Sensitivity and Specificity ofthe APC Resistance Assay in Detection of Individuals With Factor V Leiden
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
COAGULATION AND TRANSFUSION MEDICINE
Original Article
Sensitivity and Specificity ofthe APC
Resistance Assay in Detection of Individuals
With Factor V Leiden
JAMES L. ZEHNDER, MD, AND RENEE C. BENSON
Downloaded from https://academic.oup.com/ajcp/article/106/1/107/1757011 by guest on 09 December 2021
Resistance to activated protein C (APC) is the most common cause of ceiver operating characteristic (ROC) curve analysis indicated that the
familial thrombophilia. The partial thromboplastin time (PTT)-based test was of intermediate utility. There was considerable overlap in APC
test for resistance to APC has been widely employed as a screening test ratios in the range of 2 to 3 between subjects with a normal factor V
for this disorder. However, the utility of this test for screening is not genotype and heterozygotes for factor V Leiden. The authors conclude
well characterized. More than 90% of patients with resistance to APC that the APC resistance assay in its present form is not a useful screen-
have the G1691A mutation in factor V (factor V Leiden). The authors ing test for factor V Leiden heterozygotes. Until the performance of this
studied the ability of a commercial APC resistance assay to correctly assay is improved, patients should have molecular diagnostic testing
identify the factor V Leiden genotype in 130 individuals. At the recom- performed to determine their factor V Leiden status. (Key words: Re-
mended assay cut-off value of 2, the sensitivity of the APC resistance sistance to activated protein C; Factor V Leiden; Thrombophilia; Re-
assay was 50%, with a specificity of 98%. Increasing the cut-off value ceiver operating characteristics; Test utility; Test methodology) Am J
increased the sensitivity but decreased the specificity of the test. Re- Clin Pathol 1996; 106:107-111.
Activated protein C (APC) limits the clotting process by Testing for this syndrome can be performed by an in
proteolytic inactivation ofthe two clotting co-factors fac- vitro assay of resistance to activated protein C, or by iso-
tor Va and factor Villa.'"3 Resistance to activated pro- lating genomic DNA of patients, amplifying and analyz-
tein C has been shown to be a common risk factor for ing the area ofthe mutation by restriction enzyme diges-
venous thrombosis.4,5 In >90% of cases, APC resistance tion or by oligonucleotide hybridization.4-613"15 The
is due to a mutation in the factor V gene, G1691A (factor most commonly used test for APC resistance is a partial
V Leiden).6"10 This mutation does not appear to alter the thromboplastin time (PTT)-based test, reported as the
procoagulant activity of factor Va. However, the muta- ratio of a PTT performed in the presence of a standard
tion occurs at one ofthe two APC cleavage sites, resulting quantity of APC divided by the PTT without APC
in decreased inactivation of factor Va, and increases the added. It has been shown that most healthy individuals
risk of venous thrombosis in affected individuals. Horao- will have a ratio of >2, whereas those with APC resis-
zygotes for factor V Leiden have a relative risk of throm- tance were defined as having a ratio < 2.4 Because of its
bosis of 50 to 100, whereas the risk in heterozygotes is 5- simplicity this test has been widely employed as a screen-
to 10-fold higher than healthy subjects." The factor V ing test for APC resistance.
Leiden mutation appears to be relatively common with
The definitive test for this syndrome is molecular di-
a prevalence of 5% to 10% in several populations stud-
agnosis of factor V Leiden.6 Determination of factor V
ied,46 although a recent report suggests that the inci-
genotype is performed by isolating genomic DNA from
dence of this mutation in people of Japanese ancestry is
patient leukocytes. The DNA sequenceflankingthe mu-
much lower.12 This defect is the most common cause of
familial thrombophilia yet identified. tation site in the factor V gene is then amplified using
the polymerase chain reaction (PCR), and the resultant
product analyzed by restriction enzyme digestion.6 In
this way, patients can be classified as wild type (two nor-
From the Department of Pathology, Stanford University Medical mal factor V alleles), heterozygous for factor V Leiden or
Center, Stanford, California.
homozygous for factor V Leiden.
Manuscript received October 10, 1995; revision accepted January There is limited information on the sensitivity and
10, 1996.
Address reprint requests to Dr. Zehnder: Department of Pathology, specificity and performance ofthe APC resistance test in
L235, Stanford University Medical Center, Stanford, CA 94305. identifying patients with factor V Leiden. We studied the
107108 COAGULATION AND TRANSFUSION MEDICINE
Original Article
results of APC testing and factor V genotype in 130 pa- Cetus). The 267 bp amplification product was digested
tients referred for evaluation of thrombosis. with Mnl I for 60 minutes at 37 °C and the resulting frag-
ments separated by electrophoresis in a 3% agarose gel.
