AJCP  / Original Article
Diagnostic Testing Approaches for Activated Protein C
Resistance and Factor V Leiden
A Comparison of Institutional and National Provider
Practices
Juliana Perez Botero, MD,1 Julie A. Majerus,2 Ann K. Strege,2 Ryan D. Johnson, MBA, MS,3,4
Dong Chen, MD, PhD,2 and Rajiv K. Pruthi, MBBS1,2
From the 1Division of Hematology, Department of Medicine, 2Special Coagulation and Special Coagulation DNA Diagnostic Laboratories,
Department of Laboratory Medicine and Pathology, and 3The Robert D. and Patricia E. Kern Center for the Science of Health Care Delivery,
Mayo Clinic, Rochester, MN; and 4OptumLabs, Cambridge, MA.
Key Words: Factor V Leiden; Activated protein C resistance; Cost-effectiveness; Cost-benefit analysis; Thrombophilia
Am J Clin Pathol June 2017;147:604-610
DOI: 10.1093/AJCP/AQX033
ABSTRACT
Objectives:  To analyze the economic impact of testing for
activated protein C resistance (APC-R) due to factor V
Leiden (FVL) mutation with APC-R with reflexive FVL
genotyping (algorithmic approach) or genotyping alone.
Methods:  OptumLabs Data Warehouse (OLDW) data
were used to assess testing approaches. Insurance claims
for APC-R and FVL in 2013 were compared with the
Mayo Clinic database. Centers for Medicare & Medicaid
Services diagnostic fee schedules were used to assign costs.
Results:  Of 19.3 million OLDW-covered individuals,
74,242 (0.385%) received 75,608 tests: APC-R, 2,265
(2.9%); FVL genotyping, 70,619 (90.1%); and both
APC-R and FVL, 2,724 (7.0%). In total, 1,317 tests
were performed at Mayo Clinic: APC-R with reflex FVL
(1,256; 95.4%) and FVL alone (61; 4.6%). Costs per
evaluated individual and per total population (person/
year) in OLDW and algorithmic approach were $83.77 vs
$36.38 and $0.32 vs $0.14, respectively.
Conclusions:  The cost-optimized algorithmic approach
reduces health care costs.
604 Am J Clin Pathol 2017;147:604-610
DOI: 10.1093/ajcp/aqx033
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Venous thromboembolism (VTE) is a multifactorial
disease with a global incidence of 0.75 to 2.69 cases per
1,000 people per year.1 Acquired risk factors for VTE
are common,2 and activated protein C resistance (APC-
R) due to factor V Leiden (FVL) mutation is currently
the most commonly recognized hereditary risk factor
among the white population.3 Activated factors V (Va)
and VIII (VIIIa) are inactivated by activated protein C
(APC), thus limiting thrombin generation and develop-
ment of thrombosis.4,5 The c.1691G>A; pArg534Gln
mutation of the factor V gene (F5), commonly known as
FVL, affects the cleavage site for APC and confers resis-
tance to inactivation by APC, termed APC-R. Given
the incidence of VTE in the general population and the
prevalence of APC-R in the white population, labora-
tory testing to identify APC-R and/or FVL is frequently
ordered.
Laboratory evaluation for APC-R can be approached
in two ways: the first consists of initial plasma-based
screening to detect the presence or absence of APC-R;
reflexive DNA-based FVL genotyping is reserved for
those with abnormal APC-R.6,7 The second option con-
sists of direct genotyping, without assessing the presence
or absence of APC-R; this latter approach is likely the
most commonly pursued strategy. Studies demonstrating
relative costs of each approach have been published,8-10
but population-based analysis of financial implications
© American Society for Clinical Pathology, 2017. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com

of current routine clinical practice is not available. Herein
we analyzed the testing patterns from data obtained from
the OptumLabs Data Warehouse (OLDW), compared
ordering patterns with an algorithmic approach imple-
mented at our institution, and determined the potential
cost savings with the latter approach.
