Performance of a recombinant fusion protein linking coagulation factor IX with recombinant albumin in one-stage clotting assays.

Essentials Performance of the one-stage clotting (OSC) assay varies with the clotting activator used. Recombinant FIX-albumin fusion protein (rIX-FP) was reliably monitored with most OSC reagents. rIX-FP shows comparable reagent-dependent variability to other rFIX products in the OSC assay. Actin® FS and kaolin-based reagents underestimated rIX-FP activity by around 50% in the OSC assay. SUMMARY: Background Measuring factor IX activity (FIX:C) with one-stage clotting (OSC) assays, based on the activated partial thromboplastin time (APTT), is the current mainstay of diagnostic techniques for hemophilia B. Assessing the performance of new recombinant FIX (rFIX) products in OSC assays is essential, as APTT reagents from different manufacturers yield different potency estimates for rFIX. Objectives To evaluate the extent to which choice of reagent composition influences rFIX potency measurements of recombinant FIX-albumin fusion protein (rIX-FP, IDELVION) activity in OSC assays. Methods rIX-FP was added to FIX-deficient plasma, and FIX:C was assessed centrally and locally in a multicenter international field study with a variety of commercial OSC APTT reagents. Paired sample analysis of clinical samples was performed to compare values of FIX:C from local and central laboratories. In-house bioanalytical investigations with spiked samples were conducted to compare the APTT-reagent dependent variability of rIX-FP with unmodified rFIX and rFIX Fc fusion protein (rFIXFc). Results Central and local assessments of FIX:C from 10 countries and 21 participating centers showed comparable results to those from the central laboratory across the majority of 18 different APTT reagents from both clinical and spiked samples. There was a consistent underestimation of rIX-FP activity of ≈ 50% with OSC assays using Actin FS or kaolin-based APTT reagents. In the bioanalytical study, rIX-FP showed comparable variability in OSC assays to unmodified rFIX and rFIXFc. Conclusions rIX-FP activity can be accurately measured by the use of OSC assays with the majority of commercial reagents. Actin FS or kaolin-based reagents will probably lead to a 50% underestimation of activity.

International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemostasis laboratories.

Essentials AFSTYLA exhibits ≈50% underestimation in activity when the one-stage (OS) assay is utilized. A field study compared the performance of AFSTYLA with Advate in factor VIII activity assays. AFSTYLA activity can be monitored with both the chromogenic substrate and the OS assay. The consistent OS underestimation allows for a conversion factor to be applied to OS results. SUMMARY: Introduction AFSTYLA (antihemophilic factor [recombinant] single chain) is a novel B-domain truncated recombinant factor VIII (rFVIII). For AFSTYLA, an approximate 50% discrepancy was observed between results of the one-stage (OS) and chromogenic substrate (ChS) FVIII activity assays. An investigation was undertaken to test whether there is a linear relationship between ChS and OS assay results that would allow reliable clinical interpretation of results independent of the assay method used. Aims To provide confidence in future clinical monitoring, this field study investigated the performance of AFSTYLA and a full-length rFVIII (Advate® ) in FVIII activity assays routinely performed in clinical laboratories. Methods The comparison of AFSTYLA and Advate was performed in an international, multicenter and blinded field study of simulated post-infusion samples. The study documented the extent of variability between methods and laboratories and characterized the relationship between the ChS and OS assays. Results Results from 23 laboratories demonstrate that intra and interlaboratory variability in OS assays were similar for both products. When comparing within the OS assay format, there was a similar and reagent-correlated variability in response to different activators for both AFSTYLA and Advate. The OS underestimation was highly predictable and consistent across the complete range of FVIII plasma concentrations. Conclusion Post-infusion plasma AFSTYLA levels can be monitored in patients by the OS and ChS assays. The consistent and predictable difference between the two assay formats provides clinicians with adequate guidance on how to interpret the results of the OS assay using a single conversion factor.

Consensus on the standardization of terminology in thrombotic thrombocytopenic purpura and related thrombotic microangiopathies.

