Outcomes of genetic testing in adults with a history of venous thromboembolism.

Abstract

To address whether Factor V Leiden (FVL) testing alone, or in combination with prothrombin G20210A testing, leads to improved clinical outcomes in adults with a personal history of venous thromboembolism (VTE) or to improved clinical outcomes in adult family members of mutation-positive individuals.
Searches of MEDLINE, EMBASE, The Cochrane Library, the Cumulative Index to Nursing and Allied Health Literature, and PsycInfo through December 2008.
We focused on the analytic validity, clinical validity, and clinical utility of these tests. Each included article underwent double review for data abstraction and assessment of study quality. We pooled the results of studies addressing the clinical validity of these tests when there were sufficient data. Other evidence was summarized in evidence tables. We graded the evidence by adapting a scheme recommended by the Grading of Recommendations Assessment, Development and Evaluation (GRADE) Working Group by assessing the limitations affecting individual study quality, the certainty regarding the directness of the observed effects in the studies, the precision and strength of the findings, and the availability (or lack) of data to answer the relevant key question. Evidence for each sub-question was graded as high, moderate, or low.
We reviewed 7,777 titles and included 124 articles. No direct evidence addressed the primary objective. However, high-grade evidence supported the conclusion that tests for the detection of FVL and prothrombin G20210A have excellent analytic validity. Most clinical laboratories test for these mutations accurately. Heterozygosity [odds ratio (OR) =1.56 (95 percent confidence interval (CI) 1.14 to 2.12)] and homozygosity [OR=2.65 (95 percent C.I. 1.2 to 6.0)] for FVL in probands are predictive of recurrent VTE. Heterozygosity for FVL predicts VTE in family members [OR=3.5 (95 percent C.I. 2.5 to 5.0)] as does homozygosity for FVL [OR=18 (95 percent C.I. 7.8 to 40)]. Heterozygosity for prothrombin G20210A is not predictive of recurrence in probands [OR=1.45 (95 percent C.I. 0.96-2.2)]. Evidence is insufficient about heterozygosity for prothrombin G20210A in family members and insufficient about homozygosity for prothrombin G20210A. A single study supported the hypothesis that clinicians might change management based on test results. There was high-grade evidence that anticoagulation reduces recurrent events in probands with FVL or prothrombin G20210A, however, there was low-grade evidence that the relative reduction with treatment is comparable to that seen in individuals without mutations. There was moderate evidence to support the conclusion that neither harms nor benefits of testing have been demonstrated conclusively. Decision-analysis models suggest that testing may be cost-effective in select individuals.
There is no direct evidence that testing for these mutations leads to improved clinical outcomes in adults with a history of VTE or their adult family members. The literature supports the conclusion that while these assays have high analytic validity, the test results have variable clinical validity for predicting VTE in these populations and have only weak clinical utility.

Full text

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Evidence Report/Technology Assessment
Number 180


Outcomes of Genetic Testing in Adults with a History
of Venous Thromboembolism


Prepared for:
Agency for Healthcare Research and Quality
U.S. Department of Health and Human Services
http://www.ahrq.gov

Contract No. HHSA 290-2007-10061-I


Prepared by:
The Johns Hopkins University Evidence-based Practice Center

Investigators
Jodi B. Segal, M.D., M.P.H.
Daniel J. Brotman, M.D.
Ashkan Emadi, M.D., Ph.D.
Alejandro J. Necochea, M.D., M.P.H.
Lipika Samal, M.D.
Lisa M. Wilson, M.S.
Matthew T. Crim, M.Sc., M.A.
Eric B. Bass, M.D., M.P.H.











AHRQ Publication No. 09-E011
June 2009

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This report is based on research conducted by the Johns Hopkins University Evidence-based
Practice Center (EPC) under contract to the Agency for Healthcare Research and Quality
(AHRQ), Rockville, MD (Contract No. HHSA 290-2007-10061-I). The findings and conclusions
in this document are those of the author(s), who are responsible for its content, and do not
necessarily represent the views of AHRQ. No statement in this report should be construed as an
official position of AHRQ or of the U.S. Department of Health and Human Services.

