Activated protein C resistance

* Objective.-To compare functional and molecular biological tests for resistance to activated protein C (APC)/ factor V R506Q, the most common cause of familial thrombosis.

Methods.-We developed functional and molecular biological tests for resistance to APC/factor V R506Q at our institution and correlated the results for 370 patients studied by both methods. The functional method is based on addition of exogenous APC to an activated partial thromboplastin time-based assay. The molecular biological method is based on polymerase chain reaction followed by endonuclease digestion.

Results.-Considering the molecular biological test as definitive for detecting the factor V R506Q mutation, the sensitivity of the functional assay was 100%, and the specificity was 74%. The prevalence of the factor V mutation in the population studied was 12% (41 heterozygotes, two homozygotes), and the positive predictive value of the functional assay was 34%. Although a normalized sensitivity ratio (nAPC-SR) less than 0.84 is considered evidence

of resistance to APC by functional testing, we found that all patients with factor V R506Q had an nAPC-SR less than or equal to 0.71. When this alternative positive cutoff was used, the specificity of the functional test for factor V R506Q increased to 87%, and the positive predictive value increased to 52%, which constituted a significant improvement. We compared clinical findings from patients with resistance to APC with or without the presence of factor V R506Q, and found that as a group, those with factor V R506Q had a higher incidence of hypercoagulability, but fewer additional risk factors for hypercoagulability. The mechanism of resistance to APC in factor V R506Q-negative individuals is unclear, but may be related to other risk factors for hypercoagulability.

Conclusions.-The functional assay for resistance to APC is an excellent screening test for factor V R506Q, but confirmatory molecular biological testing is necessary when the functional test is positive, because of the high falsepositive rate.

(Arch Pathol Lab Med. 1998;122:325-329)

Resistance to activated protein C (APC) is a newly recognized cause of hypercoagulability that has been attributed, in the vast majority of cases, to a point mutation in the factor V gene that interferes with inactivation of factor Va by APC.1-3 Inherited hypercoagulability assoaated with APC resistance was found in more than 90% of affected families to segregate with a single point mutation at nucleotide position 1691 of the factor V gene, resulting in a guanine to adenine substitution (factor V R506Q also known as factor V Leiden).1,4 No other molecular mechanisms of resistance to APC have been identified. Resistance to APC is the most common cause of familial thrombosis and is observed in 20% to 50% of all cases.5,6 The prevalence of factor V R506Q in the general population is approximately 5%, and individuals who are heterozygous for the mutation have a sevenfold increased risk of thromboembolic events,1 whereas those who are homozygous for factor V R506Q have an 80-fold increased risk of thromboembolic events.7 Thrombosis in individuals from these two groups may be associated with additional risk factors, such as pregnancy, use of oral contraceptives, trauma, or surgery.4

Assay methods for resistance to APC include functional analysis of the intrinsic coagulation pathway and molecular genetic analysis of the factor V gene for the nucleotide 1691 substitution using molecular biological methods, such as the polymerase chain reaction (PCR) combined with endonuclease digestion.1 The functional test measures the activated partial thromboplastin time (aPTT) before and after the addition of APC, and is reported as a normalized sensitivity ratio aPTT^sub post-APC^/aPTT^sub pre-APC^ (nAPC-SR), which has been considered to be evidence of resistance to APC when decreased below 0.84.1 The normalized sensitivity ratio value is derived from comparison of a particular patients sensitivity ratio with that of a normal pool (see "Materials and Methods"). Functional testing can be performed extremely rapidly, for example, using automated coagulation instrumentation, and is inexpensive compared with molecular biological testing. Unfortunately, standard functional testing is not accurate for patients who have an elevated aPTT, including patients receiving anticoagulant therapy.4,8 Furthermore, unlike molecular biological testing, functional testing is not speafic for factor V R506Q.1 As part of our effort to institute testing for resistance to APC at our hospital, we studied the utility of functional testing for the detection of factor V R506Q by comparing results of functional and molecular biological assays for factor V R506Q in 370 consecutive patients who could be tested with both methods.