Essentials An international collaboration provides a consensus for clinical definitions. This concerns thrombotic microangiopathies and thrombotic thrombocytopenic purpura (TTP). The consensus defines diagnosis, disease monitoring and response to treatment. Requirements for ADAMTS-13 are given. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS) are two important acute conditions to diagnose. Thrombotic microangiopathy (TMA) is a broad pathophysiologic process that leads to microangiopathic hemolytic anemia and thrombocytopenia, and involves capillary and small-vessel platelet aggregates. The most common cause is disseminated intravascular coagulation, which may be differentiated by abnormal coagulation. Clinically, a number of conditions present with microangiopathic hemolytic anemia and thrombocytopenia, including cancer, infection, transplantation, drug use, autoimmune disease, and pre-eclampsia and hemolysis, elevated liver enzymes and low platelet count syndrome in pregnancy. Despite overlapping clinical presentations, TTP and HUS have distinct pathophysiologies and treatment pathways. Objectives To present a consensus document from an International Working Group on TTP and associated thrombotic microangiopathies (TMAs). Methods The International Working Group has proposed definitions and terminology based on published information and consensus-based recommendations. Conclusion The consensus aims to aid clinical decisions, but also future studies and trials, utilizing standardized definitions. It presents a classification of the causes of TMA, and criteria for clinical response, remission and relapse of congenital and immune-mediated TTP.

A computer-based model to assess costs associated with the use of factor VIII and factor IX one-stage and chromogenic activity assays.

BACKGROUND: Measurement of coagulation factor factor VIII (FVIII) and factor IX (FIX) activity can be associated with a high level of variability using one-stage assays based on activated partial thromboplastin time (APTT). Chromogenic assays show less variability, but are less commonly used in clinical laboratories. In addition, one-stage assay accuracy using certain reagent and instrument combinations is compromised by some modified recombinant factor concentrates. Reluctance among some in the hematology laboratory community to adopt the use of chromogenic assays may be partly attributable to lack of familiarity and perceived higher associated costs. OBJECTIVES: To identify and characterize key cost parameters associated with one-stage APTT and chromogenic assays for FVIII and FIX activity using a computer-based cost analysis model. METHODS: A cost model for FVIII and FIX chromogenic assays relative to APTT assays was generated using assumptions derived from interviews with hematologists and laboratory scientists, common clinical laboratory practise, manufacturer list prices and assay kit configurations. RESULTS: Key factors that contribute to costs are factor-deficient plasma and kit reagents for one-stage and chromogenic assays, respectively. The stability of chromogenic assay kit reagents also limits the cost efficiency compared with APTT testing. Costs for chromogenic assays might be reduced by 50-75% using batch testing, aliquoting and freezing of kit reagents. CONCLUSIONS: Both batch testing and aliquoting of chromogenic kit reagents might improve cost efficiency for FVIII and FIX chromogenic assays, but would require validation. Laboratory validation and regulatory approval as well as education and training in the use of chromogenic assays might facilitate wider adoption by clinical laboratories.

A critical appraisal of one-stage and chromogenic assays of factor VIII activity.

Accurate and precise potency determination by manufacturers of different types of factor VIII product (plasma-derived and recombinant FVIII [rFVIII]) is vital to clinicians and patients using FVIII concentrates. A separate, but related, requirement is ascertaining the FVIII activity levels in clinical samples for diagnosing and treating hemophilia A. The one-stage clotting assay (OSA) and the chromogenic substrate assay (CSA) are the main assays used for these measurements, with both assays being used for potency assignments, and the OSA also being widely used for clinical monitoring. Although the assays can produce concordant results, discrepancies often occur, e.g. when measuring FVIII levels in patients with mild or moderate hemophilia A, or when assaying high-purity FVIII products. Modifications to rFVIII proteins, such as B-domain deletion (BDD), and technologies for improving the pharmacokinetic profile of rFVIII may exacerbate assay discrepancies. The CSA appears to be essentially unaffected by these modifications. However, the OSA underestimates the FVIII activity levels and therapeutic potential of some further modified BDD rFVIII products, especially those conjugated to poly(ethylene glycol); the extent of the effects is dependent on the specific OSA reagents used. Although the OSA remains the preferred choice for clinical monitoring in Europe and the USA, an awareness of the limitations of that assay has prompted more laboratories to adopt the CSA.

