Stop and andexanet alfa to neutralize rivaroxaban effects.

INTRODUCTION: Investigation of factors (F) VIII and IX is common, with testing important for diagnosis or exclusion of haemophilia A or B, associated acquired conditions and factor inhibitors. Rivaroxaban, a common direct anti-Xa agent, causes significant interference in clotting assays, including substantial false reduction of factor levels. AIM: To assess whether rivaroxaban-induced interference of FVIII and FIX testing could be neutralized. MATERIALS AND METHODS: An international, cross-laboratory exercise for FVIII (n = 84) and FIX (n = 74), using four samples: (A) pool of normal plasma; (B) pool spiked with rivaroxaban (200 ng/mL); (C) rivaroxaban sample subsequently treated with 'DOAC Stop' and; (D) rivaroxaban sample treated with andexanet alfa (200 µg/mL). Testing performed blind to sample type. RESULTS: All laboratories reported normal FIX and 94% reported normal FVIII in the pool sample. Instead, 55% and 95%, respectively, reported abnormal FIX and FVIII levels for the rivaroxaban sample. DOAC Stop treatment evidenced a correction in most laboratories (100% reported normal FIX and 86% normal FVIII). Andexanet alfa provided intermediate results, with many laboratories still reporting abnormal results (59% for FVIII, 18% for FIX). We also identified reagent-specific issues. CONCLUSIONS: As expected, rivaroxaban caused false low values of FVIII and FIX. This might lead to increased testing to identify the cause of low factor levels and potentially lead to false identification of (mild) haemophilia A or B if unrecognized by clinicians/laboratories. DOAC Stop effectively neutralized the rivaroxaban effect, but andexanet alfa less so, with reagent-related effects evident, and thus, false low values sometimes persisted.

An update on quality control for the PFA-100/PFA-200.

Testing of platelet function comprises a crucial element of hemostasis assessment, particularly for investigations into bleeding and/or bruising. The Platelet Function Analyzer (PFA)-100 is the most utilized primary hemostasis-screening test system available, as recently remodeled/upgraded to the PFA-200. Internal quality control (IQC) and external quality assessment (EQA) (including proficiency testing) represent critical elements of ensuring test practice quality. Although true for all tests, IQC and EQA are logistically challenging for platelet function testing, inclusive of the PFA-100/200. We accordingly update our experience with novel yet feasible approaches to both IQC and EQA of PFA-100/200. Over the past 10 years, a total of 43 challenges have been tested, with most challenges designed to mimic moderate or severe primary hemostasis defects. The current report is restricted to the last four years and has also differentially assessed PFA-100 vs. PFA-200 EQA results to identify potential variance. Numerical results for closure times (CTs) and participant-supplied interpretive comments were analyzed. Reported CTs for each challenge were within limits of expectation, and good reproducibility was evidenced by repeated challenges. Coefficients of variation (CVs) for challenges, generally ranging from 15% to 25%, were similar or better than those obtained using native whole blood and consistent with past reports. Participant interpretations were generally consistent with test data and expectations. There was no evident difference in PFA-100 vs. PFA-200 EQA test results. The EQA material has also been successfully evaluated from the perspective of potential IQC. To conclude, IQC and EQA processes for the PFA-100/200 have been established that are highly reproducible, supporting the concept of EQA/IQC for platelet function testing, and also facilitating monitoring and improvement in its performance. In terms of EQA, PFA-100 and PFA-200 instruments appear to behave similarly.

Differential sensitivity of von Willebrand factor activity assays to reduced VWF molecular weight forms: A large international cross-laboratory study.

INTRODUCTION: von Willebrand disease (VWD), the most common inherited bleeding disorder, is due to deficiencies/defects in von Willebrand factor (VWF). Effective diagnosis requires testing for FVIII, VWF antigen and one or more VWF 'activity' assays. Classically, 'activity' is assessed using ristocetin cofactor (VWF:RCo), but collagen binding (VWF:CB) and/or other assays are used by many laboratories. This extensive international cross-laboratory study has specifically evaluated contemporary VWF activity assays for comparative sensitivity to reduction in high molecular weight (HMW) VWF, and their ability to differentiate type 1 vs 2A VWD-like samples. MATERIALS AND METHODS: A set of four samples representing step wise reduction in HMW VWF were tested by over 400 laboratories worldwide using various assays. A second set of two samples representing type 1 or type 2A VWD-like plasma was tested by a subset of 251 laboratories. RESULTS: Combined data identified some differences between VWF activity assays, with sensitivity for reduction of HMW being highest for VWF:CB and VWF:GPIbM, intermediate for VWF:RCo and VWF:GPIbR, and lowest for VWF:Ab. 'Within' method analysis identified the Stago method as the most sensitive VWF:CB assay. A large variation in inter-laboratory CV (e.g., 7-24% for the normal sample) was also demonstrated for various methods. Although performance of various methods differed significantly, most laboratories correctly differentiated between type 1 and 2 samples, irrespective of VWF activity assay employed. CONCLUSIONS: These results hold significant clinical implications for diagnosis and therapy monitoring of VWD, as well as potential future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura (TTP).

