American Specialized Coagulation Laboratory Association Research Articles

Diagnostic Testing for von Willebrand Disease: Trends and Insights from North American Laboratories over the Last Decade.

Accurate diagnosis of von Willebrand disease (VWD) depends on the quality, precision, and variability of the laboratory assays. The North American Specialized Coagulation Laboratory Association (NASCOLA) is a provider of external quality assessment (EQA) for approximately 60 specialized coagulation laboratories in North America. In this report, NASCOLA EQA data from 2010 to 2021 are reviewed for trends in methodology and precision among various assays. In particular, recent ASH ISTH NHF WFH (American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Hemophilia Federation) guidelines for diagnosis of VWD are reviewed in light of EQA data. In contrast to other geographic regions, laboratories in North America predominantly use three-assay screening panels (antigen, platelet-binding activity, and factor VIII [FVIII] activity) rather than four-assay panels (antigen, platelet-binding activity, FVIII activity, and collagen-binding activity). They also use latex immunoassays rather than chemiluminescence immunoassays, and the classic ristocetin cofactor (VWF:RCo) assay and monoclonal antibody (VWF:Ab) assay to assess VWF platelet-binding activity over newer recommended assays (VWF:GPIbM and VWF:GPIbR). Factors that may be influencing these North American practice patterns include lack of Food and Drug Administration approval of the VWF:GPIbM, VWF:GPIbR, collagen binding assays, and chemiluminescence methodologies, and the influence of the 2008 National Heart, Lung, and Blood Institute guidelines on laboratory practice. Lastly, systems-based solutions are urgently needed to improve the overall accuracy of laboratory testing for VWD by minimizing preanalytical variables and adopting assay standardization.

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

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. 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. 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. 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.

Quality of factor XI activity testing in North American Specialized Coagulation Laboratories.

The performance of factor XI activity (FXI) by laboratories in the North American Specialized Coagulation Laboratory Association proficiency testing program was analyzed. Over 10years (2003-2013), 80 samples were distributed; 33-55 laboratories participated per exercise providing 3833 total responses. Analysis was performed on numeric results and qualitative classification of results. The sample FXI levels ranged from 3.8 to 154.0 IU/dL. The overall interlaboratory average coefficient of variation (CV%) was 17.5%; the CV was higher for a sample with low (3.8 IU/dL) FXI. Results were correctly classified as abnormal (100%) for a sample with 3.8 IU/dL FXI and normal/borderline normal (97.7%) for 45 samples with 80 to<140 IU/dL FXI. The classification was heterogeneous for samples with FXI of 50 to<80 IU/dL. Six specimens were repeat-tested from 2007 to 2013. The mean FXI was not significantly different in laboratories using the same method on both exercises, suggesting good intralaboratory precision over time. Univariate analysis of data from 2011 to 2012 did not find a consistent significant difference among the activators, analyzers, calibrators, and FXI-deficient plasmas. Laboratories generally performed well in assessment of FXI based on interlaboratory precision when FXI >30 IU/dL and on classification of samples with very low or normal FXI.

Proficiency Testing Results for Heparin-Induced Thrombocytopenia in North America

Between 2010 and 2012, North American Specialized Coagulation Laboratory Association (NASCOLA) distributed five proficiency testing challenges to evaluate laboratory testing for heparin-induced thrombocytopenia (HIT). Results (n = 355) were submitted from 43 unique laboratories for 10 samples (3 positive, 2 weak positive, and 5 negative). The vast majority of results were from commercial enzyme-linked immunosorbent assay (ELISA) methods, predominantly polyvalent assays. Laboratories performed well in the classification of clear negative and positive samples. All results (100%) submitted for the five negative samples (n = 173) and 97% of immunological results submitted for the three positive samples (n = 105) were correctly classified (the incorrect responses were two borderline classifications and, from a gel-agglutination method, one negative classification). There was more difficulty in the classification of the two weak positive samples (n = 70). In one survey, 61% of results from the weak positive sample were classified as positive, while 21% were called negative, 16% were called borderline, and 2% were called inconclusive. In a second survey, 16% of results from the weak positive sample were called positive, while 56% were called negative, and 28% were called borderline. Significant interlaboratory variation was observed for ELISA results, with coefficients of variation of about 20 to 30%. We conclude that there is variability in HIT laboratory testing and that identification of weak positive samples can be challenging.