MATERIALS AND METHODS

Functional testing for resistance to APC was performed as previously described,9 based on the method of Koster and associates.10 Venous blood was collected in citrate, transported on ice, and centrifuged to obtain plasma. Plasma was assayed the same day as collected or stored at -70 deg C until the time of assay. The aPTT was performed on patient samples and on a pool of normal subjects at the time of the assay using the MDA 180 coagulation analyzer (Organon Teknika, Durham, NC) with Actin-FS reagent (Dade International, Miami, Fla). The aPTT was repeated with the normal pool, with the addition of a sufficient quantity of human APC (Hematologic Technologies Inc, Essex Junction, Vt) to prolong the baseline aPTT to 100 to 160 seconds. This amount of human APC was added to each of the patient samples. A sensitivity ratio was then calculated from the aPTT with added APC divided by the aPTT without added APC. A normalized APC sensitivity ratio (nAPC-SR) was calculated by dividing the sensitivity ratio for each patient by the sensitivity ratio of the pool of normal subjects. Patients were not tested with the functional assay if there was known concurrent treatment with heparin or oral anticoagulants or if there were findings of elevated prothrombin time or aPTT before the addition of APC.

Molecular biological analysis for the factor V R506Q mutation was performed as previously described.9 The method used was PCR analysis followed by endonuclease digestion. A 267-base pair (bp) region of the factor V gene was amplified by PCR with primers PR-6967 and PR-990.1 The PCR reaction, performed on 200-ng samples of patient DNA, was carried out in a 50-(mu)L volume containing 10 mmol/ L Tris-hydrogen chloride (pH 8.3), 1.5 mmol/L magnesium chloride, 200 (mu)mol/L of each of the four deoxynucleotide triphosphates, 4 (mu)mol/L of each of the two primers, and 1.25 U of Taq polymerase, in a Perkin Elmer Cetus model 9600 thermal cycler (Perkin Elmer Cetus, Branchburg, NJ), by a modification of the method of Don et al.1 DNA was initially denatured at 94 deg C for 4 minutes, then underwent 35 cycles of amplification, each of which had denaturation for 20 seconds at 94 deg C, 20 seconds at the annealing temperature, and 20 seconds at 72 deg C for extension. The annealing temperature decreased sequentially 1 deg C, from 65 deg C to 55 deg C during cycles 1 through 11, with all subsequent cycles annealing at 55 deg C. After 35 cycles, samples were incubated at 72 deg C for 10 minutes and then cooled at 4 deg C for 5 minutes. Control samples included water, normal DNA, factor V R506Q heterozygote DNA, and factor V R506Q homozygote DNA. A 15-(mu)L aliquot of each PCR reaction was digested with restriction endonuclease Mnl I and electrophoresed in a 2.5% agarose gel containing 0.3 (mu)g/mL ethidium bromide. The genotype of each patient was determined from the pattern of restriction fragments observed in the gel; the factor V R506Q allele produces Mnl I fragments of 67 bp and 200 bp, and the normal factor V allele produces Mnl I fragments of 37 bp, 67 bp, and 163 bp.

RESULTS

Comparison of Functional and Molecular Biological Assays for Factor V R506Q

A total of 370 patients were studied with both the functional assay for resistance to APC and the molecular biological test for factor V R506Q; the results are summarized in Table 1. Patients included young adults undergoing evaluation for thromboembolic disease, including inherited thrombophilia, as well as patients who were surgical candidates undergoing preoperative screening for hypercoagulable states. Previously, Bertina et al1 defined resistance to APC as an nAPC-SR less than 0.84 with the functional assay. The presence of factor V R506Q as detected by the molecular biological test was correlated with the presence or absence of resistance to APC on the basis of the functional assay. Because it has been shown that the functional assay for resistance to APC is not completely specific,1 we regarded the molecular biological test as the definitive test for the presence of factor V R506Q. Resistance to APC as defined by the functional assay was 100% sensitive for the presence of factor V R506Q with a specificity of 74% and a positive predictive value of 34%. The prevalence of factor V R506Q in our patient population, which included a heterogeneous group of patients, as noted above, was 12% (41 heterozygotes, two homozygotes).