The information in this report is intended to help clinicians, employers, policymakers, and others
make informed decisions about the provision of health care services. This report is intended as a
reference and not as a substitute for clinical judgment.

This report may be used, in whole or in part, as the basis for the development of clinical practice
guidelines and other quality enhancement tools, or as a basis for reimbursement and coverage
policies. AHRQ or U.S. Department of Health and Human Services endorsement of such
derivative products may not be stated or implied.

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This document is in the public domain and may be used and reprinted without permission except
those copyrighted materials noted for which further reproduction is prohibited without the
specific permission of copyright holders.


Suggested Citation:
Segal JB, Brotman DJ, Emadi A, Necochea AJ, Samal L, Wilson LM, Crim MT, Bass EB.
Outcomes of Genetic Testing in Adults with a History of Venous Thromboembolism. Evidence
Report/Technology Assessment No. 180. (Prepared by Johns Hopkins University Evidence-
based Practice Center under contract no. HHSA 290-2007-10061-I). AHRQ Publication No. 09-
E011. Rockville, MD. Agency for Healthcare Research and Quality.

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Preface

The Agency for Healthcare Research and Quality (AHRQ), through its Evidence-Based
Practice Centers (EPCs), sponsors the development of evidence reports and technology
assessments to assist public- and private-sector organizations in their efforts to improve the
quality of health care in the United States. This report was requested and funded by the Centers
for Disease Control and Prevention (CDC), Office of Public Health Genomics (OPHG). The
reports and assessments provide organizations with comprehensive, science-based information
on common, costly medical conditions and new health care technologies. The EPCs
systematically review the relevant scientific literature on topics assigned to them by AHRQ and
conduct additional analyses when appropriate prior to developing their reports and assessments.
To bring the broadest range of experts into the development of evidence reports and health
technology assessments, AHRQ encourages the EPCs to form partnerships and enter into
collaborations with other medical and research organizations. The EPCs work with these partner
organizations to ensure that the evidence reports and technology assessments they produce will
become building blocks for health care quality improvement projects throughout the Nation. The
reports undergo peer review prior to their release.
AHRQ expects that the EPC evidence reports and technology assessments will inform
individual health plans, providers, and purchasers as well as the health care system as a whole by
providing important information to help improve health care quality.
We welcome comments on this evidence report. They may be sent by mail to the Task Order
Officer named below at: Agency for Healthcare Research and Quality, 540 Gaither Road,
Rockville, MD 20850, or by e-mail to epc@ahrq.gov.

Carolyn M. Clancy, M.D.
Director
Agency for Healthcare Research and Quality

Beth A. Collins Sharp, Ph.D., R.N.
Acting Director, EPC Program
Agency for Healthcare Research and Quality

Thomas R. Frieden, M.D., M.P.H.
Director
Centers for Disease Control and Prevention









Jean Slutsky, P.A., M.S.P.H.
Director, Center for Outcomes and Evidence
Agency for Healthcare Research and Quality

Gurvaneet Randhawa, M.D., M.P.H.
Task Order Officer, Center for Outcomes and
Evidence
Agency for Healthcare Research and Quality

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Acknowledgments

The EPC thanks Renee F. Wilson, Dr. Olaide Odelola, and Ritu Sharma for their assistance
with the final assembly and formatting of this report, and Dr. Gurvaneet Randhawa for his
valuable insight.

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Structured Abstract

Objective: To address whether Factor V Leiden (FVL) testing alone, or in combination with
prothrombin G20210A testing, leads to improved clinical outcomes in adults with a personal
history of venous thromboembolism (VTE) or to improved clinical outcomes in adult family
members of mutation-positive individuals.

