COAGULATION AND TRANSFUSION MEDICINE
Review Article
A
Laboratory A p p r o a c h
of H e r e d i t a r y
to the
E v a l u a t i o n
H y p e r c o a g u l a b i l i t y
DOROTHY M. ADCOCK, MD,1-2 LOUIS FINK, MD,3 AND RICHARD A. MARLAR, PhD2'4
The concept of hypercoagulability and especially its evaluation in
the clinical laboratory has changed dramatically during the last few
years. The genetic basis and the mechanisms of the various factors
responsible for hypercoagulability are briefly reviewed with
emphasis on the most common genetic deficiencies. The major
thrust of this review centers on the cost-effective approach to examining patients with a personal or family history of venous thrombosis. Several new concepts dealing with thromboticriskare presented
with a focus on the theory that multiple factors cause thrombosis in
affected patients. A proposal for a cost-effective sequential testing
scheme for the accurate diagnosis of hereditary hypercoagulability
is discussed. The knowledge of thrombotic risk factors is evolving
rapidly, requiring the clinical laboratory to remain flexible.
Ultimately, the clinical laboratory must take a leading role in the
diagnosis of hereditary thrombotic disease by serving as the consultant to the primary caregiver by providing an up-to-date and costeffective evaluation. (Key words: Coagulation; Thrombosis;
Hypercoagulability) Am J Clin Pathol 1997,108:434-149.
The incidence of venous thrombosis in the United
States is estimated b e t w e e n 2 and 3 million per
year, of which 60,000 cases result in death. 1 If the
hemostatic system is functional, then an appropriate and limited amount of clot is formed after vascular injury. Pathogenic factors implicated in the
abnormal formation of a clot are acquired or inherited and include activation of the coagulation system, down-regulation of the endogenous coagulat i o n r e g u l a t o r y s y s t e m s , v a s c u l a r injury, a n d
endothelial cell perturbation. The mechanism and
cause of v e n o u s t h r o m b o s i s involve n u m e r o u s
components of plasma proteins, blood cells, and
blood vessels. 2
During the last 10 to 15 years, a number of genetically based defects in hemostatic proteins have been
found to occur in families with a notable history of
venous thrombosis. These genetic defects have been
grouped into a clinically related genetic disorder
termed hereditary thrombotic disease (HTD). 1,3,4
CONCEPT OF RISK FACTORS
The concept of risk for the d e v e l o p m e n t of a
thrombotic event is starting to gel into a hypothesis
of a " t w o - h i t " or " m u l t i h i t " t h e o r y in w h i c h
genetic, environmental, and acquired factors have
a major role. This concept parallels the risk-factor
concept of cardiovascular and coronary artery disease. The basic concept is that a patient with HTD
has at least one and possibly more genetic risk factors for venous thrombosis that increase the potential for developing venous thrombosis. Each factor
probably has a different risk potential. Risk poten:
tial is defined as the ability or capacity for the factor in question to cause thrombotic complications.
The amount of risk potential can vary depending
on the factor and its interaction with other factors.
M u l t i p l e genetic risk factors w i t h v a r y i n g risk
From the department of Pathology, Colorado Permanente Medical
potentials
probably occur in more severely affected
Group, Aurora, Colorado; the department of Pathology, University
of
Colorado Health Sciences Center, Denver, Colorado; the ^Department
of and families. In addition, acquired facindividuals
Pathology, Little Rock VA Medical Center, University of Arkansas School
tors (eg, effects of surgery, pregnancy, hormonal
of Medicine, Little Rock, Arkansas; and the 4Thrombosis Research
therapy, antiphospholipid syndrome, environmenLaboratory, Pathology and Laboratory Medicine Research Service,
Denver VA Medical Center, Denver, Colorado.
tal, and nutritional factors) contribute to the overall
risk p o t e n t i a l for each p e r s o n . Risk factors
Manuscript received November 26, 1996; revision accepted
are i n d e p e n d e n t factors t h a t are synergistic in
March 5,1997.
Address reprint requests to Dr Marlar: Laboratory Services
nature and that increase the risk potential for the
#113, Denver Veterans Administration Medical Center, 1055
development of venous thrombosis in the patient.
Clermont St, Denver, CO 80220.
434
ADCOCK ET AL
\j Hypercoagulability
Evaluating Heredii
The genetic risk factors remain throughout the person's life, whereas acquired factors arise periodically and may be controlled in part by the patient.