MATERIALS AND METHODS Under these conditions wild type factor V (1691G) yields
a 167 bp fragment and factor V Leiden (1691 A) yields a
Specimen Collection 200 bp fragment. On this basis, patients can be classified
as wild type factor V (only a 167 bp band is visualized),
One hundred thirty sequential patients referred for
heterozygous for factor V Leiden (167 bp and 200 bp
APC and factor V Leiden testing were studied. Patients
fragments are present), or homozygous for factor V
were excluded if they had a prolonged baseline PTT or
Leiden (only a 200 bp band is present).
Downloaded from https://academic.oup.com/ajcp/article/106/1/107/1757011 by guest on 09 December 2021
were on heparin or Coumadin therapy. Activated pro-
tein C resistance and factor V Leiden determinations
were made on all patients. Statistical Analysis
For the APC resistance test, patient samples of venous Sensitivity, specificity and ROC curves were calcu-
blood were drawn into tubes containing 3.8% sodium ci- lated using the LABROC1 program (Dr. Charles E.
trate and kept on ice. Following centrifugation at 3,000 Metz, Department of Radiology, University of Chicago),
rpm for 10 minutes at 4 °C twice, the platelet-poor which makes a maximum likelihood estimation of a bi-
plasma was aliquoted and stored at —20 °C until use. At nomial ROC curve from the continuous APC ratio data.
the time of testing, specimens were rapidly thawed at Patients were divided into true positive and true negative
37 °C. For factor V Leiden, patient blood was drawn into groups on the basis of the factor V Leiden results, which
tubes containing EDTA and refrigerated at 4 °C before constituted the gold standard in this analysis. Predictive
DNA extraction. value of a negative test was calculated as (true negatives)/
(true negatives + false negatives) X 100.
APC Resistance Assay
Activated protein C resistance was assessed using a RESULTS
commercially available kit (Coatest APC resistance,
Chromogenix AB, Sweden) according to the manufac- Patient Characteristics
turer's instructions using a fibrometer (Dataclot, Helena
Laboratories, Beaumont, TX). Normal and abnormal One hundred thirty consecutive patients were studied.
controls were performed before testing patients. All tests All were referred for evaluation of thrombosis except for
were performed in duplicate. This activated partial eight individuals who were asymptomatic family mem-
thromboplastin time (aPTT)-based test is reported as an bers of patients with factor V Leiden. The median age
APC ratio: clotting time with added APC/clotting time was 44 (range 5-87 years) 55% were female and 45%
without APC. Normal range was defined by the manu- were male. Of the 130 patients studied, 26 (20%) were
facturer as a PTT ratio > 2. heterozygous for factor V Leiden and 4 (3.1%) were ho-
mozygotes. Three patients (2.3%) had an APC ratio < 2
Factor V Leiden Assay and a normal factor V genotype. The mean APC ratio
for healthy subjects was 3.00 (standard deviation [SD]
Genomic DNA was isolated from patient leukocytes 0.78); that of heterozygotes was 2.13 (SD 0.53) and ho-
(QIAamp Blood Kit, Qiagen) collected in EDTA tubes. mozygotes 1.19 (SD 0.11). The median age of the factor
A 267 base pair (bp) fragment of the factor V gene flank- V Leiden-positive patients was 43. The median age of
ing the factor V Leiden mutation sit at position 1691 was factor V Leiden-negative patients was 45. Of the eight
amplified as described by Bertina and colleagues6 with asymptomatic family members tested, four were hetero-
the following modifications: 50 /xL of a mixture contain- zygous for factor V Leiden. The mean APC ratio of these
ing 10 mM Tris-Hcl, pH 8.3, 50 mM KC1, 1.5 raM individuals was 2.1 (range 1.8-2.4; mean of heterozy-
MgC12,0.001% gelatin, 1.25 U of Taq DNA polymerase, gotes 2.05, range 1.8-2.4; mean of healthy subjects 3.2,
150-500 ng genomic DNA and 12.5 pM of each primer range 2.2-3.4).
was heated to 80 °C, then 0.8 mM final concentration of
each nucleoside triphosphate was added. The mixture
Distribution of APC Ratios in Patients With and
was heated to 95 °C for 3 minutes and then subjected to
Without Factor V Leiden
30 cycles of 94 °C (1 minute), 55 °C (1 minute) and 72
°C (1 minute), followed by 72 °C for 7 minutes using a As shown in Figure 1, there was substantial overlap in
microprocessor controlled thermal cycler (Perkin Elmer APC ratios in patients with and without factor V Leiden.