Data sources: Searches of MEDLINE®, EMBASE®, The Cochrane Library, the Cumulative
Index to Nursing & Allied Health Literature, and PsycInfo© through December 2008.

Review methods: We focused on the analytic validity, clinical validity, and clinical utility of
these tests. Each included article underwent double review for data abstraction and assessment of
study quality. We pooled the results of studies addressing the clinical validity of these tests when
there were sufficient data. Other evidence was summarized in evidence tables. We graded the
evidence by adapting a scheme recommended by the Grading of Recommendations Assessment,
Development and Evaluation (GRADE) Working Group by assessing the limitations affecting
individual study quality, the certainty regarding the directness of the observed effects in the
studies, the precision and strength of the findings, and the availability (or lack) of data to answer
the relevant key question. Evidence for each sub-question was graded as high, moderate, or low.

Results: We reviewed 7,777 titles and included 124 articles. No direct evidence addressed the
primary objective. However, high-grade evidence supported the conclusion that tests for the
detection of FVL and prothrombin G20210A have excellent analytic validity. Most clinical
laboratories test for these mutations accurately. Heterozygosity [odds ratio (OR) =1.56 (95
percent confidence interval (CI) 1.14 to 2.12)] and homozygosity [OR=2.65 (95 percent C.I. 1.2
to 6.0)] for FVL in probands are predictive of recurrent VTE. Heterozygosity for FVL predicts
VTE in family members [OR=3.5 (95 percent C.I. 2.5 to 5.0)] as does homozygosity for FVL
[OR=18 (95 percent C.I. 7.8 to 40)]. Heterozygosity for prothrombin G20210A is not predictive
of recurrence in probands [OR=1.45 (95 percent C.I. 0.96-2.2)]. Evidence is insufficient about
heterozygosity for prothrombin G20210A in family members and insufficient about
homozygosity for prothrombin G20210A. A single study supported the hypothesis that clinicians
might change management based on test results. There was high-grade evidence that
anticoagulation reduces recurrent events in probands with FVL or prothrombin G20210A;
however, there was low-grade evidence that the relative reduction with treatment is comparable
to that seen in individuals without mutations. There was moderate evidence to support the
conclusion that neither harms nor benefits of testing have been demonstrated conclusively.
Decision-analysis models suggest that testing may be cost-effective in select individuals.

Conclusions: There is no direct evidence that testing for these mutations leads to improved
clinical outcomes in adults with a history of VTE or their adult family members. The literature
supports the conclusion that while these assays have high analytic validity, the test results have
variable clinical validity for predicting VTE in these populations and have only weak clinical
utility.

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Contents

Executive Summary.........................................................................................................................1

Evidence Report...........................................................................................................................15

Chapter 1. Introduction .................................................................................................................17
Venous Thromboembolism........................................................................................................3
Diagnosis and Treatment...........................................................................................................4
Genetic Mutations Associated with VTE: FVL and Prothrombin G20210A............................4
Methods for Identifying Mutations............................................................................................5
Objectives of this Evidence Report............................................................................................5

Chapter 2. Methods..........................................................................................................................7
Establishing a Technical Expert Panel.......................................................................................7
Analytic Framework ..................................................................................................................7
Key Questions............................................................................................................................8
Search Strategy..........................................................................................................................9
Study Selection........................................................................................................................10
Data Abstraction......................................................................................................................11
Study Quality Assessment.......................................................................................................12
Applicability ............................................................................................................................13
Data Synthesis..........................................................................................................................13
Data Entry and Quality Control...............................................................................................14
Rating the Body of Evidence...................................................................................................14