Comparison of several von Willebrand factor (VWF) activity assays for monitoring patients undergoing treatment with VWF/FVIII concentrates: improved performance with a new modified automated method.

BACKGROUND: The ability of von Willebrand factor (VWF) to bind platelet GP Ib and promote platelet plug formation is measured in vitro using the ristocetin cofactor (VWF:RCo) assay. Automated assay systems make testing more accessible for diagnosis, but do not necessarily improve sensitivity and accuracy. OBJECTIVE: We assessed the performance of a modified automated VWF:RCo assay protocol for the Behring Coagulation System (BCS(®) ) compared to other available assay methods. METHODS: Results from different VWF:RCo assays in a number of specialized commercial and research testing laboratories were compared using plasma samples with varying VWF:RCo activities (0-1.2 IU mL(-1) ). Samples were prepared by mixing VWF concentrate or plasma standard into VWF-depleted plasma. Commercially available lyophilized standard human plasma was also studied. Emphasis was put on the low measuring range. VWF:RCo accuracy was calculated based on the expected values, whereas precision was obtained from repeated measurements. RESULTS: In the physiological concentration range, most of the automated tests resulted in acceptable accuracy, with varying reproducibility dependent on the method. However, several assays were inaccurate in the low measuring range. Only the modified BCS protocol showed acceptable accuracy over the entire measuring range with improved reproducibility. CONCLUSIONS: A modified BCS(®) VWF:RCo method can improve sensitivity and thus enhances the measuring range. Furthermore, the modified BCS(®) assay displayed good precision. This study indicates that the specific modifications - namely the combination of increased ristocetin concentration, reduced platelet content, VWF-depleted plasma as on-board diluent and a two-curve calculation mode - reduces the issues seen with current VWF:RCo activity assays.

Diagnostic challenges in patients with bleeding phenotype and von Willebrand exon 28 polymorphism p.D1472H.

Exon 28 polymorphism p.D1472H is associated with significantly lower von Willebrand Ristocetin cofactor activity (VWF:RCoF) to von Willebrand antigen (VWF:Ag) ratio compared to normal, but has been reported as not conferring haemorrhagic risk. The impact of this polymorphism while assessing symptomatic patients for von Willebrand disease (VWD) has not been previously analysed. We retrospectively reviewed charts of children with clinically significant bleeding and abnormal VW panel who underwent VW exon 28 analysis. Twenty-three of 63 patients studied had p.D1472H. Of these 23 patients, 6 with borderline low VWF:RCo were given provisional diagnosis of VWD type 1 by treating physicians, which could be alternatively explained as due to the effect of p.D1472H. None of the patients with low VWF:RCo, decreased VWF:RCo/VWF:Ag ratio and p.D1472H had VWD type 2M mutations identified. This study illustrates the challenge in diagnosing VWD using ristocetin-based VW assay in symptomatic patients with p.D1472H.

An optimized fluorogenic ADAMTS13 assay with increased sensitivity for the investigation of patients with thrombotic thrombocytopenic purpura.

BACKGROUND: Most ADAMTS13 assays use non-physiological conditions (low ionic strength, low pH, barium chloride), are subject to interference from plasma proteins, hemoglobin and bilirubin, and have limited sensitivity, especially for inhibitors. OBJECTIVES: We addressed these constraints by designing a substrate that can be used in undiluted plasma. METHODS: A polypeptide was expressed in E. coli that corresponds to von Willebrand factor Gln(1599) -Arg(1668) , with mutations N1610C and K1617R and an N-terminal Gly. Substrate FRETS-rVWF71 was prepared by modifying Cys(1610) with DyLight 633 (abs 638 nm, em 658 nm) and the N-terminus with IRDye QC-1 (abs 500-800 nm). Assays were performed at pH 7.4 in 150 mm NaCl, 10 mm CaCl2 . RESULTS: Serum and plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity with FRETS-rVWF71. Neither bilirubin (≤ 20 mg dL(-1) ) nor hemoglobin (≤ 20 g L(-1) ) interfered with product detection. Assays with FRETS-rVWF71 and FRETS-VWF73 gave similar results (R(2 ) = 0.95) for plasma from 80 subjects with thrombotic microangiopathy, 22 subjects with other causes of thrombocytopenia, and 20 healthy controls. The limit of detection with FRETS-rVWF71 for ADAMTS13 activity was ≤ 0.3%. Inhibitor assays with FRETS-rVWF71 gave titers ~2.5-fold higher than with FRETS-VWF73 and clearly distinguished patients with and without inhibitors. CONCLUSIONS: FRETS-rVWF71 is suitable for ADAMTS13 assays in minimally diluted plasma or serum without interference from proteins, bilirubin or free hemoglobin in plasma. Optimized detection of ADAMTS13 inhibitors will facilitate the monitoring of antibody responses during the treatment of thrombotic thrombocytopenic purpura.