Factor VII assay performance: an analysis of the North American Specialized Coagulation Laboratory Association proficiency testing results

The performance of factor VII (FVII) assays currently used by clinical laboratories was examined in North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency tests. Data from 12 surveys conducted between 2008 and 2010, involving 20 unique specimens plus four repeat-tested specimens, were analyzed. The number of laboratories per survey was 49-54 with a total of 1224 responses. Numerous reagent/instrument combinations were used. For FVII>80 or <40U/dL, 99.5% of results (859/863) were correctly classified by laboratories as normal/abnormal. Classification of specimens with 40-73U/dL FVII was heterogeneous. Interlaboratory precision was better for normal specimens (coefficient of variation (CV) 10.7%) than for FVII<20U/dL (CV 33.1%), with a mean CV of 17.2% per specimen. Intralaboratory precision for repeated specimens demonstrated no significant difference between the paired survey results (mean absolute difference 2.5-5.0U/dL). For specimens with FVII >50U/dL, among commonly used methods, one thromboplastin and one calibrator produced results 5-6U/dL higher and another thromboplastin and calibrator produced results 5-6U/dL lower than all other methods, and human thromboplastin differed from rabbit by +7.6U/dL. Preliminary evidence suggests these differences could be due to the calibrator. For FVII <50U/dL, differences among the commonly used reagents and calibrators were generally not significant.

Critical Result Reporting: Don’t Miss the Forest for the Trees

To the Editor We read with great interest the work of Pai and colleagues,1 which surveyed the critical reporting practice among members of the North American Specialized Coagulation Laboratory Association. Critical result reporting is a contentious area of great activity. Here we would like to highlight several issues. Perhaps one …

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.

Protein C Assay Performance

To determine the performance and frequency of protein C reagents currently used by clinical laboratories, we analyzed North American Specialized Coagulation Laboratory Association (NASCOLA) protein C proficiency testing data from 6 surveys conducted in 2009 and 2010 (2009-1 to 2009-3 and 2010-1 to 2010-3). Interlaboratory coefficients of variation (CV) for commonly used reagents on a survey with normal protein C ranged from 8% to 12% for antigenic assays, from 4% to 7% for chromogenic activity assays, and from 7% to 22% for clot-based activity assays. CVs for commonly used reagents on specimens with abnormal protein C ranged from 15% to 24% for antigenic, 4% to 11% for chromogenic, and 10% to 17% for clot-based assays (averaged across 3 surveys). Some reagents were used by relatively few laboratories and therefore additional study may be needed for those reagents. For all commonly used reagents, biases were usually small and often not statistically significant. All assessed reagents were clinically accurate, and were considered acceptable options for a specialized coagulation laboratory.

Approaches to investigating common bleeding disorders: An evaluation of North American coagulation laboratory practices

Bleeding disorders commonly result from deficiencies or defects in von Willebrand factor (VWF), platelets, coagulation factors, or fibrinolytic proteins. The primary goal of our study was to assess current North American coagulation laboratory practices for diagnosing bleeding disorders, using an on-line patterns-of-practice survey of diagnostic laboratory members of the North American Specialized Coagulation Laboratory Association. The survey examined laboratory approaches to evaluating bleeding disorders, with specific questions about the tests and test panels offered and compliance to recent guideline recommendations on diagnosing von Willebrand disease (VWD) and platelet function disorders. All laboratories responding to the survey performed a prothrombin time/international normalized ratio, an activated partial thromboplastin time, and coagulation factor assays, and many tested for VWD and platelet disorders. However, few laboratories had test panels that evaluated the more common bleeding disorders and few performed some assays, including VWF multimer assessments and assays for fibrinolytic disorders. Additionally, the cutoffs used by laboratories to diagnose type 1 VWD varied considerably, with only a minority following the National Heart Lung Blood Institute recommendations. In contrast, laboratories that tested for platelet function disorders mostly complied with aggregation testing recommendations, as published in the recent North American guidelines. Our results indicate that there are some gaps in the strategies used by laboratories to diagnose bleeding disorders that might be addressed by development of further guidelines and test algorithms that emphasize evaluations for common bleeding disorders. Laboratories may also benefit from guidelines on test interpretation, and external evaluation of their bleeding disorder testing strategies.

