Diagnosis of von Willebrand disease: An assessment of the quality of testing in North American laboratories.

BACKGROUND: Laboratory diagnosis of von Willebrand Disease (VWD) is complex. Reliance on laboratory testing can be problematic as different VWD screening panels, assays and methodologies can produce analytic variability in test results. OBJECTIVES: To compare the degree of imprecision among the VWD assays and within the platelet binding activity (PBA) assays, to determine the consensus among the VWD assays for correct classification of sample results, and to determine consensus among laboratories' von Willebrand factor (VWF) multimer interpretations and final interpretations of the VWD panels. PATIENTS/METHODS: Proficiency testing results from the North American Specialized Coagulation Laboratory Association (NASCOLA) submitted by laboratories from 2010 to 2019 for all normal, type (T) 1 VWD and T2 VWD samples were analysed. RESULTS AND CONCLUSIONS: Imprecision was lowest for VWF antigen and highest for collagen binding activity (CBA) with median coefficient of variation (CV) of 12% (interquartile range (IQR) 7%) and 23% (IQR 21%) respectively. Within the VWF PBA assays, the gain-of-function mutant GP1b binding (VWF: GP1bM) methods had the least imprecision (CV 9%, IQR 10%). All assays, including the various PBA methods had excellent consensus. The majority of laboratories agreed that normal (median consensus-82%, IQR 16%) and T1 VWD (median consensus-100%, IQR 9%) samples had normal multimer distribution. Consensus among laboratories for final interpretations was excellent for normal samples (median 81%, IQR 8%), good for T1 VWD samples (median 59%, IQR 9%), and fair for T2 VWD samples (median 44%, IQR 21%). Consensus on final interpretation decreased as sample complexity increased.

Heparin-induced thrombocytopenia: An international assessment of the quality of laboratory testing.

BACKGROUND: Accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential to ensure timely treatment and prevent complications. Current diagnostic assays include enzyme-linked immunosorbent assays (ELISAs) and rapid immunoassays (RIs). RIs offer fast turnaround times but were not significantly represented in previous external proficiency testing challenges. OBJECTIVES: To use external proficiency testing to assess qualitative concordance for heparin/PF4 antibody detection. METHODS: From 2013 to 2017, the External Quality Control for Assays and Tests (ECAT) Foundation distributed 10 samples internationally. RESULTS: In total, 437 laboratories submitted 3149 results. ELISAs accounted for 1484 (47%) responses with RIs accounting for 1665 (53%) responses. RI use increased over the 5-year period. ELISAs classified 96% of both consensus positive and consensus negative samples concordantly. The coefficient of variation (CV) for positive sample optical densities (ODs) ranged from 35% to 50% when combining ELISA assay methods together. Quantitative RIs classified 97% of consensus-positive and 98% of consensus-negative samples concordantly. Qualitative RIs had a higher proportion of discordant responses and classified 88% of consensus-positive samples and 73% of consensus-negative samples concordantly. Of RIs only latex immunoassays and IgG specific chemiluminescent assays identified > 95% of samples concordantly with consensus. CONCLUSION: Quantitative RIs and ELISAs classify > 95% of samples concordantly. The ODs from different ELISA methods vary considerably and are not interchangeable. Qualitative RI use is increasing despite a greater proportion of discordant classifications. This includes a higher than expected number of negative classifications for consensus-positive samples among many RIs, challenging their use as "rule out" tests.

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.

An Assessment of the State of Current Practice in Coagulation Laboratories.

OBJECTIVES: To assess the state of current practice in coagulation laboratories regarding three pressing issues: staffing, handling Ebola specimens, and testing/billing for tests that measure direct oral anticoagulants (DOAC). METHODS: A survey and analysis of specialized coagulation laboratories in North America was conducted. RESULTS: Approximately 4,000 special coagulation tests-per-technologist-per-year was rated as either a "good" staffing level or "adequate-but-ideally-need-more" employees. Requiring technologists to perform more than that was rated as an "inadequate" staffing level. For Ebola patients, coagulation testing is mostly performed by point-of-care. Only 26.1% would perform coagulation tests for Ebola specimens within their laboratory (rather than at the bed side or a separate designated space outside the laboratory). Coagulation tests offered for Ebola patients were limited: prothrombin time (63.0% of laboratories), activated partial thromboplastin time (37.0%), D-dimer (13.0%), and fibrinogen (8.7%); 26.1% of laboratories did not offer any coagulation tests for Ebola patients. Approximately 35% of special coagulation laboratories bill for at least one laboratory test for DOACs: 33% bill for an anti-Xa calibrated with rivaroxaban, 17% bill for an anti-Xa calibrated with apixaban, and 27% bill for at least one of several tests for dabigatran. Approximately 48% do not offer any tests for DOACs. CONCLUSIONS: These results may help laboratories negotiate for additional technologists if needed, prepare for Ebola specimens, and manage the demand for laboratory tests for new DOAC anticoagulants.

External quality assessment of platelet disorder investigations: results of international surveys on diagnostic tests for dense granule deficiency and platelet aggregometry interpretation.