Critical von Willebrand factor A1 domain residues influence type VI collagen binding.

BACKGROUND: von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen. OBJECTIVES: We report here on several mutations that exclusively alter binding to type VI collagen. PATIENTS/METHODS: Healthy controls and index cases from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen. VWF gene sequencing was performed for all subjects. RESULTS: Two healthy controls and one type 1 VWD subject were heterozygous for an A1 domain sequence variation, R1399H, and displayed a selective decreased binding to type VI collagen but not types I and III. Expression of recombinant 1399H VWF resulted in absent binding to type VI collagen. Two other VWF A1 domain mutations, S1387I and Q1402P, displayed diminished binding to type VI collagen. An 11 amino acid deletion in the A1 domain also abrogated binding to type VI collagen. CONCLUSIONS: VWF:CB may be useful in diagnosis of VWD, as a decreased VWF:CB/VWF:Ag ratio may reflect specific loss of collagen binding ability. Mutations that exclusively affect type VI collagen binding may be associated with bleeding, yet missed by current VWF testing.

Comparison of type I, type III and type VI collagen binding assays in diagnosis of von Willebrand disease.

BACKGROUND: von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Collagen binding assays (VWF:CB), however, are not part of the routine work-up for von Willebrand disease (VWD). OBJECTIVES: This study presents data on collagen binding for healthy controls and VWD subjects to compare three different collagens. PATIENTS/METHODS: VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen were examined for samples obtained from the Zimmerman Program. RESULTS: Mean VWF:CB in healthy controls was similar and highly correlated for types I, III and VI collagen. The mean VWF:CB/VWF:Ag ratios for types I, III and VI collagen were 1.31, 1.19 and 1.21, respectively. In type 1 VWD subjects, VWF:CB was similar to VWF:Ag with mean VWF:CB/VWF:Ag ratios for types I, III and VI collagen of 1.32, 1.08 and 1.1, respectively. For type 2A and 2B subjects, VWF:CB was uniformly low, with mean ratios of 0.62 and 0.7 for type I collagen, 0.38 and 0.4 for type III collagen, and 0.5 and 0.47 for type VI collagen. CONCLUSIONS: Normal ranges for type I, III and VI collagen are correlated, but higher values were obtained with type I collagen as compared with types III and VI. The low VWF:CB in type 2A and 2B subjects suggests that VWF:CB may also supplement analysis of multimer distribution. However, these results reflect only one set of assay conditions per collagen type and therefore may not be generalizable to all collagen assays.

Limitations of the ristocetin cofactor assay in measurement of von Willebrand factor function.