Towards harmonization of external quality assessment/proficiency testing in hemostasis.

Quality in diagnostic testing represents a key target of laboratory medicine, for which an assurance around the quality of testing is expected from all involved in the process. Laboratories attempt to assure the quality of their testing by various processes, but especially by performance of internal quality control and external quality assessment (EQA). This is especially true for tests of hemostasis and coagulation. EQA in general provides information on test accuracy and on evaluation of long-term laboratory performance. EQA providers support laboratory performance by various means, including distribution of material for testing of analytes ("proficiency testing"), educational support through expert advice, distribution of publications or case series. Participation in EQA is often a laboratory accreditation requirement. This review aims to identify some of the strengths and weaknesses of EQA, and targets attempts towards harmonization of EQA practice, in order to achieve the best outcome for participant laboratories and, thus, for patients and their clinical care providers.

Lack of grading agreement among international hemostasis external quality assessment programs.

: Laboratory quality programs rely on internal quality control and external quality assessment (EQA). EQA programs provide unknown specimens for the laboratory to test. The laboratory's result is compared with other (peer) laboratories performing the same test. EQA programs assign target values using a variety of methods statistical tools and performance assessment of 'pass' or 'fail' is made. EQA provider members of the international organization, external quality assurance in thrombosis and hemostasis, took part in a study to compare outcome of performance analysis using the same data set of laboratory results. Eleven EQA organizations using eight different analytical approaches participated. Data for a normal and prolonged activated partial thromboplastin time (aPTT) and a normal and reduced factor VIII (FVIII) from 218 laboratories were sent to the EQA providers who analyzed the data set using their method of evaluation for aPTT and FVIII, determining the performance for each laboratory record in the data set. Providers also summarized their statistical approach to assignment of target values and laboratory performance. Each laboratory record in the data set was graded pass/fail by all EQA providers for each of the four analytes. There was a lack of agreement of pass/fail grading among EQA programs. Discordance in the grading was 17.9 and 11% of normal and prolonged aPTT results, respectively, and 20.2 and 17.4% of normal and reduced FVIII results, respectively. All EQA programs in this study employed statistical methods compliant with the International Standardization Organization (ISO), ISO 13528, yet the evaluation of laboratory results for all four analytes showed remarkable grading discordance.

Overview of Hemostasis and Thrombosis and Contribution of Laboratory Testing to Diagnosis and Management of Hemostasis and Thrombosis Disorders.

Hemostasis is a complex and tightly regulated process whereby the body attempts to maintain a homeostatic balance to permit normal blood flow, without bleeding or thrombosis. When this balance is disrupted, due to trauma or underlying congenital bleeding or thrombotic disorders, clinical intervention may be required. To assist clinicians in diagnosing and managing affected patients, hemostasis laboratories offer an arsenal of tests, both routine (screening) and more specialized (diagnostic). In general, screening assays are used to screen for hemostasis-related disease or to monitor or measure the effect of anticoagulant therapy, which may be applied to treat patients with recent thrombosis or at risk of thrombosis. Diagnostic assays are used to diagnose or exclude specific hemostasis-related diseases, and in some cases, to monitor or measure the effect of anticoagulant therapy, or alternatively procoagulant therapy that may be applied to those at risk of bleeding. This chapter provides an overview of hemostasis and thrombosis with respect to laboratory tests that may be applied to affected patients.

Explaining and reducing the variation in inter-laboratory reported values for International Normalised Ratio.

INTRODUCTION: Monitoring of vitamin K antagonist (VKA) therapy is usually achieved using the International Normalised Ratio (INR). However, despite international standardisation, there remains considerable concern regarding ongoing high levels of inter-laboratory variation, as generated by different laboratories using the same homogeneous plasma sample. Notably, significant discrepancies continue to be evidenced in external quality assessment (EQA) environments, prompting additional investigations to determine causes and to identify potential inconsistencies of practice. MATERIALS AND METHODS: Several investigations involving all 580 participants in the Haemostasis program of the RCPAQAP Haematology were undertaken from 2009 to 2016, gathering details of methodology, and comparative assessments of INR values differentially obtained directly from participants versus values calculated using raw data for PT, ISI and MNPT provided by the same participants. RESULTS: Up to 6% of laboratories reported substantially different INR results compared to results calculated using differentially provided ISI, MNPT and PT data in 6 out of 8 surveys in 2009, highlighting discrepancies in ISI and MNPT values reported vs used by laboratories. Subsequent highlighting of issues to laboratories led to significant improvements in later surveys, with <1% of laboratories yielding different values in 2012, 2013 and 2016. CONCLUSIONS: Our study identified that pre- or post- analytical errors explained a large proportion of inter-laboratory variation in INR. These errors can lead to serious clinical consequences if such data discrepancies are applied to patients, with incorrectly reported INRs potentially leading to altered warfarin therapy. Further education in the importance of the INR process appears warranted.