The quality of platelet aggregation and dense granule deficiency testing is important for diagnosing platelet function disorders. After a successful pilot exercise on diagnosing platelet dense granule deficiency by electron microscopy (EM), the North American Specialized Coagulation Laboratory Association (NASCOLA) has launched regular external quality assurance (EQA) for dense granule EM, as well as for the interpretation of platelet aggregation findings. EQA records were analyzed to assess performance. For EM EQA, between 2009 and 2011, there was excellent performance in distinguishing normal from dense granule-deficient samples and good (>70%) agreement on classifying most electron dense structures in platelets. For aggregation EQA, some normal variants were misclassified and overall case interpretations were more acceptable for rare disorders than for common findings. NASCOLA experiences with these EQAs indicate that there is a need to improve the quality of platelet disorder evaluations. For aggregometry interpretations, deficits in performance could be addressed by translating guideline recommendations into practice.

Assessment of a cohort of primarily pediatric patients with a presumptive diagnosis of type 1 von Willebrand disease with a novel high shear rate, non-citrated blood flow device.

BACKGROUND: A precise approach to the diagnosis of von Willebrand disease (vWD) remains elusive. One important reason is that vWD is a blood flow-related disorder: a vW Factor-platelet GPIb binding defect exists in this condition under the high shear-rate (> 1000 sec-1 in whole blood; > 3000 sec-1 in PRP) conditions of physiologic blood flow which exist in the arterioles of mucous membranes, from which most bleeding in vWD occurs. METHODS: We therefore studied 28 patients (mean 18.9 yrs) with vWD, diagnosed according to the 2007 NHLBI clinical guidelines, and 26 healthy controls (mean 17.5 yrs). Blood was collected into a plastic tube containing 4 U/ml FC dalteparin, 1.75 µg/ml of the Tab (anti-CD41) monoclonal antibody directed against platelet GPIIb, and 1.0 µg/ml of an ALEXA 555-conjugated rabbit anti-mouse second antibody. Within 30-90 min, the blood was then withdrawn at 667 and 1330 sec(-1) through a special flow chamber allowing for real-time epifluorescence digital videomicroscopy of platelets interacting with a microfibrillar collagen substrate. With MetaMorph software (Universal Imaging) we quantified the percent area (PA) covered by and total volume (TV) of adherent platelet aggregates within a 435 µm × 580 µm field of view. RESULTS: At 667 sec(-1) after 1 min PA and TV were similar for patients and controls, but at 1330 sec(-1) PA was 9.32 ± 4.21 (mean ± SD) for patients, a value lower (p < 0.001) than the 12.8 ± 3.39 for controls. TV was (1.43 ± 0.91) x 10(4) for patients, a value also lower (p < 0.001) than the (2.22 ± 0.77) x 10(4) for controls. PA or TV was below the 2.5th percentile for controls in 10 patients (36%) and both PA and TV were below the 2.5th percentile in eight. CONCLUSIONS: The novel flow device found that PA and TV were significantly reduced under high shear stress in vWD patients compared to normal controls. However, there was some overlap between the vWD and the control group, suggesting that some vWD patients had normal platelet adhesion/aggregation under the conditions studied. Further study with a higher shear rate appears indicated.

Laboratory assessment of factor VIII inhibitor titer: the North American Specialized Coagulation Laboratory Association experience.

Quantification of inhibitory antibodies against infused factor VIII (FVIII) has an important role in the management of patients with hemophilia A. This article summarizes results from the largest North American FVIII inhibitor proficiency testing challenge conducted to date. Test samples, 4 negative and 4 positive (1-3 Bethesda units [BU]/mL), were distributed by the ECAT Foundation in conjunction with the North American Specialized Coagulation Laboratory Association and analyzed by 38 to 42 laboratories in 2006 and 2007. Whereas laboratories were able to distinguish between the absence and presence of low-titer FVIII inhibitors, the intralaboratory coefficient of variation was high (30%-42%) for inhibitor-positive samples, and the definition of lower detection limits of the assay was variable (0-1 BU/mL). Most laboratories performed the Bethesda assay with commercially supplied buffered normal pooled plasma in a 1:1 mix with patient plasma. These data provide information for the development of consensus guidelines to improve FVIII inhibitor quantification.

Performance evaluation of the CellaVision DM96 system: WBC differentials by automated digital image analysis supported by an artificial neural network.

We evaluated the CellaVision DM96 (CellaVision AB, Lund, Sweden), an automated digital cell morphology and informatics system for peripheral blood smears. Technologists agreed with 82% of the instrument's preclassifications. Correlation coefficients between final results released from the CellaVision and results obtained by direct microscopy were 0.96 (all neutrophils), 0.94 (lymphocytes), 0.88 (segmented neutrophils), 0.73 (eosinophils), 0.69 (bands), and 0.67 (monocytes). After correction for statistically and clinically insignificant variations, the CellaVision DM96 had 95% sensitivity and 88% specificity for immature myeloid cells. It was 100% sensitive and 94% specific for blasts, and 100% sensitive and 97% specific for unusual WBCs and nucleated RBCs. Advantages of the CellaVision DM96 over direct microscopy include the ability to review slides from a remote location, consultation and quality control on a cell-by-cell basis, and potential labor savings.