The mean nAPC-SR was 0.49 +/- 0.09 (range 0.29-0.71) for patients with factor V R506Q and 0.68 +/- 0.12 (range 0.40-0.83) for patients with resistance to APC lacking the factor V R506Q mutation (P

Clinical Findings in Patients With Resistance to APC and Factor V R506Q

We were able to obtain clinical histories and/or additional laboratory data for 38 of the 43 patients with resistance to APC and factor V R506Q; the findings are summarized in Table 2. Patients ranged in age from 26 to 79 years (mean 47 years), and included nine men and 29 women. Of 28 patients for whom pertinent clinical history could be obtained, 23 (82%) had a previous or subsequent history of hypercoagulability (follow-up times ranged from 1 to 12 months), including deep venous thrombosis (11 cases), thrombophlebitis (5 cases), pulmonary embolism (3 cases), or other thrombotic events (4 cases). Eight (35%) of the 23 hypercoagulable patients were found to have at least one additional risk factor for hypercoagulability, including hyperlipidemia, diabetes, and malignant neoplasms.12 A total of 13 (46%) of 28 patients with resistance to APC and factor V R506Q were found to have at least one additional risk factor for hypercoagulability.

Clinical Findings in Patients With Resistance to APC Lacking Factor V R506Q

Clinical history and/or additional laboratory data were available for 76 of the 83 patients with resistance to APC lacking factor V R506Q; the findings are summarized in Table 2. Patients ranged in age from 22 to 89 years (mean 59 years), and included 15 men and 61 women. Thirty-one (41%) of 76 patients had a previous or subsequent history of hypercoagulability (with follow-up ranging from 1 to 18 months), including deep venous thrombosis (11 cases), thrombophlebitis (3 cases), pulmonary embolism (9 cases), or other thrombotic events (8 cases). Twenty-four (77%) of 31 hypercoagulable patients were found to have at least one known risk factor for hypercoagulability, including pregnancy, estrogen administration, hyperlipidemia, recent surgery, diabetes, and malignant neoplasms.12 A total of 61 (80%) of 76 patients with resistance to APC but lacking factor V R506Q were found to have at least one known risk factor for hypercoagulability.

COMMENT

In this study we compared and correlated functional testing for resistance to APC and molecular biological testing for factor V R506Q in a large group of patients. Factor V R506Q is recognized as the mechanism of resistance to APC in more than 90% of cases of familial thrombophilia,4 and no other molecular mechanisms for resistance to APC have been identified. Resistance to APC was originally defined by Bertina and associates1 as an nAPC-SR less than 0.84, which is 1.96 SD below the mean nAPC-SR in 100 healthy controls after outlier removal. Because of this definition, the functional assay for resistance to APC is not completely specific for factor V R506Q; 20% of individuals with an nAPC-SR less than 0.84 lacked the factor V R506Q mutation in the study of Bertina et al,l including 50% of age- and sex-matched controls with nAPC-SR in the 0.70 to 0.83 range. In a subsequent study of 422 thrombophilia patients and 472 controls by Bertina and associates,6 38% of subjects with nAPC-SR less than 0.84 lacked the factor V R506Q mutation, including all 41 patients and 16 controls with nAPC-SR in the 0.70 to 0.83 range. In the current study, we also found that the 0.84 cutoff for resistance to APC was not specific for factor V R506Q, with an even greater number of false-positive individuals at the 0.84 cutoff (Table 1). This difference is due in part to differences in patient populations studied. The present study included a significant number of older presurgical patients in contrast to the younger thrombophilia patients and agematched controls in the studies of Bertina and associates. In addition, there was an increased relative number of patients with nAPC-SR in the 0.70 to 0.83 range in the present study compared with that of Bertina et al.1 Another factor that may account for differing false-positive rates is the use of different aPTT reagents (Actin-FS for the present study; Cephrotest, Nycomed Pharma, Oslo, Norway, for the studies by Bertina et al). The range of etiologies of resistance to APC in the absence of factor V R506Q is unclear and requires additional investigation. As part of their original study of resistance to APC, Bertina et all found that an nAPC-SR less than 0.70 was specific for factor V R506Q; 100% of individuals below this cutoff value for the functional test were homozygous or heterozygous for factor V R506Q, similar to our 87% specificity in the present study for individuals with an nAPC-SR less than or equal to 0.71. In a subsequent report, Bertina and associates noted that none of 797 individuals with an nAPC-SR greater than 0.70 had factor V R506Q. Family studies were performed for some of these factor V R506Q-negative individuals, and none had evidence of an inherited defect.6 Based on these results, it can be concluded that more than 90% of cases of resistance to APC are due to factor V R506Q, and that the functional test for resistance to APC can serve to identify all of these patients because of its 100% sensitivity. By lowering the positive cutoff of resistance to the APC functional test to 0.71, the specificity for factor V R506Q can be significantly improved. Other laboratories may wish to institute a lower positive cutoff value for the functional test if they obtain similar results.