A.J.C.P. -July 19%ZEHNDER AND BENSON 109
Utility ofAPC Resistance Assay
Downloaded from https://academic.oup.com/ajcp/article/106/1/107/1757011 by guest on 09 December 2021
All Patients
Excluding Factor V Leiden Homozygous
£
FlG. 1. Results of the activated protein C resistance assay in all patients
tested. Foreground: patients documented to have factor V Leiden
(G1491 A). Background: patients with normal factor V genotype.
0.2 A
False Positive Fraction (1-Specificity)
Fifty-four percent of patients (14 of 26) who were hetero-
zygous for factor V Leiden had APC ratios > 2. FIG. 2. Receiver operating characteristic (ROC) curves for the activated
protein C resistance assay in identifying patients with factor V Leiden.
Squares: all patients; diamonds: excluding factor V leiden homozy-
ROC Curves for the APC Ratio Test gotes. The area under the curve is 0.863 with estimated standard devia-
tion (SD) 0.043 for all patients and 0.838 with estimated SD 0.047 ex-
The ROC curves for all patients and patients not on cluding the four patients who were homozygous for factor V Leiden.
anticoagulant therapy is shown in Figure 2. The area un- The ROC curve for the APC resistance assay is characteristic of an in-
der the curve is 0.863 with estimated SD 0.043 for all termediate utility test (>0.9 being highly useful, 0.9 being highly useful,110 COAGULATION AND TRANSFUSION MEDICINE
Original Article
in our population was 20%, in close agreement with the test is 100%, assuming that both chromosome 1 factor V
incidence of this disorder in other unselected series of alleles are amplified in all patients. This test has addi-
thrombosis patients.'' Although only four patients were tional advantages over the APC resistance test of not be-
homozygotes for factor V Leiden, the APC resistance test ing affected by Coumadin therapy or prolonged baseline
appears to be very sensitive and specific for detecting PTTs in patients on heparin or those with lupus antico-
these individuals, with a mean ratio of 1.19. However, agulants. A disadvantage of the molecular test in its cur-
most patients with factor V Leiden are heterozygotes, rent form is that it is labor intensive. However, automa-
and in this group the APC ratios demonstrate a signifi- tion of PCR-based analysis systems or single base
cant overlap with the normal range, resulting in a low mismatch detection hybridization systems on ELISA
sensitivity (50%) at the recommended cut-off value of 2 plates promise to streamline this type of analysis in the
Downloaded from https://academic.oup.com/ajcp/article/106/1/107/1757011 by guest on 09 December 2021
and the test ROC curve in the intermediate utility future and make these tests better suited for screen-
range.16 Increasing the cut-off increases the sensitivity of ing. 1 ' 2 " Another disadvantage of molecular testing for
the APC resistance test, but has the undesirable effect of factor V Leiden would be the failure to identify the 10%
increasing the number of false-positive tests as indicated to 20% of patients with resistance to APC who have a
by the decrease in specificity depicted in Figure 3. Inspec- normal factor V genotype. The significance of APC resis-
tion of Figures 1 and 3 reveals that at a ratio of >3.0, the tance in the setting of a normal factor V genotype is un-
diagnosis of factor V Leiden could be excluded with a clear at present. To date, no mutations at the other APC
high degree of certainty (predictive value of a negative cleavage site of factor Va or in factor Villa have been
test result = 97% for all patients). described to account for APC resistance in these patients.
Thus, the standard APC resistance test is useful in It is possible that some of these patients will have resis-
identifying the small number of factor V Leiden homo- tance to APC on the basis of an as yet undefined mecha-
zygotes and in excluding the diagnosis of factor V Leiden nism. As the risk of thrombosis and the mechanism(s) of
in the 36% of healthy subjects with a ratio of >3.0. How- thrombosis if present are unknown at in these patients,
ever, the test does not clearly discriminate between factor they remain an intriguing and important population to
V Leiden heterozygotes and healthy subjects. This leaves identify both for clinical and research purposes.
the majority of patients (64% of healthy subjects and 54%
of those with factor V Leiden; 60% overall) in a gray area Acknowledgments. The authors thank Dr. P. Joanne Cornbleet for
her critical review of the manuscript.
between a ratio of 2.0-3.0, requiring a confirmatory test,
the molecular diagnosis of factor V Leiden. As such, the
APC resistance assay in its present form is not a useful REFERENCES
screening test. It is possible that variants of the PTT- 1. Esmon CT. The roles of protein C and thrombomodulin in the
based assay using normalized ratios may more accu- regulation of blood coagulation. J Biol Cham 1989:264:4743-
rately separate heterozygotes from healthy subjects.61314 4746.