Materials and Methods
Study Setting, Design, and Patient Population
OLDW
We conducted a retrospective analysis of administra-
tive claims data from OLDW, which includes more than
100 million privately insured and a number of Medicare
Advantage enrollees throughout the United States.11 The
database contains longitudinal data from geographically
diverse regions across the United States, with greatest
representation from the South and Midwest. The data-
base contains professional, facility, and outpatient med-
ication service claims for covered individuals enrolled
in the health plans.12 The data include enrollee infor-
mation (insurance plan, sex, age race/ethnicity, dates of
eligibility) and medical claims (including International
Classification of Diseases, 9th Revision, Clinical
Modification [ICD-9-CM] diagnosis codes; ICD-9 pro-
cedure codes; Current Procedural Terminology, ver-
sion 4 [CPT-4] procedure codes; Healthcare Common
Procedure Coding system procedure codes; sites of ser-
vice codes; and provider specialty codes). Because this
portion of the study involved analysis of preexisting,
de-identified data, the Mayo Clinic Institutional Review
Board deemed it exempt from institutional review board
approval.
We identified all claims for FVL and an APC-R
clot-based assay performed between January 2013 and
December 2013 (calendar year 2013). If a test was per-
formed multiple times in one day, we only counted it
as one per day. To determine if the two tests were per-
formed alone or in combination, we looked out 14 days
from the initial test. We restricted the cohort to patients
with continuous medical enrollment for the entire 2013
calendar year. Descriptive summary of testing patterns
is reported.
Special Coagulation Laboratory, Mayo Clinic
(Rochester, MN)
After institutional review board approval, we searched
the Mayo Clinic Laboratory Information systems for all
results of the APC-R and FVL mutation tests performed
© American Society for Clinical Pathology
AJCP  / Original Article
for patients seen in the Mayo Clinic (Rochester, MN) over
the same time period (calendar year 2013). These tests
were ordered on patients seen in the primary care clinics
and multiple different specialties within the Mayo Clinic
campus. The data were cross-referenced with a Special
Coagulation Laboratory database that maintains infor-
mation on all patients seen at the Mayo Clinic on whom
special coagulation testing is performed.
Assay Method
The OLDW includes claims identified by CPT-4
codes for laboratory tests performed but does not include
data on performing laboratory or assay method.
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For the Special Coagulation Laboratory at the Mayo
Clinic in Rochester, blood samples were collected in
3.2% sodium citrate tubes (Becton Dickinson, Franklin
Lakes, NJ) centrifuged to prepare platelet-poor plasma
as per Clinical & Laboratory Standards Institute guide-
lines13 and either assayed within 4 hours of specimen
collection or frozen at –70°C until assay performance.
The APC-R assay was performed using the COATEST
APC Resistance V kit (Chromogenix/Instrumentation
Laboratory, Bedford, MA) following the manufacturer’s
instructions.
An initial activated partial thromboplastin time
(aPTT) was performed in a standard fashion (incubating
patient plasma with phospholipids containing an activa-
tor followed by recalcification). The second aPTT was
performed as in the previous assay except that a standard-
ized amount of human APC was included in the recalci-
fication solution (APC-aPTT), which would be expected
to be prolonged compared with baseline aPTT. A  ratio
of APC-aPTT to aPTT (APC-R ratio) was calculated
and reported. In both aPTT assays, patient plasma was
diluted with factor V–deficient plasma. As with all assays,
an in-house reference range needed to be established or at
least the manufacturers’ reported reference range needed
to be verified.14 We established the reference range in our
laboratory by performing the assay in 120 healthy individ-
uals, correlating results with the FVL assay; an APC-R
ratio of less than 2.3 is considered abnormal and indic-
ative of APC-R. Using this cutoff, in our laboratory, we
had a sensitivity and specificity of 100% for the FVL
mutation (data not shown).
The FVL assay was performed using the Invader
factor V assay (Hologic, Santa Clara, CA) following
the manufacturer’s instructions. The APC-R ratio does
not consistently discriminate between heterozygous and
homozygous FVL; thus, given the differing risks of VTE,
reflexive FVL testing is typically performed for confirma-
tion of zygosity.