Recently Voelkerding and associates13 and Zehnder and Bensonl14 reported results of functional testing for resistance to APC using the Coatest APC resistance kit (Chromogenix AB, Molndal, Sweden) in comparison with molecular biological testing. In both studies, there was overlap between normal individuals and those with factor V R506Q by functional testing. In the former study, the sensitivity of the functional test for factor V R506Q was 81% in a patient population being evaluated for hypercoagulability,13 whereas in the latter study the sensitivity of the functional test was 50%.14 Voelkerding et al,13 concluded that comparing functional with molecular testing can guide the individual laboratory in establishing cutoff values for screening with the functional test. Zehnder and Benson14 concluded that the functional test kit is not a useful screening method for factor V R506Q. A study comparing the Coatest APC resistance kit to an in-house method developed at University Hospital, Leiden, showed considerable discordance between the commercial and in-house methods.ls Based on our results and those of Bertina et al, we conclude that validated inhouse functional assay methods are capable of serving as screening tests for factor V R506Q and have significant advantages compared with universal molecular biological testing for factor V R506Q. However, each laboratory needs to examine its own results, to determine the incidence of false negatives with the functional screening test employed, before instituting a testing algorithm as presented in this report.

As part of our effort to initiate testing for resistance to APC/factor V R506Q at our institution, we compared the clinical findings for all patients with resistance to APC with and without the presence of factor V R506Q. As a group, those with factor V R506Q tended to be younger, had a greater associated number of hypercoagulable events, and had fewer known risk factors for hypercoagulability compared with individuals lacking the factor V R506Q mutation (Table 2). Although mechanisms of resistance to APC in factor V R506Q-negative patients are not clear, the possible association of factor V R506Q-negative resistance to APC with other risk factors for hypercoagulability12 deserves further investigation. Because of the high incidence of other risk factors for hypercoagulability in factor V R506Q-negative patients with resistance to APC, it may be useful to screen patients who fall into this category for these risk factors. However, as a group, factor V R506Q-negative patients with resistance to APC had a lower incidence of hypercoagulability compared with factor V R506Q patients.

A point of confusion in comparing resistance to APC and factor V R506Q is the finding by Zoller et al4 that three thrombosis-prone families with resistance to APC lacked the presence of factor V R506Q, suggesting another inherited cause of resistance to APC. However, no additional mechanisms for resistance to APC have become evident since that study. Also, the finding of factor V R506Q-negative heritable resistance to APC was limited to 3 out of 50 families studied, as noted above, representing less than 10% of patients studied.4

Based on our experience comparing functional testing for resistance to APC and molecular biological testing for factor V R506Q, we conclude that the functional test is an excellent screening test for factor V R506Q because of its simplicity, low cost, speed, and 100% sensitivity for factor V R506Q. In our laboratory, the cost per test for materials to perform the functional screening test amounts to less than 20% of the cost per test for materials to perform the confirmatory molecular biological test. Confirmatory molecular biological testing is necessary when the functional test is positive because of the high false-positive rate, although this can be significantly decreased by lowering the positive cutoff of the functional test to nAPC-SR less than or equal to 0.71. A drawback of the functional assay is that it is not accurate for patients receiving anticoagulants and other patients with an elevated PTT.4,8 The functional test is also more sensitive to preanalytic sample handling than is DNA analysis.