Critical Values in the Coagulation Laboratory

Critical values are vital to safe clinical and laboratory practice. To address the lack of information on critical values in coagulation, pattern-of-practice surveys were distributed to members of the North American Specialized Coagulation Laboratory Association. More than 70% of respondents had critical values for commonly performed tests. Median values were as follows: prothrombin time, more than 37 seconds; international normalized ratio, more than 5; activated partial thromboplastin time, more than 100 seconds; and fibrinogen level, less than 100 mg/dL. Critical value reporting generated a significant workload, with up to 15% of these tests yielding critical results. The median time to report critical values was 7 minutes for inpatients. Despite the lack of guidelines surrounding critical values in coagulation, this survey confirms that laboratories have reasonable and uniform practices. It also provides critical value medians and ranges for a wide range of tests. Laboratories without critical values or in the process of reviewing their values may find this survey of their peers useful.

Von Willebrand Factor Assay Proficiency Testing Continued

To the Editor I was very interested to read the recent report from Chandler and colleagues1 on the issue of von Willebrand factor (vWF) assay proficiency testing from the perspective of the North American Specialized Coagulation Laboratory Association (NASCOLA). This excellent report described the experience from North America, in part confirming previous observations from Europe and Australia.2–4 Among the considered findings, the vWF ristocetin cofactor (vWF:RCo) assay yielded the highest imprecision among all vWF assays, and the vWF collagen binding (vWF:CB) assay yielded best overall usefulness in terms of identifying type 2 von Willebrand disease (vWD). As partly explored in the report,1 proficiency testing in Australasia is largely provided by the Royal College of Pathologists of Australasia (RCPA) Quality Assurance programs (QAPs). Notably, the RCPA Haematology QAP has been investigating the issue of proficiency testing in vWF assays for more than a decade, first reporting in this field in 1999,5,6 and most recently in 2008.7 Notably, more than 65 samples have been distributed to participating laboratories, representing a wide diversity of sample types Table 1. Although an update of our findings is due, this correspondence is not …

Von Willebrand Disease Laboratory Diagnosis

von Willebrand disease (vWD) is an autosomal disorder characterized by episodic mucocutaneous bleeding.1 vWD may be the most common inherited bleeding disorder; however, estimates of its prevalence range from 0.01% to 1.0%, depending on the definition and population basis.2 The clinical diagnosis of vWD is complex because symptoms vary within kindred and over time. Furthermore, the most common symptoms, epistaxis, menorrhagia, ecchymoses, and bleeding after dental extraction, occur nearly as frequently in healthy people as in people diagnosed with vWD identified by laboratory detection of known mutations.3 Owing to the difficulties associated with the diagnosis of vWD, the National Heart, Lung, and Blood Institute (NHLBI) sponsored an expert panel that started to plan clinical practice guidelines in the spring of 2004. The panel sought expert consultation and review by several professional organizations such as the American Society of Hematology, the Centers for Disease Control and Prevention, the Food and Drug Administration, and the North American Specialized Coagulation Laboratory Association (NASCOLA). Their final recommendations became available in 2007.1 von Willebrand factor (vWF) is a compound plasma protein that circulates as small (∼2 million Da) to large (∼20 million Da) multimers. vWF, particularly its large multimers, coats the exposed fibrillar collagen of damaged adventitia. The bound vWF expands (“unrolls”) and binds platelet membrane receptor glycoprotein (GP) Ib/V/IX, enabling platelets to adhere to vascular injury sites in arterioles and capillaries. vWD researchers consider the level of vWF activity to be directly proportional to the availability of large vWF multimers, a consideration supported by their presence in plasma samples from healthy subjects and their partial absence from some patients with vWD, as demonstrated using vWF multimeric electrophoresis.4 vWF also binds and stabilizes coagulation factor VIII (FVIII). Binding to collagen, platelets, and FVIII relies on ligand-specific receptors confined to specific …

Development of North American Consensus Guidelines for Medical Laboratories That Perform and Interpret Platelet Function Testing Using Light Transmission Aggregometry

Platelet function testing is important for the diagnostic evaluation of common and rare bleeding disorders. Our study goals were to promote best practices and reduce unnecessary testing variances by developing North American guidelines on platelet function testing. Guidelines were developed by consensus for expert recommendations (minimum level for approval, 70%) that included recommendations on the evaluation and interpretation of light transmission platelet aggregometry (LTA). To assess consensus, medical opinions on recommendations were gathered from diagnostic laboratories that perform LTA, in collaboration with the Quality Management Program-Laboratory Services (QMP-LS) in Ontario, Canada (10 laboratories), and the North American Specialized Coagulation Laboratory Association (NASCOLA; 47 laboratories, 5 overlapping the QMP-LS group). Adequate consensus was achieved for all and 89% of recommendations for the QMP-LS and NASCOLA groups, respectively. The recommendations adopted provide North American laboratories with additional guidance on platelet function testing, including how to interpret LTA abnormalities.