Chapter 3. Results..........................................................................................................................15
Results of the Search................................................................................................................15
Description of types of studies retrieved ...........................................................................15
Key Questions 1. Association of FVL testing alone, or in combination with prothrombin
G20210A testing, with improved clinical outcomes in adults with a personal history of
VTE, or in adult family members of mutation-positive individuals..................................15
Key Question 2: Analytic validity of tests to identify FVL and prothrombin G20210A
mutations............................................................................................................................17
Detection of FVL...............................................................................................................24
Detection of prothrombin G20210A..................................................................................25
Simultaneous detection of FVL and prothrombin G20210A mutations............................25
External quality assurance ................................................................................................25
Summary of evidence for Key Question 2.........................................................................31
Key Question 3. Clinical validity of testing for FVL and/or prothrombin G202010A ...........32
FVL and recurrent venous thrombosis in probands...........................................................32
Summary of evidence for Key Question 3 (probands) ......................................................39
FVL and prothrombin G20210A and venous thrombosis in family members..................40
Association of FVL and prothrombin G20210A with venous thrombosis during
pregnancy and the post-partum period.........................................................................46
Age and VTE among family members with mutations .....................................................47

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Other high-risk periods......................................................................................................48
Summary of evidence for Key Question 3 (family members)...........................................48
Key Question 4. Clinical utility of testing for FVL and/or prothrombin G202010A..............49
Effect of testing on clinicians’ management (KQ 4a) .......................................................49
Effect of testing and resultant management on VTE-related outcomes (KQ 4b)..............50
Effect of testing and results on other outcomes (KQ 4c)...................................................53
Cost-effectiveness of FVL and prothrombin G20210A testing in the case of probands
and their relatives (KQ4d) ...........................................................................................57
Summary of evidence for Key Question 4.........................................................................63

Chapter 4. Discussion....................................................................................................................65
Key Question 1. Association of FVL testing alone, or in combination with prothrombin
G20210A testing lead to improved clinical outcomes in adults with a personal history of
VTE, or in adult family members of mutation-positive individuals..................................65
Key Question 2. Analytic validity of tests to identify FVL and prothrombin G20210A
mutations ...........................................................................................................................65
Key Question 3. Clinical validity of testing for FVL and prothrombin G20210A
mutations............................................................................................................................66
Testing probands................................................................................................................66
Testing family members ....................................................................................................67
Key Question 4. Clinical utility of testing for FVL and prothrombin G20210A mutations....68
Effect of testing on clinicians’ management......................................................................68
Effect of testing and resultant management on VTE-related outcomes ............................68
Effect of testing and results on other outcomes.................................................................69
Cost effectiveness of FVL and prothrombin G20210A testing in the care of
probands and their relatives.........................................................................................69
Summary of the Evidence........................................................................................................71
Limitations of this Report........................................................................................................73
Implications for Future Research.............................................................................................74
Analytic validity.................................................................................................................75
Clinical validity..................................................................................................................75
Clinical utility....................................................................................................................76

References......................................................................................................................................77

Acronyms.......................................................................................................................................99

Figures
Figure 1. FVL and prothrombin testing for prevention of venous thromboembolic
events: analytic framework..................................................................................................8
Figure 2. Summary of literature search (number of articles)...................................................16
Figure 3. Odds ratios for VTE for probands with heterozygous FVL mutation, as
compared to probands without the mutation......................................................................34
Figure 4. Odds ratios for VTE for probands with homozygous FVL mutation, as
compared to probands without the mutation......................................................................35
Figure 5. Odds ratios for VTE for probands with mixed FVL mutation, as

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Figure 6. Odds ratios for VTE for probands who were double heterozygous for the
FVL and prothrombin G20210A mutations, as compared to probands without
the mutation .......................................................................................................................37
Figure 7. Odds ratios for VTE for probands who were heterozygous for prothrombin
G20210A, as compared with probands without the mutation............................................38
Figure 8. Odds ratios for VTE for probands with unspecified prothrombin G20210A
Mutation, as compared with probands without the mutation.............................................39
Figure 9. Odds ratios for VTE for family members with heterozygous FVL mutation, as
compared to family members without the mutation..........................................................42
Figure 10. Odds ratios for VTE for family members with homozygous FVL , as compared
to family members without the mutation...........................................................................43
Figure 11. Odds ratios for VTE for family members with unspecified FVL , as compared
to family members without the mutation...........................................................................44
Figure 12. Odds ratios for VTE for family members with double heterozygous mutations,
as compared to family members without the mutation......................................................45
Figure 13. Odds ratios for VTE for family members with unspecified prothrombin
G20210A mutation, as compared to family members without the mutation.....................46
compared to probands without the mutation......................................................................36

