Systematic review and meta-analysis of within-subject and between-subject biological variation data of coagulation and fibrinolytic measurands.

OBJECTIVES: The diagnosis and monitoring of bleeding and thrombotic disorders depend on correct haemostatic measurements. The availability of high-quality biological variation (BV) data is important in this context. Many studies have reported BV data for these measurands, but results are varied. The present study aims to deliver global within-subject (CVI) and between-subject (CVG) BV estimates for haemostasis measurands by meta-analyses of eligible studies, by assessment with the Biological Variation Data Critical Appraisal Checklist (BIVAC). METHODS: Relevant BV studies were graded by the BIVAC. Weighted estimates for CVI and CVG were obtained via meta-analysis of the BV data derived from BIVAC-compliant studies (graded A-C; whereby A represents optimal study design) performed in healthy adults. RESULTS: In 26 studies BV data were reported for 35 haemostasis measurands. For 9 measurands, only one eligible publication was identified and meta-analysis could not be performed. 74% of the publications were graded as BIVAC C. The CVI and CVG varied extensively between the haemostasis measurands. The highest estimates were observed for PAI-1 antigen (CVI 48.6%; CVG 59.8%) and activity (CVI 34.9%; CVG 90.2%), while the lowest were observed for activated protein C resistance ratio (CVI 1.5%; CVG 4.5%). CONCLUSIONS: This study provides updated BV estimates of CVI and CVG with 95% confidence intervals for a wide range of haemostasis measurands. These estimates can be used to form the basis for analytical performance specifications for haemostasis tests used in the diagnostic work-up required in bleeding- and thrombosis events and for risk assessment.

How Do Laboratories Perform von Willebrand Disease Diagnostics and Classification of von Willebrand Disease Patients? Results from External Quality Data and an International Survey.

BACKGROUND: Reduced or dysfunctional von Willebrand factor (VWF) may lead to von Willebrand disease (VWD), which is a common inherited bleeding disorder. VWD is classified into three major types: type 1 is a partial quantitative deficiency of VWF, type 3 is a complete quantitative deficiency of VWF, and type 2 consists of qualitative abnormalities of VWF. To arrive at a correct VWD diagnosis, multiple tests and a correct interpretation of these tests are needed. AIM: The aim of the present study was to gain insight into the approach of laboratories toward VWD diagnosis. METHODS: Data from four samples of the external quality assessment (EQA) VWF surveys of the ECAT (External Quality Control for Assays and Tests) were evaluated. Furthermore, results were analyzed of a questionnaire that was sent to hemostasis laboratories about VWD diagnostic approaches. RESULTS: For most EQA samples, the majority of participants indicated the correct classification. However, 6 to 60% indicated another classification. For all samples, significant differences in VWF results were observed between the correct and incorrect classifications. The questionnaire demonstrated that the testing approach varied between the laboratories, especially for parameters that were essential for discrimination between VWD type 1 and healthy individuals, as well as the cutoff values used to discriminate VWD types 1 and 2. CONCLUSIONS: Diagnosis of VWD is heterogeneous in diagnostic approach, guidelines, and cutoff values within large ranges of VWF results between laboratories. Harmonization of approaches and increased accuracy of VWF measurements may help to establish a correct diagnosis.

How Do Laboratories Perform von Willebrand Disease Diagnostics and Classification of von Willebrand Disease Patients? Results from External Quality Data and an International Survey.

BACKGROUND: Reduced or dysfunctional von Willebrand factor (VWF) may lead to von Willebrand disease (VWD), which is a common inherited bleeding disorder. VWD is classified into three major types: type 1 is a partial quantitative deficiency of VWF, type 3 is a complete quantitative deficiency of VWF, and type 2 consists of qualitative abnormalities of VWF. To arrive at a correct VWD diagnosis, multiple tests and a correct interpretation of these tests are needed. AIM: The aim of the present study was to gain insight into the approach of laboratories toward VWD diagnosis. METHODS: Data from four samples of the external quality assessment (EQA) VWF surveys of the ECAT (External Quality Control for Assays and Tests) were evaluated. Furthermore, results were analyzed of a questionnaire that was sent to hemostasis laboratories about VWD diagnostic approaches. RESULTS: For most EQA samples, the majority of participants indicated the correct classification. However, 6 to 60% indicated another classification. For all samples, significant differences in VWF results were observed between the correct and incorrect classifications. The questionnaire demonstrated that the testing approach varied between the laboratories, especially for parameters that were essential for discrimination between VWD type 1 and healthy individuals, as well as the cutoff values used to discriminate VWD types 1 and 2. CONCLUSIONS: Diagnosis of VWD is heterogeneous in diagnostic approach, guidelines, and cutoff values within large ranges of VWF results between laboratories. Harmonization of approaches and increased accuracy of VWF measurements may help to establish a correct diagnosis.