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.

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 laboratory's 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.

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/proficiency testing and internal quality control for the PFA-100 and PFA-200: an update.

Platelet function testing is an essential component of comprehensive hemostasis evaluation within the framework of bleeding and/or bruising investigations, and it may also be performed to evaluate antiplatelet medication effects. Globally, the platelet function analyzer (PFA)-100 (Siemens Healthcare, Marburg, Germany) is the most used primary hemostasis-screening instrument and has also been recently remodeled/upgraded to the PFA-200. The PFA-100 is sensitive to a wide range of associated disorders, including platelet function defects and von Willebrand disease (VWD), as well as to various antiplatelet medications. The PFA-100 is also useful in therapy monitoring, especially in VWD. External quality assessment (EQA) (or proficiency testing) and internal quality control (IQC) are critical to ensuring quality of test practice, inclusive of all hemostasis tests. However, both EQA and IQC for platelet function testing, including the PFA-100, is logistically challenging, given theoretical requirements for production, storage, and shipment of large volumes of "stabilized" normal and pathological blood/platelets covering both normal function plus a wide variety of potential defects. We accordingly describe the development and testing of novel feasible approaches to both EQA and IQC of PFA-100/PFA-200 instruments, whereby a range of formulated "platelet function antagonist" materials are utilized. For EQA purposes, these are distributed to participants, and citrated normal whole blood collected on site is then added locally, thereby creating test material that can be locally evaluated. Several exercises have been conducted by the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) over the past 6 years. A total of 26 challenges, with most designed to mimic moderate to severe primary hemostasis defects, have been tested in 26 to 50 laboratories depending on the year of dispatch. Numerical results for PFA-100/PFA-200 closure times (CTs) and interpretive comments supplied by participants are analyzed by the RCPAQAP. During this period, reported CTs for each challenge were within limits of expectation and good reproducibility was evidenced by repeated challenges. Coefficients of variation (CVs) generated for challenges using the two major PFA-100/PFA-200 cartridge types (collagen/adenosine diphosphate and collagen/epinephrine) are always similar to those obtained using native whole blood, and in general range from 15 to 25%. Interpretations are also in general consistent with expectations and test data provided by laboratories. The EQA created material has also been assessed within the context of possible IQC material. In conclusion, EQA and IQC processes for the PFA-100/PFA-200 have been developed that include highly reproducible test challenge processes, not only supporting the concept that EQA/IQC is possible for platelet function testing but also providing a valuable mechanism for monitoring and improving laboratory performance in this area.

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.

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.

Proficiency testing/external quality assurance for the PFA-100(®).

BACKGROUND: Platelet function testing is integral to haemostasis investigations and the Platelet Function Analyser-100 (PFA-100(®)) is globally the most utilised primary haemostasis-screening instrument. External Quality Assurance (EQA) (or proficiency testing) is critical to ensuring quality of test practice, but EQA for platelet function is logistically challenging and actual test-challenges generally not possible. METHODS: A novel approach was therefore developed whereby a range of formulated test tubes are distributed to EQA participants to which citrated normal whole blood collected on site is added, thereby creating test material that can be locally evaluated. Several exercises have been conducted over the past four years (total of 18 challenges, most designed to mimic an aspirin effect or a mild or severe primary haemostasis defect, tested in 26-47 laboratories). RESULTS: Numerical results for PFA-100(®) closure times (CTs) and interpretive comments provided by participants were analysed. Reported CTs for each challenge were within limits of expectation and good reproducibility was evidenced by repeated challenges. Coefficients of variation (CVs) generated for two PFA-100(®) cartridge types (C/ADP and C/Epi) for challenges [median (range): 14.8 (3.9-29.5) and 13.9 (0.6-29.5)] was similar to those obtained using native whole blood [15.6 (14.2-18.9) and 17.3 (13.5-20.5)]. Interpretations were in general also consistent with expectations and test data provided by laboratories. CONCLUSIONS: In conclusion, an EQA process for the PFA-100(®) has been developed that includes a highly reproducible test-challenge process, not only proving the concept is possible for platelet function testing, but also providing a valuable mechanism for monitoring and improving laboratory performance.

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).