Summary Tables
Table 1. List of additional exclusion criteria by Key Question...............................................11
Table 2. Analytic validity of testing for the FVL mutation.....................................................18
Table 3. Details of studies on analytic validity of testing for FVL mutation ..........................22
Table 4. Analytic validity of testing for prothrombin G20210A.............................................26
Table 5. Details of studies on analytic validity of testing for prothrombin G20210A ............28
Table 6. Analytic validity of simultaneous detection (multiplex) of FVL and prothrombin
G20210A mutations ..........................................................................................................29
Table 7. Details of studies on analytic validity of simultaneous detection (multiplex) of
FVL and prothrombin G20210A mutations ......................................................................30
Table 8. Pooled results describing risk of recurrent VTE for probands with VTE .................33
Table 9. Pooled results describing the risk of VTE for family members of probands with
VTE comparing those with a mutation to those without a mutation.................................40
Table 10. Four studies examining the effect of a VTE-related outcomes of changes in
therapy based on testing for FVL or prothrombin G20210A.............................................51
Table 11. Studies examining the effect of FVL and prothrombin G20210A
testing on non-VTE-related outcomes.................................................................................54
Table 12. Summary of studies evaluating the cost-effectiveness of testing for FVL and/or
prothrombin G20210A mutations .......................................................................................58
Table 13. Strength of the evidence regarding Key Questions 1 and 2: overarching question
and analytic validity ............................................................................................................71
Table 14. Strength of the evidence regarding Key Question 3: clinical validity.....................72
Table 15: Strength of the evidence regarding Key Question 4: clinical utility .......................73

Appendixes
Appendix A: Technical Experts and Peer Reviewers
Appendix B: Hand-searched Journals

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Appendix C: Detailed Search Strategies
Appendix D: Excluded Articles
Appendix E: Data Review Forms
Appendix F: Evidence Tables



Appendixes and Evidence Tables for this report are provided electronically at
http://www.ahrq.gov/downloads/pub/evidence/pdf/gentestvt/fvl.pdf.