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Perez Botero et al  / Hereditary Platelet Disorders Testing
❚Figure 1❚  Activated protein C resistance (APC-R) reflex
algorithm. FVL, factor V Leiden.
APC-R Algorithm/Profile
All orders for laboratory tests at the Mayo Clinic in
Rochester are entered into an electronic orders system.
For the APC-R/FVL evaluation, the default option avail-
able to providers is to select the APC-R reflex algorithm,
termed the APCR-R profile. In this approach, after an ini-
tial APC-R screening assay is performed, the FVL assay
is performed only for an abnormally low APC-R ratio
(≤2.3) or for an indeterminate APC-R ratio ❚Figure 1❚. An
indeterminate APC-R ratio may occur in plasma samples
that have a prolonged baseline aPTT, for example, due to
lupus anticoagulants, excess heparin or direct thrombin
inhibitors, and so on. A prolonged baseline aPTT renders
the APC-R ratio invalid. Direct DNA-based FVL assay
is also available for providers to order; there are no “hard
stops” in the orders system.
Statistical Analysis
The data were entered into Microsoft Excel (Microsoft,
Redmond, WA) and descriptive statistics were calculated.
Laboratory test costs of APCR and FVL were obtained
from the Centers for Medicare & Medicaid Services (CMS)
2014 Clinical Diagnostic Laboratory Fee Schedule for
appropriate CPT codes (85307 and 81241, respectively).15
We used the national limit from the fee schedule, which is a
percentage of the median of all local fee schedule amounts
for each test. These costs were then used to calculate total
costs for testing in our OLDW and Mayo Clinic cohorts.
The total costs were used to measure cost per patient and
extrapolated to determine the cost potential savings per
100,000 standardized population.
Results
OLDW
Of the 100 million cumulative patients in the data-
base, 19.3 million unique individuals met our study
606 Am J Clin Pathol 2017;147:604-610
DOI: 10.1093/ajcp/aqx033
❚Figure 2❚  OptumLabs data warehouse testing pathway
results based on defined testing events for 2013. APC-R,
activated protein C resistance; FVL, factor V Leiden. a2,724
each for a total of 5,448 test orders.
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criteria of continuous coverage over the study period (cal-
endar year 2013). After application of our testing criteria,
which included grouping of tests into testing episodes,
defined as a 14-day window after the initiation of a per-
formed test to determine if a patient had only one of the
tests or both, and the criteria of continuous enrollment
for 2013, it was revealed that of a total of 19.3 million
unique individuals with continuous coverage in 2013, a
total of 78,332 tests were performed on 74,242 (0.385%)
unique individuals. Approximately 15% were 60 years or
older, and in approximately 15%, age was not available;
56% of the total study sample were female.
Frequency of testing consisted of the APC-R test
alone (2,265; 2.9%), FVL testing alone (70,619; 90.1%),
and both APC-R and FVL tests (2,724; 7.0%), resulting
in an approximate ratio of APC-R to FVL of 1:15. Of
those having both the APC-R and FVL tests, more than
half (54.6%) of the APC-R tests were followed up with
FVL, and only a small number of FVL tests were fol-
lowed up by APC-R (0.2%) ❚Figure 2❚.
Mayo Clinic Special Coagulation Laboratory Database
Over the study period, 379,762 unique individuals
received care at the Mayo Clinic in Rochester, for whom
a total of 1,317 (0.347%) tests for assessment of APC-R/
FVL were ordered and performed. The APCR-R was per-
formed on a total of 1,256 (95.4%) patients, whereas the
FVL assay alone was performed on 61 (4.6%), resulting in
an APCR-R to FVL test ratio of approximately 20:1. Of
the 1,256 APCR-R tests, 38 (3.0%) had the FVL ordered
and performed simultaneously; review of clinical notes
revealed no documentation of the rationale for ordering
both the assays simultaneously. During the study period,
the performing laboratory did not have systems in place
to prevent duplicate test performance.
