Activated protein C resistance

Objectives.-To evaluate a modification of a commercially available reagent kit (COATEST APC Resistance Kit) for functional activated protein C (APC) resistance testing, and to determine the ability of the modified assay to demonstrate APC resistance in patients receiving warfarin.

Design.-Functional APC resistance testing was performed using both the first-generation COATEST APC Resistance Kit and a modified, or second-generation, version of the COATEST assay that uses predilution of the patient sample with factor V-deficient plasma. Factor V genotyping for APC resistance (FV R506Q) was performed using a well-characterized polymerase chain reaction-restriction fragment length polymorphism method.

Setting.-University medical center.

Patients.-Seventy-three individuals referred for hypercoagulability testing who were not receiving warfarin therapy and 29 patients with a history of venous thrombosis who were receiving warfarin therapy.

Main Outcome Measures.-Sensitivity and specificity as

determined by comparing functional APC resistance to the FV R506Q genotype.

Results.-In 73 patients referred for hypercoagulability testing, but not receiving warfarin therapy, a sensitivity of 0.86 and a specificity of 0.75 were obtained with the firstgeneration COATEST assay. In contrast, a sensitivity and specificity of 1.0 were obtained when the second-generation COATEST assay was employed. In 29 patients receiving warfarin, the first-generation assay exhibited a sensitivity and specificity of 0.88 and 0.95, respectively, whereas the sensitivity and specificity for the second-generation assay was 1.0.

Conclusions.-Predilution of patient plasma with factor V-deficient plasma results in improved sensitivity and specificity of the COATEST APC Resistance Kit, thus offering a simple modification to enhance APC resistance determination in the routine clinical laboratory setting.

Arch Pathol Lab Med. 1998;122:430433)

Proteolytic degradation of factor Va by activated protein C (APC) limits clot formation following activation of the coagulation cascade.1,2 Recently, a point mutation in the factor V gene (FV R506Q or FV Leiden) was identified that renders factor Va relatively resistant to proteolytic degradation by APC.3s The mutation is present in 3% to 5% of the general population and is associated with a lifelong 5- to 10-fold increase in the relative risk of venous thrombosis.3 9-tl Greater than 90% of APC resistance in individuals is due to FV R506Q, and the mutation is the most common genetic cause of inherited thrombophilia identified to date.12,13

The diagnosis of APC resistance is made by functional testing or inferred through identification of FV R506Q. Most functional assays for APC resistance are based on the observation that individuals with this trait exhibit a diminished anticoagulant response to exogenous APC as detected by the activated partial thromboplastin time (aPTT) clotting assay.16 The results are expressed as an APC sensitivity or "resistance" ratio (hereinafter referred to as APC ratio), defined as the ratio of the patients aPTT in the presence of exogenous APC to that obtained in the absence of exogenous APC. First-generation functional assays for APC resistance, based on aPTT measurements, are characterized by the following important limitations: (1) an overlap in APC ratios between normal individuals and individuals heterozygous for FV R506Q413,17 and (2) the inability to evaluate patients with prolonged aPTT values due to anticoagulant therapy. As a result, genotyping for FV R506Q is needed on a significant number of patients to establish the diagnosis of APC resistance.

For functional APC resistance testing, our laboratory has used a first-generation, commercially available assay (COATEST APC Resistance Kit). Previously, we reported a sensitivity and specificity of 0.81 and 0.88, respectively, for the first-generation COATEST assay with respect to its ability to demonstrate APC resistance due to FV R506Q. The objectives of the current study were to evaluate a modified, or second-generation, version of the COATEST assay that uses predilution of patient samples with factor V-deficient plasma, and to determine the performance of the modified assay in patients receiving warfarin.

METHODS

Human Subjects

Specimens analyzed in this study were obtained in accordance with guidelines approved by the Human Subjects Review Committee of the University of Wisconsin Hospital and Clinics. The study population was composed of patients referred to our coagulation laboratory for hypercoagulability testing. Patients were excluded from the study if they were receiving heparin or if they tested positive for a lupus anticoagulant.