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.

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.

International external quality assessment for measurements of direct oral anticoagulants: results and recommendations.

There is limited information regarding the performance of tests for direct oral anticoagulants (DOACs). To generate more knowledge, the accuracy of DOAC tests were evaluated using external quality assessment data from multiple years. This data demonstrated a good correlation for the tests with a small overall interlaboratory variability (10% for dabigatran, rivaroxaban and apixaban and 12% for edoxaban). The greatest differences between the various reagents were observed for rivaroxaban, especially for concentrations below 100 ng/ml. In conclusion, the results show overall reliable DOAC levels with some differences between reagent groups. Important finding: clinical decision criteria could be affected by the choice of reagent.

Quality performance for indirect Xa inhibitor monitoring in patients using international external quality data.

Objectives Chromogenic anti-activated factor X (FXa) assays are currently the "gold standard" for monitoring indirect anticoagulants. However, anti-FXa has been shown to vary according to the choice of reagents. In the present study, the performance of anti-FXa measurement was evaluated in order to gain more insight into the clinical applications. Furthermore, the longitudinal coefficient of variation (CV) was studied to investigate whether there is improvement over time. Methods Laboratory tests results were evaluated for samples spiked with unfractionated heparin (UFH), low-molecular-weight-heparin (LMWH), fondaparinux and danaparoid sodium. External quality assessment (EQA) data from multiple years were used from more than 100 laboratories. Results Comparison of the results for all methods showed significant differences in measured values between the frequently used methods (ANOVA: p < 0.001). The largest differences were observed for LMWH and UFH measurements. These differences may be caused by differences in method composition, such as the addition of dextran sulphate. Substantial interlaboratory variation in anti-FXa monitoring was observed for all parameters, particularly at low concentrations. Our results showed that below 0.35 IU/mL, the CVs for UFH and LMWH increase dramatically and results below this limit should be used with caution. Conclusions Our study demonstrates that the choice of the anti-FXa method is particularly important for UFH and LMWH measurement. The variation in measurements may have an effect on clinical implications, such as therapeutic ranges. Furthermore, the longitudinal EQA data demonstrated a constant performance and, in at least 50% of the cases, improvement in the CV% of the anti-Xa results over time.

Determination of sigma score based on biological variation for haemostasis assays: fit-for-purpose for daily practice?

Background Internal quality control (QC) rules for laboratory tests can be derived from analytical performance specifications (APS) using the six-sigma method. We tested the applicability of this paradigm to routine haemostasis measurements. Methods Three laboratories using different instruments and reagents calculated sigma scores for their prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen and antithrombin (AT) measurements. Sigma scores were calculated using biological variation (BV) data from the literature in combination with internal and external QC data. Results Wide ranges in sigma scores for the PT (0.1-6.8), APTT (0.0-4.3), fibrinogen (1.5-8.3) and AT (0.1-2.4) were observed when QC data was combined with the minimum, median and maximum value of BV data, due in particular to a large variation in within-subject and between-subjects coefficients of variation. When the median BV values were applied, most sigma scores were below 3.0, for internal QC data; 75% and for external QC data; 92%. Conclusions Our findings demonstrate that: (1) The sigma scores for common haemostasis parameters are relatively low, and (2) The application of the six-sigma method to BV-derived APS is hampered by the large variation in published BV data. As the six-sigma concept is based on requirements for monitoring, and many haemostasis tests are only designed for diagnostic purposes, a fit-for-purpose APS is needed to achieve clinically relevant quality goals.

Searching for avidity by chemical ligation of combinatorially self-assembled DNA-encoded ligand libraries.

DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial screening platform for avidity interactions, the optimal bivalent aptamer design (examplar ligands) for human alpha-thrombin is determined in a single round of selection and the DNA scaffold replaced with minimal impact on the final design.

Fibrinogen determination according to Clauss: commutability assessment of International and commercial standards and quality control samples.

BACKGROUND: Many clinical laboratories use a clotting rate assay according to Clauss for the determination of fibrinogen in citrated plasma. The aim of the present study was to assess the commutability of the current International Standard for fibrinogen (coded 09/264), three commercial fibrinogen standards, and 10 freeze-dried plasma quality control samples from various sources. METHODS: Clotting rate assays according to Clauss were performed on three automated instruments (Sysmex CA1500, STA-Rack Evolution and ACL-Top 700), using three commercial thrombin reagents (Siemens, Stago, and Instrumentation Laboratory). Relationships between the results obtained with the three instruments were determined with 25 fresh-frozen plasma samples obtained from patients. The deviations of the assay results obtained with the freeze-dried samples were compared with the deviations obtained with the fresh-frozen samples, according to approved CLSI guideline C53A. RESULTS: Freezing and thawing had no influence on the assay results. There were significant differences in the mean assay results (fibrinogen, g/L) for the fresh-frozen plasma samples between the three automated instruments: 2.51 (STA-Rack Evolution), 2.25 (ACL-Top 700) and 2.20 (Sysmex CA1500). Similar differences were observed for several freeze-dried plasma samples. Some freeze-dried plasma samples, including the International Standard, were out of the 95% confidence interval for the relationship between STA-Rack Evolution and Sysmex CA1500. CONCLUSIONS: Some freeze-dried plasmas including the international standard for fibrinogen are not commutable among automated instruments for fibrinogen clotting rate assays according to Clauss. Our results have consequences for all interested parties in the traceability chain (WHO, industry, external quality assessment schemes, clinical laboratories).

Risk factor analysis of cerebral white matter hyperintensities in children with sickle cell disease.

Sickle cell disease (SCD) is complicated by silent cerebral infarcts, visible as white matter hyperintensities (WMHs) on magnetic resonance imaging (MRI). Both local vaso-occlusion, elicited by endothelial dysfunction, and insufficiency of cerebral blood flow (CBF) have been proposed to be involved in the aetiology. We performed an explorative study to investigate the associations between WMHs and markers of endothelial dysfunction and CBF by quantifying WMH volume on 3.0 Tesla MRI. We included 40 children with HbSS or HbSß(0) thalassaemia, with a mean age of 12.1 ± 2.6 years. Boys demonstrated an increased risk for WMHs (odds ratio 4.5, 95% confidence interval 1.2-17.4), unrelated to glucose-6-phosphate dehydrogenase deficiency. In patients with WMHs, lower fetal haemoglobin (HbF) was associated with a larger WMH volume (regression coefficient = -0.62, R2 = 0.5, P = 0.04). Lower ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) levels were associated with lower CBF in the white matter (regression coefficient = 0.07, R2 = 0.15, P = 0.03), suggesting that endothelial dysfunction could potentially hamper CBF. The findings of our explorative study suggest that a high level of HbF may be protective for WMHs and that endothelial dysfunction may contribute to the development of WMHs by reducing CBF.

Mutation and ADAMTS13-dependent modulation of disease severity in a mouse model for von Willebrand disease type 2B.

Von Willebrand disease (VWD)-type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

Impact of COVID-19 pandemic on the quality of test output in haemostasis laboratories.

INTRODUCTION: The high incidence of thrombotic events in patients with COVID-19 affects health care worldwide and results in an increased workload in haemostasis laboratories due to more frequent testing of D-dimer, haemostatic parameters and anti-Xa tests. However, the impact of this increase in assay requests on the quality of performance in haemostasis laboratories remains unclear. In this study, the impact of the COVID-19 pandemic on the quality of performance and management of haemostasis laboratories was evaluated. METHODS: The impact on the quality of performance was studied using external quality assessment data from 2019 to 2020 derived from ECAT surveys. A questionnaire was sent to Dutch haemostasis laboratories to identify challenges and management strategies. Furthermore, the number of assays performed in 2019 and 2020 was supplied by four Dutch hospitals, located in regions with different disease incidence. RESULTS: No differences in response rate nor the quality of the measurements were observed between the EQA surveys in 2019 and 2020. The questionnaire results showed a large increase of >25% in the number of test requests for anti-Xa, D-dimer and fibrinogen assays in 2020 compared to 2019. Extreme peaks in test requests were also observed in the four evaluated hospitals. Additionally, 84% of the respondents indicated that they had experienced increased work pressure, and increased sick leave was observed in 71% of the participating laboratories. CONCLUSIONS: The enormous increase in test requests, especially for D-dimer assays and anti-Xa activity, did not affect the quality of performance within haemostatic laboratories during the COVID-19 pandemic.

