Comparison of Thrombophilia Assay Results for the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Plasma Standard from Different External Quality Assessment Providers-for the External Quality Assurance in Thrombosis and Haemostasis Group.

External quality assessment (EQA) is used to evaluate laboratory performance in tests of hemostasis; however, some esoteric tests are performed by too few centers in any one EQA program to allow valid statistical assessment. To explore the feasibility of pooling data from several EQA providers, an exercise was carried out by the External Quality Assurance in Thrombosis and Haemostasis group, using the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee (SSC) plasma standard for thrombophilia screening assays. Six EQA providers took part in this exercise, distributing the SSC plasma standard as a "blinded" sample to participants for thrombophilia tests between November 2020 and December 2021. Data were collected by each provider, anonymized, and pooled for analysis. Results were analyzed as overall results from each EQA provider, and by kit/method-specific comparisons of data from all providers pooled together. For each parameter, median results and range were determined. Over 1,250 sets of data were returned in the six EQA programs. The overall medians (all data pooled) were <4% of the assigned values for each parameter with the exception of protein C activity by clot-based assay. Method-related differences in median results were observed for free protein S antigen and protein S activity-a pattern seen across data from the different EQA providers. Antithrombin antigen results reported in mg/dL provided an example where small numbers of results for a single EQA provider may be supplemented by pooling data from multiple providers with good agreement seen among results reported by the different EQA providers. This study demonstrated that a multicenter EQA provider collaboration can be carried out and demonstrated benefit for assays with smaller number of participants. In addition, results showed good agreement with the assigned values of the SSC plasma standard. Further exercises for tests performed by only small numbers of laboratories can be planned.

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.

Diagnostic laboratory standardization and validation of platelet transmission electron microscopy.

Platelet transmission electron microscopy (PTEM) is considered the gold standard test for assessing distinct ultrastructural abnormalities in inherited platelet disorders (IPDs). Nevertheless, PTEM remains mainly a research tool due to the lack of standardized procedures, a validated dense granule (DG) count reference range, and standardized image interpretation criteria. The aim of this study was to standardize and validate PTEM as a clinical laboratory test. Based on previously established methods, we optimized and standardized preanalytical, analytical, and postanalytical procedures for both whole mount (WM) and thin section (TS) PTEM. Mean number of DG/platelet (plt), percentage of plts without DG, platelet count (PC), mean platelet volume (MPV), immature platelet fraction (IPF), and plt light transmission aggregometry analyses were measured on blood samples from 113 healthy donors. Quantile regression was used to estimate the reference range for DG/plt, and linear regression was used to assess the association of DG/plt with other plt measurements. All PTEM procedures were standardized using commercially available materials and reagents. DG interpretation criteria were established based on previous publications and expert consensus, and resulted in improved operator agreement. Mean DG/plt was stable for 2 days after blood sample collection. The median within patient coefficient of variation for mean DG/plt was 22.2%; the mean DG/plt reference range (mid-95th %) was 1.2-4.0. Mean DG/plt was associated with IPF (p = .01, R2 = 0.06) but not age, sex, PC, MPV, or plt maximum aggregation or primary slope of aggregation (p > .17, R2 < 0.02). Baseline ultrastructural features were established for TS-PTEM. PTEM was validated using samples from patients with previously established diagnoses of IPDs. Standardization and validation of PTEM procedures and interpretation, and establishment of the normal mean DG/plt reference range and PTEM baseline ultrastructural features, will facilitate implementation of PTEM as a valid clinical laboratory test for evaluating ultrastructural abnormalities in IPDs.

Are laboratories following published recommendations for lupus anticoagulant testing? An international evaluation of practices.

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.

Comparison of coagulation factor XIII content and concentration in cryoprecipitate and fresh-frozen plasma.

BACKGROUND: For patients with plasma coagulation factor XIII (pFXIII) deficiency, recommended means of replacement include infusions of fresh-frozen plasma (FFP), cryoprecipitate, or (where available) factor (F)XIII concentrates. Quantitative differences in pFXIII concentration in FFP and cryoprecipitate are not well defined and were, therefore, the subject of this study. STUDY DESIGN AND METHODS: FFP and cryoprecipitate (10 bags each from blood group O donors) were analyzed to quantify pFXIII activity and antigen. Coagulation FVIII, fibrinogen, and von Willebrand factor (VWF) were also quantitated. RESULTS: Mean (+/-SD) pFXIII activity in cryoprecipitate and FFP bags was 60 +/- 30 and 288 +/- 77 U per bag, respectively, and pFXIII antigen and activity levels were concordant. Other comparisons (mean +/- SD) between cryoprecipitate and FFP, respectively, were as follows: coagulation FVIII activity, 133 +/- 37 and 265 +/- 83 U per bag; fibrinogen content (Clauss kinetic assay), 183 +/- 44 and 725 +/- 199 mg per bag; VWF antigen content, 181 +/- 53 and 218 +/- 70 U per bag; VWF ristocetin cofactor activity, 168 +/- 34 and 221 +/- 65 U per bag; VWF collagen-binding activity, 164 +/- 40 and 208 +/- 71 U per bag; and fluid (plasma) volumes per bag, 21.3 +/- 2.7 and 245 +/- 29 mL. CONCLUSION: In contrast to other cryoprecipitable coagulation proteins, pFXIII is only mildly enriched in cryoprecipitate when compared with FFP (approx. two- to threefold). Although both products can provide effective pFXIII replacement, FFP may be preferred when infusion volume is not a major consideration and pFXIII concentrates are not available. VWF is substantially enriched in cryoprecipitate (approx. ninefold compared with its concentration in FFP), with VWF activity content exceeding that of FVIII by approximately 26 percent on average.