© American Society for Clinical Pathology

❚Figure 3❚  Mayo Clinic Special Coagulation Laboratory database testing pathway results for 2013. APC-R, activated protein
C resistance; FVL, factor V Leiden. aFVL was ordered in addition to APC-R with reflex FVL and was unnecessary given the
wild-type results.
For those having the APCR-R, 1,023 (81.0%) of 1,256
patients had a normal APC-R ratio, and thus FVL was not
indicated or performed; all 38 patients who had the APCR-R
and FVL ordered simultaneously had a normal APC-R
ratio and were wild type (negative) for the FVL mutation.
Of the 1,256 undergoing APCR-R, in three patients
with a reduced APC-R, reflexive FVL testing was can-
celled once results of previous FVL testing performed
elsewhere were brought to the attention of the laboratory;
the APC-R ratio was indeterminate in 119 patients, thus
prompting reflexive FVL testing; and 114 (9.1%) had an
abnormally low APC-R ratio indicative of the presence
of APC resistance. Reflexive FVL testing, to genotype
this cohort, demonstrated that 109 (96.0%) of 114 were
heterozygous. One (0.9%) of 114 was homozygous; this
patient, who was wild type, was a recipient of an ortho-
topic liver transplant and presumably received an allograft
from a patient who was a FVL carrier, resulting in a dis-
crepancy between the APC ratio and the FVL result.
The 61 patients receiving FVL testing as the only test
(34/61; 55.8%) were wild type, 26 (42.6%) of 61 were het-
erozygous, and one (1.6%) of 61 was homozygous. In 50
of these orders, there was no mention of ordering ratio-
nale in the medical record; six patients were taking anti-
coagulants that interfered with the APC-R assay, and in
seven patients, confirmation of zygosity was the indica-
tion ❚Figure 3❚.
Total Cost of Testing Scenarios
Using the 2014 CMS Clinical Diagnostic Fee Schedule
for CPT codes,15 standard reimbursement rates were applied
© American Society for Clinical Pathology
AJCP  / Original Article
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to create cost scenarios for the testing patterns seen in the
OLDW and the Mayo Clinic Special Coagulation Laboratory
Database. Comparisons of the cost per evaluated individ-
ual and also the cost per individual in the total population
were compared, and the savings opportunity when using an
APC-R profile testing approach was calculated.
Given that the intent of this analysis was to demon-
strate the cost savings using the APCR-R profile, the
cost of testing in the Mayo Clinic Special Coagulation
Laboratory Database (outlined in ❚Table 1❚) was restricted
to those patients who only had the APCR-R pro-
file ordered. As mentioned above, 38 patients had the
APCR-R profile and FVL tests ordered, and 61 patients
had the FVL testing ordered.
Based on the pattern of testing, the cost per evalu-
ated individual was $83.77 and $36.38 in the OLDW and
Mayo Clinic, respectively; the cost-savings opportunity
was $47.39 per evaluated individual. When the data were
normalized, the total cost of testing per 100,000 indi-
viduals was $0.32 and $0.14 in the OLDW and Mayo
Clinic populations, respectively, with a resulting cost sav-
ings opportunity of $0.18 per population individual and
$18,245.02 per 100,000 ❚Table 2❚.
Discussion
In general, laboratory tests can be broadly catego-
rized as screening and diagnostic assays; the former are
typically used to screen for the presence or absence of dis-
ease, whereas the latter provide confirmatory evidence (or
lack thereof) of disease. The optimal choice of assay will
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Perez Botero et al  / Hereditary Platelet Disorders Testing

❚Table 1❚ ❚Table 2❚
Factor V Leiden Testing Type, Costs, and Cost Savings in the OptumLabs Data Warehouse and Mayo Clinic Factor V Leiden
OptumLabs Data Warehouse and Mayo Clinic in 2013a Testing Type, Costs, and Cost Savings in a Standardized
Populationa
OptumLabs Data Mayo
Population Warehouse Clinic Standardized Population
(100,000)
Covered population 19,261,389 NA
Evaluated individuals 74,242b 1,256c OptumLabs Mayo
APC-R assay alone (without 2,265 1,023d Characteristic Data Warehouse Clinic
FVL)
Standardized population 100,000 100,000
APC-R and FVL 2,724
Evaluated individuals 385 385
FVL alone 70,619
Total APC-R tests 26 385
APC-R reflex to FVL (APC-R NA 233
Total FVL tests 380 71
profile)
Total APC-R reimbursements $540.98b $8,050.35
Total APC-R tests 4,989 1,256
Total FVL reimbursements $31,708.78 $5,954.38
Total FVL tests 73,343 233e
Total reimbursements $32,249.76 $14,004.73
Total APC-R reimbursements $104,319.99f $26,262.96
Cost per evaluated individual $83.77 $36.38

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Total FVL reimbursements $6,114,605.91 $19,425.21
Total reimbursements $6,218,925.90 $45,688.17
Cost per evaluated individual $83.77 $36.38
Savings opportunity per $47.39
evaluated individual
APC-R, activated protein C resistance; FVL, factor V Leiden; NA, not
applicable.