Functional Analysis of APC Resistance

The details of the first-generation assay for APC resistance have been reported previously.'1 Briefly 9 volumes of venous blood were mixed with 1 volume of 0.1 mol/L trisodium citrate and centrifuged at 2000g for 15 minutes at room temperature. The platelet-poor plasma was removed and stored at -70 deg C until used. For all samples, plasma was separated and frozen within 3 hours of phlebotomy. The response of each plasma sample to APC was determined with the COATEST APC Resistance Kit (Chromogenix, Molndal, Sweden), containing a 0.025 mol/L calcium chloride (CaCl2) solution, human APC colyophilized with CaCl2 and an aPTT reagent containing a mixture of synthetic phospholipids with colloidal silica as contact activator. Determinations were performed according to the manufacturer's guidelines. For each determination, equal volumes (50 muL) of aPTT reagent and plasma samples were incubated at 37 deg C for 5 minutes, followed by the addition of an equal volume of either CaCl2 or APC/CaCI2. The time to clot formation was determined in duplicate using an MLA Electra 900c Automatic Coagulation Timer (Medical Laboratories Automation, Pleasantville, NY). Results were expressed as APC ratios (clotting time obtained using the solution APC/CaCl2 divided by the clotting time obtained with CaCl, alone). The second-generation functional assay was performed as described above, with the exception that prior to APC resistance determination, patient plasma was prediluted 1: 5 volumetrically with plasma deficient in factor V that contains the heparin neutralizer Polybrene (V-Def Plasma, Chromogenix). Determination of FV R506Q Genotype Factor V genotyping for FV R506Q was performed as previously described.18

RESULTS

Comparison of First- and Second-Generation Functional Assays

Seventy-three individuals referred for hypercoagulability testing, but not receiving warfarin therapy, met our inclusion criteria. Plasma samples from these individuals were analyzed by both first- and second-generation APC resistance assays, and factor V genotypes were determined. The results of these analyses are shown in Fig 1. Of the 73 subjects, 36 were genotypically normal and had APC ratios ranging from 1.7 to 3.2 (mean 2.33, SD 0.37) with the first-generation test and from 2.1 to 3.4 (mean 2.72, SD 0.35) with the second-generation test. Thirty-four individuals heterozygous for FV R506Q exhibited APC ratios ranging from 1.4 to 2.5 (mean 1.84, SD 0.25) with the first-generation assay and from 1.5 to 1.9 (mean 1.73, SD 0.12) with the second-generation assay. Three individuals were homozygous for FV R506Q; these individuals had APC ratios of 1.1 and 1.3 in the first-generation assay and 1.2 and 1.3 in the second-generation assay.

Comparison of First- and Second-Generation Assays in Patients Receiving Warfarin

Twenty-nine patients with a history of venous thrombosis receiving warfarin therapy were available for analysis (Fig 2). Genotyping showed that 21 of the 29 patients were wild-type, whereas 7 were heterozygous and 1 was homozygous for FV R506Q. Using the first-generation assay, the 21 patients with wild-type genotype exhibited a range of APC ratios from 2.0 to 3.3 (mean 2.47, SD 0.32). The range of APC ratios in the seven patients heterozygous for FV R506Q was 1.5 to 2.4 (mean 1.80, SD 0.30). Using the second-generation assay, the 21 patients with wild-type genotype exhibited a range of APC ratios from 2.5 to 3.5 (mean 3.15, SD 0.25). The range of APC ratios in the seven patients heterozygous for FV R506Q was 1.7 to 1.9 (mean 1.78, SD 0.07).