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Executive Summary

Introduction

Venous thromboembolism (VTE) refers to pathologic thrombosis in the venous circulation.
Although the most frequent venous thromboembolic event is deep venous thrombosis in the
veins of the legs, thromboses can also occur in the veins of the upper extremities, pelvis,
abdomen, and cerebral venous sinuses. Pulmonary embolism is the main life-threatening
complication of deep vein thrombosis, in which a portion of the venous thrombus is carried to
the pulmonary arteries by blood flow, potentially obstructing the pulmonary vasculature.
Treatment begins with short-term use of a parenteral anticoagulant (and sometimes thrombolytic
therapy) and then usually continues with a vitamin K antagonist, most commonly warfarin. The
duration of therapy depends on whether the patient is considered to have continuing risk factors
for recurrence.
Much effort has been devoted to quantifying the risk of recurrent thrombosis. If the patient
has a persistent risk factor for thrombosis, anticoagulant therapy is often continued, sometimes
for the life of the patient. The clinician and patient also try to reduce exposure to any modifiable
risk factors and may use mechanical or pharmacological means of preventing thrombosis at high-
risk times, such as during hospitalization or pregnancy.
The question of whether a patient has ongoing risk leads to the subject of this report, the
value of testing individuals who have had a venous thromboembolic event for Factor V Leiden
(FVL) and prothrombin G20210A. The FVL mutation is a base change (from G to A at position
1691) in the gene coding for the Factor V protein; the resulting amino acid substitution
eliminates one of three activated protein C cleavage sites in Factor V. As a result, Factor V is
inactivated to a lesser extent and persists longer in the circulation, leading to more thrombin
generation. In the United States, a single FVL allele is present in about 5, 2.2, and 1.2 percent of
the Caucasian, Hispanic, and African American populations, respectively. The prothrombin
(Factor II) mutation is the second most common inherited risk factor for thrombosis. The
mutation in the prothrombin G20210A gene is associated with an elevation of prothrombin levels
to about 30 percent above normal in heterozygotes and to 70 percent above normal in
homozygotes. In the United States, the prevalence of this allele is 1.1 percent in Caucasians and
Hispanics and 0.3 percent in African Americans.
The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) initiative was
established by the Office of Public Health Genomics at the Centers for Disease Control and
Prevention (CDC) to address the increasingly urgent need for timely and objective information
that would allow health care providers and payers, policymakers, and consumers to identify
genetic tests that are safe and useful and to provide guidance on their appropriate use in practice,
based on available evidence. At their request, we reviewed the evidence regarding the value of
testing for these mutations in two specific populations: (1) individuals who have had a venous
thromboembolic event (probands), and (2) their family members.

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Methods

The overarching question we were asked to address (Key Question [KQ] 1) was: Does FVL
testing, alone or in combination with prothrombin G20210A testing, lead to improved clinical
outcomes (e.g., avoidance of a recurrent VTE) in adults with a personal history of VTE or to
improved clinical outcomes (e.g., avoidance of an initial VTE) in adult family members of
mutation-positive individuals? Are testing results useful in medical, personal, or public health
decision making? To address this question, we reviewed the literature regarding these tests’
analytic validity, clinical validity, and clinical utility when used in probands with VTE and in
their family members.

The other KQs were as follows:

KQ2 What is the evidence regarding the analytic validity of existing diagnostic tests for the
FVL mutation and the prothrombin G20210A mutation, specifically their analytic
sensitivity and specificity, reproducibility, and robustness (sources of variability)?

KQ3a What is the evidence that the presence of FVL alone, prothrombin G20210A alone, or the
two in combination predicts the risk of recurrent VTE in individuals (probands) who have
had VTE and predicts the risk of VTE in the probands’ family members who have been
tested? Does the testing add predictive information beyond clinical data?

KQ3b What is the evidence that demographic or clinical factors modify the relationship between
the presence of FVL or prothrombin G20210A and the risk of VTE?

KQ4a What is the evidence that clinicians manage patients differently based on the results of
testing for FVL or prothrombin G20210A? How do clinicians manage anticoagulation of
individuals who have had testing, as compared to those who have not had testing? What
other diagnostic tests do clinicians order or not order, based on testing results? What
recommendations do clinicians make regarding other therapies and exposures, based on
testing results?

KQ4b What is the evidence that testing, and the resultant management, reduces VTE related-
outcomes or has other benefits in individuals who have had VTE or in the probands’
family members who have been tested?

KQ4c What is the evidence of harms to individuals with VTE or to the probands’ family
members who are tested for FVL or prothrombin G20210A as a result of testing or as a
result of changed management based on the test results?

KQ4d What is the evidence that testing for FVL alone, prothrombin G20210A alone, or the two
tests in combination is a cost-effective strategy when caring for a patient with VTE or a
family member of a proband?