a
Values are presented as numbers unless otherwise indicated.
b
In total, 74,242 tested out of 19,261,389 covered individuals with continuous
enrollment for 2013.
c
In total, 1,256 (95.4%) of 1,317 were ordered as APC-R with reflex to FVL if
indicated.
d
APCR ratio was normal and thus FVL was not indicated or performed.
e
A total of 114 with abnormal APC-R ratio and 119 with an indeterminate
APCR ratio.
f
Per Clinical Diagnostic Fee Schedule 2014: APC-R, Current Procedural
Terminology (CPT) code 85307 = $20.91; FVL, CPT code 81241 = $83.37.
vary with the performance characteristics of the assay (eg,
sensitivity, specificity, and positive and negative predictive
values), as well as population prevalence of disease; lower
cost screening assays with high sensitivity are likely more
cost-effective when population prevalence of disease is low,
whereas a diagnostic test as an initial test may be more
appropriate with a high population prevalence of disease.
In coagulation testing, an algorithmic approach con-
sisting of screening and reflexive confirmatory testing is
felt to provide the most cost-effective approach,16 but for-
mal analysis of cost savings is lacking. For detecting the
presence or absence of APC-R/FVL, initial plasma-based
assays provide a lower cost option for screening but have
limitations. Several variables may affect results of coagu-
lation testing. Preanalytical variables that may result in an
indeterminate or nondiagnostic APC-R assay include pres-
ence of lupus anticoagulants or other coagulation factor
inhibitors that affect the aPTT and medications, such as
direct thrombin and anti-Xa inhibitors, that interfere with
coagulation assays. However, for most testing scenarios,
optimization of the APC-R assay results in high sensitiv-
ity.10,17 While molecular testing is not significantly affected
by the preanalytical variables that affect coagulation tests,
higher complexity of testing likely makes it less cost-effec-
tive when applied as a screening test in populations with a
Savings opportunity per evaluated $47.39
individual
Cost per population total $0.32 $0.14
Savings opportunity per population — $0.18
individual
Annual savings opportunity per — $18,245.02
100,000 covered lives
APC-R, activated protein C resistance; FVL, factor V Leiden.
a
Values are presented as numbers unless otherwise indicated.
b
Per Clinical Diagnostic Fee Schedule 2014: APC-R, Current Procedural
Terminology (CPT) code 85307 = $20.91; FVL, CPT code 81241 = $83.37.
low prevalence of disease. The prevalence of FVL varies
between 15% in white European populations3 and 5.3%
in white Americans, whereas its prevalence is typically
less than 1% to 2% in other nonwhite populations.18 The
pretest probability of FVL positivity is likely to be lower
when testing the general population19 compared with test-
ing selected patient populations (eg, patients with VTE).8
In this study, using administrative claims data repre-
senting a broad sample of privately insured people in the
United States, we confirmed the ordering practice patterns
reported from single-institution studies.20 Although data
from the OLDW do not provide the results of testing or
the indications for testing, the comparative data from our
institution confirm the modest prevalence of APC-R/FVL
in the tested population and suggest that an algorithmic
approach to APC-R/FVL testing is likely to be cost-effec-
tive. In addition to being more cost-effective, from a clin-
ical standpoint, the APC-R is more clinically meaningful.