2. Esmon CT. Molecular events which control the protein C antico-
Recently, a tissue factor-dependent factor V assay has agulant pathway. Thromb Haemost 1993;70:29-33.
been developed and reported to reliably separate factor 3. Heijboer H, Brandjes D, Buller HR, Sturk A, ten Cate JW. Defi-
V Leiden heterozygotes from healthy subjects;19 PTT- ciencies of coagulation-inhibiting and fibrinolytic proteins in
outpatients with deep vein thrombosis. N Engl J Med 1990; 323:
based assays diluting the patient plasma into factor V de- 1512-1516.
ficient plasma are reported to have less overlap between 4. Svennson PJ, Dahlback B. Resistance to activated protein C as a
basis for venous thrombosis. N EnglJ Med 1994;330:517-522.
healthy subjects and factor V leiden heterozygotes as well 5. Dahlback B, Carlsson M, Svensson PJ. Familial thrombophilia
as reliably test individuals on oral anticoagulants.2021 due to a previously unrecognized mechanism characterized by
Until such a test is available and performance validated, poor anticoagulant response to activated protein C: Prediction
of a cofactor to activated protein C. Proc Nail Acad Sci USA
given the overlap of APC ratios in factor V Leiden het- 1993;90:1004-1008.
erozygotes and healthy subjects using the first-generation 6. Bertina RM, Koeleman BPC, Roster T, et al. Mutation in blood
Chromogenix APC resistance assay, it is important to coagulation factor V associated with resistance to activated pro-
tein C. Nature 1994;369:64-67.
document factor V Leiden status of patients with APC 7. Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B.
ratios in the gray area of 2.0-3.0 with molecular diagnos- Activated protein C resistance caused by Arg506Gln mutation
tic testing. in factor Va. Lancet 1994;343:1362-1363.
8. Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke
An alternative strategy is to perform the factor V JP, Bertina RM. Venous thrombosis due to poor response to
Leiden molecular test initially in patients suspected of activated protein C: Leiden thrombophilia study. Lancet
inherited thrombophilia. In this study, 20% of patients 1993;342:1503-1506.
9. Voorberg J, Roelse J, Koopman R, et al. Association of idiopathic
were found to have the mutation. As the factor V Leiden thromboembolism with a single point mutation at Arg506 of
test is by definition the standard, the sensitivity of the factor V. Lancet 1994;343:1535-1536.
A.J.C.P.-July 1996ZEHNDER AND BENSON m
Utility of APC Resistance Assay
10. Zoller B, Dahlback B. Linkage between inherited resistance to ac- 16. Swets J A. Measuring the accuracy of diagnostic systems. Science
tivated protein C and factor V gene mutation in venous throm- 1988;240:1285-1293.
bosis. Lancet 1994;343:1536-1538. 17. Zweig MH. ROC analysis: Assessment of diagnostic accuracy in
11. Dahlback B. New molecular insights into the genetics of throm- the laboratory. Lab Med 1991;22:708-711.
bophilia: Resistance to activated protein C caused by Arg506 to 18. Zweig MH, Campbell G. Receiver-operating characteristic (ROC)
Gin mutation in factor V as a pathogenic risk factor for venous plots: A fundamental evaluation tool in clinical medicine. Clin
thrombosis. Thromb Haemost 1995;74:139-148. Chem 1993;39:561-577.
12. Takamiya O, Ishida F, Kodaira H, Kiyoshi K. APC resistance and 19. Le DT, Griffin JH, Greengard JS, Mujumdar V, Rapaport SI. Use
Mnl 1 genotype of coagulation factor V are rare in Japanese pop- of a generally applicable tissue factor-dependent factor V assay
ulation. Thromb Haemost 1995; 74:996. to detect activated protein C-resistant factor Va in patients re-
ceiving warfarin and in patients with a lupus anticoagulant.
13. Rodgers GM. Activated protein C resistance and inherited throm- Blood 1995;85:1704-1711.
bosis. Am J Clin Path 1994; 103:261-262. 20. Tosetto A, Rodeghiero F. Diagnosis of APC resistance in patients
14. de Ronde H, Bertina RM. Laboratory diagnosis of APC resistance: on oral anticoagulant therapy. Thromb Haemost 1995; 73:732-
Downloaded from https://academic.oup.com/ajcp/article/106/1/107/1757011 by guest on 09 December 2021
A critical evaluation of the test and the development of diagnos- 733.
tic criteria. Thromb Haemost 1994;72:880-886. 21. Andersson N-E, Andersson M, Andras M, Rosen S. High discrim-
15. Rosen S, Johansson K, Lindberg K, Dahlback B. Multicenter eval- ination for FVQ506 also in OAC patients, with Coatest APC
uation of a kit for activated protein C resistance using plasmas resistance after predilution in FV-deficient plasma. Blood
from healthy individuals. Thromb Haemost 1994;72:255-260. 1995; 86:912a (Abstr).
Vol. 106-No. 1You can also read