BACKGROUND: Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in von Willebrand factor (VWF) and diagnosed by a disproportionate decrease in VWF ristocetin cofactor activity (VWF:RCo) as compared with VWF antigen (VWF:Ag). OBJECTIVE: We report here on the spurious diagnosis of VWD in a patient with a sequence variation in the ristocetin-binding domain of VWF. PATIENTS/METHODS: The index case had a VWF:RCo of 11 IU dL(-1), with VWF:RCo/VWF:Ag ratio of 0.09. DNA sequencing revealed a novel P1467S mutation in a known ristocetin-binding region of the A1 domain. Because of the discrepancy between the laboratory findings, consistent with type 2M VWD, and the patient's lack of bleeding symptoms, further studies were performed to determine whether this mutation affected VWF function or merely reduced its ability to interact with ristocetin. RESULTS: Studies with recombinant VWF showed normal platelet binding with botrocetin, but a significant decrease in binding in response to ristocetin. Ristocetin-induced binding to recombinant GPIb was also absent, but normal binding was seen when a gain-of-function GPIb construct was used in the absence of ristocetin. VWF function under shear stress was normal when analyzed with a cone and plate(let) analyzer. CONCLUSIONS: The decreased VWF:RCo seen with the P1467S sequence variation likely represents an artifact as a result of the use of ristocetin to measure VWF activity. The normal VWF function in other assays correlates with the lack of hemorrhagic symptoms, and suggests the need for more physiologically relevant assays of VWF function.

Urgent reversal of warfarin with prothrombin complex concentrate.

BACKGROUND: When life-threatening bleeding occurs in patients on warfarin, timely reversal becomes imperative. In the USA, warfarin effect is commonly reversed with fresh frozen plasma (FFP). The use of FFP is complicated by delays in correction, volume overload and often, inadequate correction. OBJECTIVE: Evaluate the feasibility and efficacy of a protocol for rapid administration of prothrombin complex concentrate (PCC) in the setting of the urgent need for reversal of warfarin. METHODS/PATIENTS: We instituted a policy for rapid delivery and administration of PCC. Appropriate patients received 25-50 U kg(-1) of PCC. The prothrombin time (PT)/International Normalized Ratios (INR) was recorded before and immediately after dosing, and 24 h postdosing. Patients requiring surgical interventions were cleared for the operating room (OR) immediately. Fifty-eight patients were treated, with a median age of 75.5 years (range 26-92). RESULTS: The median INR on presentation was 3.8 (1.4-52.8). Immediately following PCC administration the median INR was 1.3 (0.9-5.7), only two patients with INRs exceeding 2.0. The benefit was maintained at 24 h with a median INR of 1.5 (1.1-3.4). Four patients experienced thrombotic events during their hospitalization, (two deep vein thrombosis, two non-q-wave myocardial infarction) although none was attributed to PPC therapy. CONCLUSIONS: PCC administration is an effective treatment modality for the correction of warfarin anticoagulation in the urgent setting. Advantages over FFP include more timely correction, absence of volume overload and potentially more complete correction. Broader use of PCC in this setting appears to be appropriate.

Measurement of von Willebrand factor cleaving protease (ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas.

BACKGROUND: ADAMTS-13 is a von Willebrand factor (VFW)-cleaving protease. Its congenital or acquired deficiency is associated with thrombotic thrombocytopenic purpura (TTP) and more rarely with the hemolytic uremic syndrome. We report on a survey evaluating 11 methods for ADAMTS-13 measurement performed in different labs. DESIGN: Two plasmas, one normal and one from a patient with familial TTP, were mixed at the co-ordinating center to prepare 6 plasmas with 0%, 10%, 20%, 40%, 80% and 100% ADAMTS-13 levels. Each plasma was aliquoted and assembled into sets of 60 (coded from 1 to 60), each containing 10 copies of the original 6 plasmas. Plasmas were frozen and shipped in dry ice to 10 labs with a common frozen reference plasma. Laboratories were asked to measure ADAMTS-13 with their methods. Results were sent to the coordinating center for statistical analysis. RESULTS: Of the 10 methods performed under static conditions 9 were quantitative and one was semiquantitative. One method performed under flow conditions evaluated the extent of cleavage of endothelial cell-derived ultralarge VWF string-like structures and expressed results as deficient, normal, or borderline. Linearity (expected-vs-observed levels), assessed as the squared correlation coefficient, ranged from 0.98 to 0.39. Reproducibility, expressed as the coefficient of variation for repeated measurements, ranged from < 10% to 83%. The majority of methods were able to discriminate between different ADAMTS-13 levels. The majority were able to detect the plasma with 0% level and some of them to discriminate between 0% and 10%. Overall the best performance was observed for three methods measuring cleaved VWF by ristocetin cofactor, collagen binding, and immunoblotting of degraded multimers of VWF substrate, respectively. The poor interlaboratory agreement of results was hardly affected by the use of the common standard. The method performed under flow conditions identified the plasmas with 0%, 10%, 20% and 40% activity as deficient in 7, 5, 1 and 3 of the 10 replicate measurements. The plasmas with 80% and 100% were identified as normal in all of the 10 replicate measurements. CONCLUSIONS: The survey shows varied performance, but supports an optimistic view about the reliability of current methods for ADAMTS-13.