A combinatorial approach for the design of complementarity-determining region-derived peptidomimetics with in vitro anti-tumoral activity.

The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity-determining regions (CDRs) from gastrin17 (pyroEGPWLEEEEEAYGWMDF-NH(2)) antibodies as starting material for cyclic peptide synthesis in a microarray format. Gastrin17 is a trophic factor in gastrointestinal tumors, including pancreatic cancer, which makes it an interesting target for development of therapeutic antibodies. Screening of microarrays containing bicyclic peptidomimetics identified a high number of gastrin binders. A strong correlation was observed between gastrin binding and overall charge of the peptidomimetic. Most of the best gastrin binders proceeded from CDRs containing charged residues. In contrast, CDRs from high affinity antibodies containing mostly neutral residues failed to yield good binders. Our experiments revealed essential differences in the mode of antigen binding between CDR-derived peptidomimetics (K(d) values in micromolar range) and the parental monoclonal antibodies (K(d) values in nanomolar range). However, chemically derived peptidomimetics from gastrin binders were very effective in gastrin neutralization studies using cell-based assays, yielding a neutralizing activity in pancreatic tumoral cell lines comparable with that of gastrin-specific monoclonal antibodies. These data support the use of combinatorial CDR-peptide microarrays as a tool for the development of a new generation of chemically synthesized cyclic peptidomimetics with functional activity.

Monitoring unfractionated heparin therapy: Lack of standardization of anti-Xa activity reagents.

INTRODUCTION: One of the main advantages of using anti-Xa instead of activated partial thromboplastin time in monitoring of unfractionated heparin (UFH) therapy relies on its hypothesized standardization, with a unique therapeutic range defined to be 0.30 to 0.70 IU/mL. The aim of the present study was to compare the inter-reagent agreement of anti-Xa activity. METHODS: Citrate tubes were obtained from 104 inpatients on UFH. Plasma samples were stored frozen in aliquots at -70°C before being shipped to three accredited coagulation laboratories to be evaluated for anti-Xa activity using their routine assay(s). Pooled normal plasmas spiked with dilutions of the 6th International Standard of UFH to achieve anti-Xa activities up to 1.0 IU/mL were evaluated using the same techniques. RESULTS: In the plasmas from patients on UFH, the median anti-Xa activity ranged from 0.37 IU/mL with one reagent to 0.57 IU/mL with another; results were in between (0.45 IU/mL) using two other reagents. Comparisons of results obtained using the different reagents demonstrated unacceptable bias up to 0.24 IU/mL between some reagents (41% difference). The concordance as whether anti-Xa activities measured using different reagents were within or outside the therapeutic range was between 0.411 and 0.939 (kappa). Similar discrepancy was demonstrated for anti-Xa activities when evaluating normal plasma spiked with the International Standard. A discrepancy of the same order of magnitude was demonstrated in the 2017 External Quality Assessment Program provided by the External Quality Control in Assays and Tests exercises. CONCLUSIONS: The reported discrepancy between test results obtained using different anti-Xa assays clearly suggests a lack of standardization of that assay with potentially significant impact on the patients' anticoagulation.

Characterization of gastrin-cholecystokinin 2 receptor interaction in relation to c-fos induction.