Hepatocytes are the main source for circulating factor V,
whereas FVL testing is performed on peripheral blood
leukocyte genomic DNA. Thus, patients with FVL under-
going orthotopic liver transplantation or hematopoietic
stem cell transplant may have discrepant APC-R and FVL
results.21-24 Thus, the APC-R may be a better reflection of
increased risk of thrombosis than FVL genotype by itself
and hence more useful in clinical decision making.9
From a cost standpoint, as we have demonstrated,
the two testing approaches have very different financial

608 Am J Clin Pathol 2017;147:604-610 © American Society for Clinical Pathology

DOI: 10.1093/ajcp/aqx033

implications. Costs for the APC-R profile are $36.38 per
evaluated individual, which on a large scale and taken
over the entire population cost approximately $0.14 per
covered individual. By comparison, the testing pattern
demonstrated in the OLDW results in a cost of $83.77
and $0.32 per evaluated individual and per population
member, respectively, an approximately twofold higher
payer cost. Our analysis is based only on patients under-
going testing with the APCR-R profile, whereas FVL
was ordered in 38 and 61 patients in addition to the
APCR-R and as an initial test, respectively. Based on
an analysis of the actual practice, the cost per evaluated
individual would increase to $40.82, and the annual
savings per 100,000 covered lives would decrease to
$15,715.05.
A small proportion (3.1%) of patients in the OLDW
had both APC-R and FVL testing, and of those, 92.1%
had both APC-R and FVL ordered on the same day. From
the data available, it was not possible to determine whether
those who had APC-R and FVL ordered the same day
were sent to laboratories that offer the APC-R profile or
whether both assays were ordered more for convenience
of the patient, reducing the need for a return visit for fol-
low-up FVL testing, or simply a lack of understanding of
the information received from the assays. Our data and
that of others demonstrate that in approximately 80%
of tests, FVL testing is not needed.25 Extrapolating the
OLDW data, where approximately 0.385% of the cov-
ered population underwent APC-R/FVL evaluation, to
a national level, we estimate that approximately one mil-
lion individuals are tested for APC-R/FVL annually. In
addition to appropriate patient selection, a cost-effective
approach to testing is essential.
Limitations to this data analysis warrant discussion.
This OLDW data set identifies only outpatient testing
scenarios, and the symptomatology of the evaluated
patients, including results of testing and population
prevalence of APC-R/FVL in the evaluated individu-
als, remains unknown. In addition, the 14-day window
may have precluded the inclusion of additional APC-R
or FVL tests, but the impact on the cost analysis is
not likely to be significant. In addition, while we have
applied CMS reimbursement rates, commercial insurers
may have different arrangements that can offer different
financial conclusions to this analysis. In addition, this
study does not address the impact of testing for APC-R/
FVL on patient care, thus precluding a formal cost-ef-
fectiveness analysis.
What can be done to encourage proper test utiliza-
tion for APC-R/FVL? Several parallel interventions may
ensure long-term success. Educating ordering providers,
although a daunting task given the diversity of ordering
© American Society for Clinical Pathology
AJCP  / Original Article
providers, is critical but unlikely to suffice. Recent “choos-
ing wisely” initiatives from specialty societies are steps in
the right direction.26 The electronic orders systems can
be programed to assist the provider in choosing the right
test and help reduce duplicate orders. In addition, ceding
control of optimal testing to the performing laboratory
is an option that has been successful in our institution
but may pose challenges to reference laboratories, and
finally changing test reimbursement rates or noncoverage
of tests is a more drastic option. The latter would likely
increase out-of-pocket expenses for the patient.
In conclusion, we demonstrate the cost savings of an
algorithmic approach using an optimized plasma-based
assay for detection of APC resistance rather than the
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DNA-based FVL assay.
Corresponding author: Rajiv K. Pruthi, MBBS, Mayo Clinic
and Foundation, 200 First St SW, Rochester, MN 55905; pruthi.
rajiv@mayo.edu.
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