Genotyping of factor V G1691A (Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes.

BACKGROUND: The factor V G1691A Leiden (FVL) mutation is the most common known hereditary risk factor for venous thrombosis. METHODS: Third Wave Technologies, Inc. (Madison, WI) has developed a new microtiter plate-based assay that does not require PCR, restriction digestion, or gel electrophoresis. This technology system, termed the Invader(TM) assay, utilizes a 5' "invading" oligonucleotide and a partially overlapping 3' "signal" oligonucleotide, which together form a specific structure when bound to a complementary genomic DNA template. A thermostable flap endonuclease cleaves this structure, releasing the 5' flap from the signal oligonucleotide. Increased temperature and an excess of the signal probe enable multiple probes to be cleaved for each target sequence present without temperature cycling. The cleaved probes then direct cleavage of a secondary probe, which is 5' end-labeled with fluorescein but is quenched by an internal dye. Upon cleavage, the fluorescein-labeled product is detected using a standard fluorescence plate reader. Genotypes are determined by net wild-type/mutant signal ratio. RESULTS: Complete concordance was observed, after resolution of four discordances, when 1369 individuals (1264 wild type, 102 heterozygous, 3 homozygous) were FVL genotyped by both the Invader assay and by allele-specific PCR. CONCLUSION: We conclude that FVL genotyping using invasive cleavage of oligonucleotide probes is a rapid and reliable alternative to genotyping by more traditional PCR-based methods.

A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microtiter plate fluorescence resonance energy transfer (FRET) assay.

The goal of this multicenter study was to evaluate the second-generation Invader technology for detecting the factor V (Leiden) mutation directly from genomic DNA of different sample types. Invader assay results were compared with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or allele-specific PCR (AS-PCR) analysis. The Invader assay is a PCR-independent methodology that uses a microtiter plate format. In the assay, a specific upstream Invader oligonucleotide and a downstream probe hybridize in tandem to a complementary DNA template and form a partially overlapping structure. The Cleavase VIII enzyme recognizes and cuts this structure to release the 5' flap of the probe. This flap then serves as an Invader oligonucleotide to direct cleavage of a fluorescence resonance energy transfer (FRET) probe in a second invasive cleavage reaction. Cleavage of this FRET probe results in the generation of a fluorescent signal. The results of the Invader assay were 99.5% concordant with the PCR-based methods. Of the 372 samples tested once, only two gave discordant results (one from operator error and one from unknown causes), but were concordant on retesting. These results indicate that a simple microtiter plate-based Invader assay can reliably genotype clinical patient samples for the factor V (Leiden) point mutation directly from genomic DNA without prior target amplification.

Allelic distribution of the glycoprotein Ia (alpha2-integrin) C807T/G873A dimorphisms among caucasian venous thrombosis patients and six racial groups.

Two linked silent dimorphisms, 807 C --> T (Phe224) and 873 G --> A (Thr246) within the glycoprotein Ia (GPIa) gene have been correlated with low and high platelet receptor density, respectively, and associated with vascular disease. A multiplexed allele-specific PCR assay was used to determine the GPIa 807T/873A allele frequency among 331 Caucasian venous thrombosis patients and 3571 unrelated individuals belonging to six different racial groups. The 807T/873A allele frequencies were 54%, 51%, 39%, 39%, 38%, 34% and 30% among Native Americans, Hispanics, Caucasians, Caucasian venous thrombosis patients, Asian Indians, African-Americans, and Koreans, respectively. Significant differences in the GPIa allele frequency among racial groups were revealed which emphasized the need for appropriate controls in studies evaluating the association of GPIa genotype to vascular disease.