Recently, alternative methods of performing the functional assay for resistance to APC have been reported. Le and associates16 reported a one-stage tissue factor-dependent factor V assay in the presence or absence of APC, which is not affected by the presence of heparin or warfarin. However, these authors studied only a small number of patients with factor V R506Q (n = 18) and 95 normal controls. Kapiotis et al17 studied a modified Coatest APC resistance kit that employed predilution of patient samples with factor V-deficient plasma plus a heparin neutralizer. In their study of 87 patients (32 with factor V R506Q and 55 normal controls), the functional assay exhibited 100% sensitivity and specificity for factor V R506Q and was suitable for use in patients undergoing treatment with heparin or warfarin. However, 50% of patients with an elevated PTT due to the presence of a lupus anticoagulant (who were presumably negative for factor V R506Q) tested positive for resistance to APC with this assay method.17 These alternative methods may prove useful for resistance to APC testing in a wider range of patients, including patients receiving anticoagulant therapy, than may be reliably studied with the currently described functional assay.

We thank Ellen Goonan, MT(ASCP), and Susan Lemire, MT(ASCP), for their expert technical assistance.

References

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2. Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterization of the molecular defect in factor V(R506Q). J Biol Chem. 1995;270:4053-4057.

3. Heeb MJ, Kojima Y, Greengard JS, Griffin JH. Activated protein C resistance: molecular mechanisms based on studies using purified Gln5o-Factor V. Blood. 1995;85:3405-3411.

4. Zoller B, Svensson PJ, He X, Dahlback B. Identification of the same Factor V gene mutation in 47 of 50 thrombosis-prone families with inherited resistance to activated protein C. J Clin Invest. 1994;94:2521-2524.

5. Svennson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med. 1994;330:517-522.

6. Bertina RM, Reitsma PH, Rosendaal FR, Vandenbroucke JP. Resistance to activated protein C and Factor V Leiden as risk factors for venous thrombosis. Thromb Haemost.1995;74:449-453.

7. Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in patients homozygous for Factor V Leiden (activated protein C resistance). Blood. 1995;85:1504-1508.

8. De Ronde H, Bertina RM. Laboratory diagnosis of APC-resistance: a critical evaluation of the test and the development of diagnostic criteria. Thromb Haemost. 1994;72:880-886.

9. Hirsch DR, Mikkola KM, Marks PW, Fox EA, Dorfman DM, Ewenstein BM, Goldhaber SZ. Pulmonary embolism and deep venous thrombosis during pregnancy or oral contraceptive use: prevalence of factor V Leiden. Am Heart /. 1996;131:1145-1148.

10. Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden thrombophilia study. Lancet. 1993;342:1503-1506.

11. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. Touchdown PCR to circumvent spurious priming during gene amplification. Nucl Acids Res. 1991;19:4008-4009.

12. Bauer KA. Natural anticoagulants and the prethrombotic state. In: Han

din RI. Lux SE, Stossel TP, eds. Blood: Principles and Practice of Hematology. Philadelphia, Pa: JB Lippincott Co; 1995:1319-1339.

13. Voelkerding KV, Wu L, Williams EC, et al. Factor V R506Q gene mutation analysis by PCR-RFLP: optimization, comparison with functional testing for resistance to activated protein C, and establishment of cell line controls. Am J Clin Pathol.1996;106:100-106.

14. Zehnder JL, Benson RC. Sensitivity and specificity of the APC resistance assay in detection of individuals with Factor V Leiden. Am J Clin Pathol. 1996; 106:107-111.

15. Legani C, Palareti G, Biagi R, et al. Activated protein C resistance: a comparison between two clotting assays and their relationship to the presence of the factor V Leiden mutation. Br J HaematoL 1996;93:694-699.

16. Le DT, Griffin JH, Greengard JS, Mujumdar V, Rapaport Sl. Use of a generally applicable tissue factor-dependent Factor V assay to detect activated protein C-resistant Factor Va in patients receiving warfarin and in patients with a lupus anticoagulant. Blood. 1995;85:1704-1711.

17. Kapiotis S, Quehenberger P, Jilma B, et al. Improved characteristics of aPC-resistance assay: Coatest aPC resistance by predilution of amples with Factor V deficient plasma. Am J Clin Pathol.1996;106:588-593.

Accepted for publication December 4, 1997. From the Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass.

Presented in part at the fall meeting of the American Society of Clinical Pathologists and the College of American Pathologists, San Diego, Calif, September 28-October 4, 1996.

Reprint requests to Department of Pathology, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115 (Dr Dorfman).

Copyright College of American Pathologists Apr 1998
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