Our comprehensive search included electronic and hand searching. We searched five
databases, MEDLINE® (1950 through May 2008), EMBASE® (1974 through December 2008),

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The Cochrane Library (Issue 2, 2008), the Cumulative Index to Nursing & Allied Health
Literature (CINAHL®; 1982 through December 2008) and PsycInfo©, to identify primary
literature on the analytic validity, clinical validity, and clinical utility of testing for FVL and
prothrombin G20210A.
Two independent reviewers, from among six study team members, conducted title scans in
parallel. The title review was designed to capture as many studies as possible that reported on the
analytic validity, clinical validity, and clinical utility of testing for FVL and prothrombin
G20210A. All titles potentially addressing these issues were promoted to the abstract review
phase.
Abstracts were reviewed independently by two investigators. Abstracts were excluded if the
investigators agreed that the article: (1) was not relevant to any of the key questions; (2) did not
include any human data; (3) contained no original data; (4) was not conducted in adults; and (5)
was not published in English. Differences of opinion were resolved through consensus
adjudication.
Full articles selected for review underwent another independent parallel review by two
investigators. In addition to the exclusion criteria used for the abstract review, there were
additional exclusion criteria for each KQ. For the question about clinical validity of the
mutations (KQ 3), we included only prospective studies of probands, although we permitted
retrospective studies of family members because we anticipated few prospective studies. Each
article underwent double review by study investigators for full data abstraction and assessment
of study quality. We used a sequential review process in which the primary reviewer completed
all data abstraction forms, and the second reviewer checked the first reviewer’s data abstraction
forms for completeness and accuracy. Reviewer pairs were formed to include personnel with
both clinical and methodological expertise. All information from the article review process was
entered directly into the SRS 4.0 database.
The primary outcome extracted from the studies of analytic validity was concordance
between the test and the reference test, as that was most often reported. When there were
sufficient data (three or more studies) and the studies were qualitatively homogeneous with
respect to key variables (population characteristics, study duration, mutation status, and length of
follow-up), we conducted meta-analyses for the studies addressing the clinical validity of the
tests. When it was inappropriate to combine studies quantitatively, we qualitatively summarized
the results. For pooling, we used the number of events and count of the patients under
observation in each group. We calculated a pooled estimate of the odds ratio for VTE in
probands and separately in family members. We used a random effects model with the
DerSimonian and Laird method for calculating between-study variance.
At the completion of our review, we graded the quantity, quality, and consistency of the best
available evidence addressing the KQs by adapting an evidence-grading scheme recommended
by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE)
Working Group. To assess the quantity of evidence, we focused on the number of studies with
the strongest design. We also assessed the quality and consistency of the best available evidence,
including assessment of the limitations affecting individual study quality (using the individual
study quality assessments), certainty regarding the directness of the observed effects in the
studies, the precision and strength of the findings, and the availability (or lack) of data to answer
the KQ. We classified evidence bodies as: (1) “high” grade, indicating confidence that further
research is very unlikely to change our confidence in the estimated effect in the abstracted
literature; (2) “moderate” grade, indicating that further research is likely to have an important

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impact on our confidence in the estimates of effects and may change the estimates in the
abstracted literature; (3) “low” grade, indicating the further research is very likely to have an
important impact on confidence in the estimates of effects and is likely to change the estimates in
the abstracted literature.


Results

We reviewed 7,777 titles and included 124 articles in the review of one or more of the KQs.

Key Question 1

We identified no evidence to directly address KQ1, which required studies that directly tested
the impact of testing on patient outcomes. The articles addressing the questions below,
particularly KQ4, provided indirect evidence to answer KQ1.

Key Question 2

The conventional “gold standard” method for FVL and prothrombin G20210A detection is
the sequencing of the specific genetic region of the gene of interest. However, a number of other
reference methods are used instead because of the complexity and high costs associated with
sequencing. Many studies that we reviewed used polymerase chain reaction and restriction
fragment length polymorphism (PCR-RFLP) or allele-specific polymerase chain reaction (AS-
PCR) assays as their reference standards, both of which are considered by the Food and Drug
Administration to be acceptable reference standards.

Detection of FVL. Forty-one studies compared at least two methods for FVL detection. The
majority of studies demonstrated 100 percent concordance between the experimental method and
the reference method. The least concordance was seen in a study using electrochemical
genosensors, which had a concordance of only 93 percent.