Protein S testing in patients with protein S deficiency, factor V Leiden, and rivaroxaban by North American Specialized Coagulation Laboratories.

In 2010-2012, the North American Specialized Coagulation Laboratory Association (NASCOLA) distributed 12 proficiency testing challenges to evaluate laboratory testing for protein S (PS). Results were analysed to assess the performance of PS activity, PS free antigen, and PS total antigen testing. Statistical analysis was performed on the numeric results and qualitative classification submitted for each method. There were 2,106 total results: 716 results from PS activity assays, 833 results from PS free antigen assays, and 557 results from PS total antigen assays. The three assay types performed well in the classification of five normal samples and nine abnormal samples, although certain PS activity methods were more likely to classify normal samples as abnormal and one PS total antigen assay was more likely to classify abnormal samples as normal. PS activity methods were affected by interfering substances such as heterozygous or homozygous factor V Leiden mutation (underestimation) and the anticoagulant drug rivaroxaban (overestimation). In conclusion, NASCOLA laboratories using a variety of PS assays performed well in the classification of clearly normal and abnormal samples. Laboratories performing PS activity assays should be aware of potential interferences in samples positive for FV Leiden or containing certain anticoagulant medications.

Posttransplantation thrombotic thrombocytopenic purpura: a single-center experience and a contemporary review.

OBJECTIVE: To assess the activity of von Willebrand factor-cleaving protease (vWF-CP) in patients with thrombotic thrombocytopenic purpura (TTP) complicating bone marrow transplantation (BMT) and peripheral blood stem cell transplantation (PBSCT). PATIENTS AND METHODS: From March 1, 1999, to June 30, 2001, allogeneic and autologous hematopoietic stem cell transplantation was performed in 118 and 400 patients, respectively. We reviewed risk factors for development of posttransplantation TTP and measured vWF-CP activity during active TTP in 10 recipients. RESULTS: The incidence of TTP after allogeneic and autologous transplantation was 6.8% (8/118) and 0.25% (1/400), respectively. Among the allogeneic transplant recipients, the incidence of TTP after nonmyeloablative (NMA) PBSCT, matched unrelated donor BMT, and sibling BMT or PBSCT was 15.4% (2/13), 11.8% (2/17), and 4.5% (4/88), respectively. Of the 10 patients with TTP, 9 (90%) had received extensive prior therapy, including autologous transplantation in both NMA recipients. Acute graft-vs-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate in most affected patients. The vWF antigen level was elevated in all patients, and no patients showed evidence of vWF-CP deficiency. During active TTP, 6 patients had grade II-IV acute GVHD, 1 had extensive chronic GVHD, and 4 had cytomegalovirus viremia. Risk factor analysis for development of TTP showed that transplant type (NMA and matched unrelated donor) and source of stem cells (bone marrow vs peripheral blood stem cell) were significant. CONCLUSIONS: Posttransplantation TTP was not found to be associated with severe vWF-CP deficiency. The elevated levels of vWF antigen are consistent with diffuse endothelial injury likely because of multiple interacting factors such as extensive prior therapy, GVHD, cyclosporine, and reactivation of cytomegalovirus. The disorder appears to be more frequent among patients with, or at risk for, acute GVHD, suggesting a possible role in the pathogenesis. Nonmyeloablative transplantation does not appear to confer a lesser risk, possibly for these reasons.

Molecular analysis in a patient with severe factor VII deficiency and an inhibitor: report of a novel mutation (S103G).

Congenital factor VII (FVII) deficiency is an autosomal recessive bleeding disorder with variable phenotypic correlation between FVII activity and bleeding risk. We report a novel mutation of the FVII gene that creates the amino acid change Ser 103 to Gly, which resulted in severe FVII deficiency with reduced FVII antigen. This mutation in the heterozygous form was also present in a mildly affected, unrelated patient. We also report on the natural history of an FVII inhibitor in the patient with severe FVII deficiency.