Lupus Anticoagulant Testing

Lupus anticoagulant (LAC) testing is important for evaluating patients with antiphospholipid syndromes and hypercoagulable states. We reviewed results of proficiency testing challenges (n = 5) distributed by the North American Specialized Coagulation Laboratory Association to examine LAC testing performed by participating laboratories. The activated partial thromboplastin time (APTT) and dilute Russell viper venom time (dRVVT) constituted major testing methods. In screening studies, LAC-sensitive APTT methods were more sensitive to weak LAC than dRVVT-based methods but less specific. In confirmatory testing, dRVVT methods performed better, but performance was LAC-dependent. The highest false-negative confirmatory test results were obtained for the platelet neutralization procedure. Noncompliance with recommendations for LAC testing by the International Society on Thrombosis and Haemostasis was high (8%-38%), with the majority of noncompliant laboratories failing to report results of mixing studies. These data provide new insights into LAC testing in North America and identify opportunities for standardization.

Laboratory Assessment of Factor VIII Inhibitor Titer: The North American Specialized Coagulation Laboratory Association Experience

Laboratory Assessment of Factor VIII Inhibitor Titer: The North American Specialized Coagulation Laboratory Association Experience

Critical Values in the Coagulation Laboratory: Results of a Survey of the North American Specialized Coagulation Laboratory Association.

Abstract 1415Poster Board I-438 Introduction:Critical laboratory values (CVs) are those that indicate the patient is in danger of death unless corrective action is initiated immediately. CVs are an important feature of laboratory practice parameters, standards of regulatory agencies (including the Joint Commission's National Patient Safety Goals), and national and institutional quality improvement studies. However, there has been no research into CVs in coagulation laboratories. Our objectives were to determine: 1) The current policies on CVs in specialized coagulation laboratories 2) Current patterns of practice for reporting CVs for coagulation parameters; and 3) If there is any consensus, based on practice, on CVs for coagulation tests. Methods:A web-based survey on CVs was distributed to members of the North American Specialized Coagulation Laboratory Association in February 2009. Results:38 laboratories participated. 97% had CVs for coagulation tests in place. 85% of laboratories used local opinion or consensus to set their CV policies, likely reflecting a lack of published data. 9% of laboratories were unsure where their CVs came from. CV reporting practices were fairly uniform. 97% of surveyed laboratories authorized technologists to report CVs, and only a minority (<40%) involved senior technologists or laboratory physicians in reporting. CV policies at almost all sites (97%) authorized the patient's nurse or MD, or the ordering MD to receive CV results. Medical students, clerical staff, or uninvolved staff nurses were rarely authorized to receive CVs (3%, 13%, and 37%, respectively).Policies in all participating laboratories required documentation of CV communication. Telephone was the primary method of communication, with 39% of sites used electronic broadcast or forced print as an adjunct. Inconsistency was noted in strategies to handle repeat CVs on the same patient, ranging from reporting all CVs, to reporting worsening CVs, to not reporting CVs on certain patient populations (e.g. critical FVIII:C levels in known hemophiliacs). After-hours communication of CVs on outpatients and referred clients was identified as challenging. All sites first attempted to telephone the patient's on-call MD, and if unsuccessful, sent a report by facsimile, contacted the Laboratory Chief of Service, or delayed reporting until the next day.When laboratories were asked about their CV values, over 80% had CVs for the INR (mean > 5, range > 2 - 6), aPTT (mean > 100 sec, range > 45 - 150 sec) and fibrinogen level (mean < 90 mg/dL, range < 50 - 100 mg/dL). Fewer laboratories (< 25%) had CVs for less commonly performed coagulation tests, including anti-Xa levels, factor and inhibitor assays, and thrombophilia testing. These CVs were more heterogeneous. Conclusion:This survey identified several potential areas for improvement in the way specialized coagulation laboratories handle CVs. Consensus data would assist laboratories in standardizing their policies, and establishing benchmarks for CV reporting and policy review. It would also ensure that a laboratory's CVs are in line with those of its peers, and are clinically relevant. The workload associated with CVs for coagulation assays is unknown, and it is possible that some of the current reporting strategies lead to inefficiencies and delays (e.g. reporting CVs for tests that are not truly life threatening; lack of established policies for after-hours handling of CVs when an on-call physician cannot be reached). Our study identifies an important need for further evaluation and standardization of CVs for coagulation tests. Disclosures:No relevant conflicts of interest to declare.