COMMENT

Functional APC resistance testing has become an important part of the laboratory evaluation of hypercoagulability syndromes. More than 90% of individuals with APC resistance carry a point mutation in the factor V gene (FV R506Q or FV Leiden) that renders factor Va relatively resistant to proteolytic degradation by APC.3 The availability of both functional and genotypic approaches for APC resistance testing offers diagnostic options for the individual clinical laboratory. In our setting, we have recommended functional testing as the initial approach when feasible. When APC resistance is detected, we have recommended follow-up genotyping for confirmation of FV R506Q. This approach not only verifies the presence or absence of FV R506Q, but also detects individuals homozygous for FV R506Q, who are at greatest risk for thrombosis. This algorithm offers an overall cost savings to our patient population as the total cost to perform functional testing, in our laboratory, is approximately one third that of genotyping.

In our initial application of this algorithm,' functional testing was performed with the commercially available, first-generation COATEST APC Resistance Kit. We reported previously that the sensitivity and specificity of the COATEST APC Resistance Kit was 0.81 and 0.88, respectively, with respect to identifying APC resistance due to FV R506Q. These levels of sensitivity and specificity resulted in our performing a significant number of molecular analyses that resulted in normal genotype determinations. The current study was undertaken to determine if improvement in sensitivity and specificity of the COATEST APC Resistance Kit could be achieved by predilution of patient plasma with factor V-deficient plasma. This in vitro modification was first suggested by Sun et al19 and was subsequently confirmed to improve the performance of functional assays for APC resistance and to allow determination of APC resistance in patients on anticoagulant therapy.20-22

In the current series of 73 patients referred for hypercoagulability testing, we obtained a sensitivity of 0.86 and a specificity of 0.75 with the first-generation COATEST assay for APC resistance when we used the manufacturer's guidelines of defining APC resistance as a ratio of 2.0 or less (as done in our previous studies). In contrast, a sensitivity and specificity of 1.0 were obtained when the second-generation COATEST assay, which incorporates predilution with factor V-deficient plasma, was employed. In 29 patients receiving warfarin, the first-generation assay exhibited a sensitivity and specificity of 0.88 and 0.95, respectively, compared with a sensitivity and specificity of 1.0 for the second-generation assay.

Based on our findings, we supplanted the first-generation COATEST assay with the second-generation COATEST assay in our diagnostic coagulation laboratory. To further evaluate the performance of the second-generation assay in routine diagnostic use, we genotyped patients whose APC resistance ratios were 2.5 or less during a 6month period. In this approach, we assumed that APC resistance ratios of 2.6 or greater would be genotype normal for FV R506Q. During the 6-month period, different lots of reagents for functional testing were used. Of 91 patients evaluated by both functional and genotyping approaches, only a single patient that was genotype normal would have been classifed as APC resistant when applying the criteria of a ratio of 2.0 or less as defining resistance (data not shown).

As a result of this study, we have revised our recommended diagnostic algorithm. Previously, with the firstgeneration COATEST assay, we recommended genotype confirmation on individuals whose APC ratios were 2.5 or less. Using the second-generation COATEST assay, we now recommend genotyping those patients with APC ratios at or below our revised "cutoff" value of 2.1. This revised algorithm has resulted in a decrease in our volume of molecular testing for FV R506Q, offering cost savings to both our patients and clinical laboratory.

While characterized by improved sensitivity and specificity, the second-generation COATEST assay is still limited in that it is not recommended for use with plasma samples with prolonged aPTTs due to either the presence of heparin or lupus anticoagulants. Recently, a chromogenic assay based on direct measurement of the effect ol APC on the activity of factor Va in prothrombin activation has been evaluated for clinical use.23 The chromogenic assay appears to be highly sensitive and specific for demonstrating APC resistance due to FV R506Q, and can be used to assay plasma from individuals receiving heparin or with lupus anticoagulants. In its current format, however, the chromogenic assay requires more technical steps and reagents in comparison with the second-generation COATEST assay. It is likely, therefore, that many routine coagulation laboratories will continue to use the COATEST assay format for APC resistance determination. Fox those laboratories, the second-generation COATEST assay is an attractive approach for enhancing APC resistance determination.

References

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