Detection of prothrombin G20210A. The concordance rates between the experimental methods
and the reference standards for the detection of prothrombin G20210A were 100 percent in
nearly all of the 23 studies.

Simultaneous detection of FVL and prothrombin G20210A. All 12 studies that employed
multiplex technologies for the simultaneous detection of the two mutations reported 100 percent
concordance between this approach and the matched reference standard.

Quality assurance. We identified three studies that addressed external quality assurance or
laboratory performance relative to the gold standards. One described the results from the United
Kingdom National External Quality Assessment Scheme (UK NEQAS) Thrombophilia
Screening Program. Two hundred eighty centers participated in the thrombophilia screening
exercises. For the centers performing genetic analysis for FVL and the prothrombin mutation, an

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error rate of 3 to 6 percent was identified, with both transcription-related and analytical errors
observed.
Another study described results from the Royal College of Pathologists of Australasia
external quality assurance (QA) program. This program sent 133 DNA samples with known
mutations to laboratories in 10 separate surveys. Of 3,799 responses, the overall successful
identification rate was 98.6 percent. Success rates in identifying specific mutations were 98.1
percent for FVL and 98.8 percent for prothrombin G20210A.
Finally, a survey was organized by the Subcommittee on Hemostasis of the Italian
Committee for Standardization of Laboratory Tests (CISMEL). The authors concluded that
regular quality control programs are warranted to identify the causes of failures to correctly
identify specimens.

Key Question 3

Probands. Twenty-two articles examined the rates of recurrent venous thromboembolism in
individuals with a history of VTE (probands) and the FVL mutation.
We pooled 13 studies comparing probands with heterozygous FVL to probands without this
mutation, yielding an odds ratio for recurrence of 1.56 (95 percent confidence interval [CI], 1.14
to 2.12). The annualized event rates among heterozygous individuals were 3.1 percent to 7.5
percent in three studies. Seven studies described event rates in probands homozygous for FVL
and those without mutations. The number of homozygous individuals ranged from 1 to 11 across
studies. The odds ratio of recurrence was 2.65 (95 percent CI, 1.2 to 6.0).
Eight studies did not specify if the participants were heterozygous or homozygous for FVL.
The pooled odds ratio (from 3 studies) associated with this unspecified status was 1.56 (95
percent CI, 0.75-3.2), very similar to the odds ratio for known heterozygous individuals.
We identified 18 articles that examined rates of recurrent VTE in probands with the
prothrombin G20210A mutation. Nine of these compared individuals heterozygous for the
mutation to individuals without mutation. The pooled odds ratio was 1.45 (95 percent CI, 0.96-
2.2). Two studies described individuals homozygous for this mutation, but there were only 3
individuals with homozygosity. None of the three had a recurrent thrombosis.
Seven studies did not specify whether the individuals had homozygous or heterozygous
prothrombin G20210A. The pooled odds ratio in the four studies with available data was 0.73
(95 percent CI, 0.37-1.4).
Doubly heterozygous individuals (with FVL and the prothrombin G20210A mutations) had
an odds ratio of 4.8, with a wide confidence interval (95 percent CI, 0.5 to 46.)
When we separately evaluated patients with idiopathic VTE as the index event, we found that
the odds ratio associated with heterozygous FVL was close to one (1.17 [95 percent CI, 0.63-
2.18]).

Family members. We identified 17 articles that evaluated the occurrence of venous thrombosis
in family members of probands. The majority of these were retrospective studies, although three
included a prospective component. Nine studies described results for family members who were
heterozygous for FVL. Six studies contributed to the pooled odds ratio, which was 3.5 (95
percent CI, 2.5 to 5.0). Homozygosity for FVL among family members had a pooled odds ratio
of 18 (95 percent CI, 7.8 to 40) in the five studies pooled. The six studies that did not specify