The interaction of gastrin with the cholecystokinin 2 (CCK2)/gastrin receptor has been studied extensively in relation to gastric acid secretion. However, not much is known about the contribution of individual amino acids of gastrin interacting with the CCK2 receptor, when gastrin is acting as a tumor growth factor. The purpose of the present study was to determine the significance of each individual amino acid residue of human gastrin-17 with respect to CCK2 receptor-mediated cell proliferation. Activation of this receptor was assessed using an in vitro bioassay based on gastrin-induced expression of a c-fos-luciferase reporter, transfected in AR42JB13 and Colo 320 cells, a rat pancreatic and human colorectal cell line respectively. Gastrin-17 dose dependently increased c-fos induction in both cancer cell lines. L365,260, a known CCK2 receptor antagonist, completely blocked the gastrin signal, demonstrating the specificity of this assay. We demonstrated for the first time that four carboxy-terminal amino acids of gastrin-17 are essential for activation of the CCK2 receptor with respect to c-fos induction. Also other residues of gastrin-17, notably glycine-2 for the rat CCK2 receptor and glutamic acid 8-10 and tyrosine-12 for the human receptor, were found to be important, although to a lesser extent. Alanine-substitution variants of each of the four carboxy-terminal amino acids of gastrin-17 showed strongly reduced receptor activation but did not act as competitive inhibitors of gastrin-17. Identification of the essential role of the carboxy-terminal tetrapeptide of gastrin-17 in CCK2 receptor-mediated c-fos induction indicates that gastrin inhibitory therapeutic strategies should mainly be targeted toward this region of gastrin.

Designing antibodies for the inhibition of gastrin activity in tumoral cell lines.

Gastrin and its derivatives are becoming important targets for immunotherapy of pancreatic, gastric and colorectal tumors. This study was conducted to design antibodies able to block gastrin binding to the gastrin/cholecystokinin-2 (CCK-2) receptor in order to delay tumor growth. The authors have used different gastrin molecules, combined with the diphtheria toxoid, to generate and select human single chain variable fragments (scFvs) as well as mouse monoclonal antibodies and scFvs against different regions of gastrin. There was a remarkable conservation in the antibody repertoire against gastrin, independently of the approach and the species. The germlines most frequently used in gastrin antibody formation were identified. Three different epitopes were identified in the gastrin molecule. The resulting mouse monoclonal antibodies and scFvs were analyzed for gastrin neutralization using Colo 320 WT cells, which overexpress the CCK-2 receptor. The gastrin neutralizing activity assay showed that N-terminal specific mouse monoclonal antibodies were more efficient to inhibit proliferation of Colo 320 WT cells than the anti-C terminal antibodies. Moreover, the human antigastrin scFvs obtained in this study inhibited significantly the proliferation of Colo 320 tumoral cells. These findings should contribute to a more rational design of antibody-based antigastrin therapies in cancer, including passive administration of human antibodies with blocking activity.

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).

Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg(593) to Cys substitution.

Inhibitory antibodies develop in approximately 25% of patients with severe hemophilia. A following treatment with factorVIII. In E-16KO or E-17KO mice, in which the factor VIII gene has been inactivated by insertion of a neo cassette, inhibitors develop following administration of factor VIII. Here, we describe the generation of transgenic mice expressing human factor VIII-R593C (huFVIII-R593C). Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of transgenic mice revealed that human factor VIII-R593C was expressed in the liver. Transgenic mice were crossed with factor VIII-deficient mice (E-16KO mice). In plasma of E-16KO mice antibodies were detected after five serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levels of antibodies. No antibody secreting cells were observed in either spleen or bone marrow of huFVIII-R593C/E-16KO mice. Also, factor VIII-specific memory B cells were not observed in the spleen of huFVIII-R593C/E-16KO mice. Analysis of T cell responses revealed that splenocytes derived of E-16KO mice secreted IL-10 and IFN-gamma following restimulation with factor VIII in vitro. In contrast, no factor VIII-specific T cell responses were observed in huFVIII-R593C/E-16KO mice. These results indicate that huFVIII-R593C/E-16KO mice are tolerant to intravenously administered factor VIII. It is anticipated that this model may prove useful for studying immune responses in the context of factor VIII gene therapy.

Successful treatment of a noninhibitory antibody-mediated acquired factor X deficiency in a patient with marginal-zone lymphoma.

Prolonged clotting times were observed in a patient with spontaneous hemorrhage. Analysis showed severe factor X deficiency due to clearance by a noninhibitory antibody. Lymphadenopathy identified on imaging led to diagnosis of marginal B-cell lymphoma. Treatment of lymphoma with rituximab and chlorambucil resulted in complete disappearance of the bleeding disorder.