Results of an External Proficiency Testing Exercise on Platelet Dense-Granule Deficiency Testing by Whole Mount Electron Microscopy

Performance on specialized diagnostic tests for platelet disorders, including dense-granule deficiency, is rarely evaluated by external quality assessment (EQA). Members of the North American Specialized Coagulation Laboratory Association that evaluate platelet dense-granule deficiency commonly use whole-mount electron microscopy (EM) methods. This observation led us to develop a pilot EQA survey with standardized EM images and clinical samples on grids from a healthy control subject and a subject with dense-granule deficiency. The survey participants were 8 centers, including 2 with no experience in platelet whole mount EM. All participants, including inexperienced sites, correctly interpreted findings for the normal and dense-granule-deficient platelets. Among experienced sites, agreement was excellent (>82%) on platelet structures to count or not count as dense granules. Participants indicated that future EQA challenges should include clinical samples on grids and standardized images. This is the first report that platelet EM can be assessed by EQA.

Laboratory Assessment of Factor VIII Inhibitor Titer

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.

Are laboratories following published recommendations for lupus anticoagulant testing?

Laboratory tests for lupus anticoagulants (LA) are commonly performed to evaluate thrombosis or suspected phospholipid antibody syndromes. To determine current LA testing practices, and if they conform to published recommendations, two questionnaires were distributed to clinical laboratory members of the North American Specialized Coagulation Laboratory Association (NASCOLA) and the ECAT Foundation (ECAT). The first and second questionnaires were completed by 113 and 96 laboratories, respectively. Commonly performed LA tests included the dilute Russell's viper venom time, LA sensitive activated partial thromboplastin time and hexagonal phospholipid test. Although some laboratories did single LA tests if requested, the majority complied with published recommendations: to use platelet poor plasma for LA tests; to use two or more screening tests, representing different assay principles, and one assay having a low phospholipid concentration to exclude LA; to confirm LA phospholipid dependency by the method giving an abnormal LA screen; to document the inhibitor activity on pooled normal plasma; and not to use phospholipid antibodies to confirm LA. A minority (<35%) followed the recommendations to exclude factor deficiencies and factor inhibitors as the cause of an abnormal LA test. After participating, 32% of laboratories had changed practices and 20% indicated that they would be changing practices. While most laboratories generally follow published guidelines for LA testing, few follow recommendations to evaluate for other coagulation abnormalities. Questionnaires may be helpful quality initiatives to improve compliance with laboratory testing guidelines and recommendations.

Laboratory testing for heparin-induced thrombocytopenia is inconsistent in North America: A survey of North American specialized coagulation laboratories

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. As HIT is considered a clinico-pathologic entity, laboratory practices have an important role in diagnosing or excluding HIT. It was the objective of this study to assess the current status of laboratory testing for HIT in North America. An online survey consisting of 67 questions related to laboratory testing for HIT was developed by the North American Specialized Coagulation Laboratory Association (NASCOLA), and distributed to its 59 members. The survey included queries about HIT test ordering practices, HIT immunoassay and activation assays performed, and reporting practices. Data was collected from the 44 NASCOLA laboratories who responded. Of these sites, 88% performed immunoassays for HIT, commonly using commercial assays. However, sites varied in practices related to use of controls, immunoglobulin class of antibody detected, and in result interpretation and reporting. Platelet activation assays for HIT were performed by 36% of sites, commonly using assays of serotonin release (50%) or heparin-induced platelet aggregation (43%). Sites varied in the use of washed platelets versus platelet-rich plasma, controls, and heparin concentrations. This survey is the first comprehensive assessment of patterns of practice in HIT testing among diagnostic coagulation laboratories in North America. We observed site-specific variability of testing methods encompassing all stages of testing, including pre-analytical handling, testing methodologies, and result interpretation and reporting. The variability in HIT platelet activation assay methods among institutions indicates a need for proficiency testing to assess assay performance, and for consensus guidelines on HIT laboratory testing.