These genetic and acquired factors are additive,
substantially increasing the potential risk for the
development of venous thrombosis. The concept of
the interaction of n u m e r o u s factors (genetic and
a c q u i r e d ) to p r o d u c e a p h e n o t y p e of v e n o u s
thrombosis is familiar to geneticists and is termed
multifactorial trait.5 Multifactorial trait is the comb i n e d effect of m u l t i p l e g e n e s a n d p o s s i b l y
acquired factors to produce the observed phenotype. Because the concept of multifactorial trait
relates to H T D , w e h a v e t e r m e d the s y n d r o m e
thrombotic threshold trait (R.A.M., J a n u a r y 1997,
unpublished data). The thrombotic threshold trait
concept is a more accurate description of the interplay of genetics and acquired factors in families
with a high incidence of venous thrombosis. The
details and interactions of the genetic risk factors
a n d the a c q u i r e d factors h a v e n o t been clearly
defined, and many of the factors remain unknown.
The nidus for this concept is just beginning to be
realized; details of the factors and their interactions
will be developing during the next several years.
The remainder of this article details the concept of
multiple interactions and how we can clinically
assess patients who have HTD or how best to evaluate for thrombotic threshold trait.
KNOWN DEFECTS ASSOCIATED WITH HTD
Knowledge of the interactions between the DNA
sequence and the functional deficiencies associated
with certain mutations has facilitated mapping of
the genes and domains for specific functions. Some
DNA mutations (polymorphisms) are apparently
prevalent in the p o p u l a t i o n and have normal or
near-normal function. To accomplish m a p p i n g , a
distinction must be made between nucleotide substitutions not associated with a phenotypic change
and the mutations that result in disease. At present,
the only genetic analysis for HTD in widespread
use in clinical laboratories is for the G—»A mutation
at base 1691 in the factor V L e i d e n gene, which is
associated with activated protein C (APC) resist a n c e . 6 - 9 D u r i n g the n e x t s e v e r a l y e a r s , t h e r e
undoubtedly will be increased genetic testing for
HTD. The following examples describe some of the
genotypic alterations in APC resistance (APC-R),
protein C (PC) deficiency, protein S (PS) deficiency,
and antithrombin III (AT) deficiency. In most cases
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435
the defect was elucidated by sequencing at least
part of the affected gene.
Factor VUideu
H e r e d i t a r y r e s i s t a n c e to APC-R h a s b e e n
described as an autosomal variable penetrance syndrome for thrombophilia and is the most common
c a u s e for i n h e r i t e d v e n o u s t h r o m b o s i s in
whites. 9 - 1 1 The genetic basis for APC-R has been
defined as a point mutation in the factor V gene at
codon 506, the site where APC cleaves and inactivates the factor Va procoagulant. 6 - 1 2 Factor V has
been m a p p e d to chromosome lq21-25 and has 25
exons. 13 - 14 One or both copies of the factor V gene
from patients with resistance to APC thus carry a
single G to A missense mutation in exon 10 at base
1691, which leads to a conversion of arginine 506 to
glutamine. 6 - 9 After activation, this mutated factor
V a heavy chain cannot be cleaved by APC 1 2 ; its
procoagulant activity therefore persists, and the
risk of venous thrombosis increases substantially.
The increased thrombotic risk associated with the
heterozygous factor V mutation is approximately 5
to 10 times the thrombotic risk for the normal popu l a t i o n . 1 5 - 1 7 The r i s k for p a t i e n t s c a r r y i n g a
homozygous factor V mutation is increased 50- to
100-fold. 9 Up to one-half of all patients with a history of thromboembolism have a functional resistance to APC; most of these defects are attributable
to the major factor V mutation at codon 506 (factor
V L e j d e n ). 1 8 The frequency of the mutant allele in the
general population in Western countries is approximately 2% to 7%. 19 ' 20 The mutation is lower in Asia
Minor and is much lower in Africa, Southeast Asia,
and Australia and in Native American Indians. 2 1 , 2 2
Activated protein C resistance is approximately 10
times more common in the United States than the
other k n o w n genetic defects associated with
venous thrombosis. Because of the relatively high
gene frequency, occurrence of APC resistance in
patients with other genetic and acquired risk fact o r s for t h r o m b o p h i l i a is n o t u n c o m m o n . 2 3 - 3 1
W h e n v a r i o u s c o m b i n a t i o n s occur, the affected
p a t i e n t s m a y h a v e an e a r l i e r o n s e t of v e n o u s
thromboses and a marked increase in the risk for
venous thromboses. This disorder can be detected
by genetic analysis or p l a s m a - b a s e d screening.
Genetic analysis has the a d v a n t a g e over plasma
screening because the results can indicate the diagn o s i s of APC r e s i s t a n c e w h e n the p a t i e n t s are
r e c e i v i n g a n t i c o a g u l a n t s or i n h i b i t o r s of t h e
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COAGULATION AND TRANSFUSION MEDICINE
Review
activated partial thromboplastin time (APTT) test
are present. 3 2 The availability of a rapid, direct,
molecular examination of the mutation at the DNA
level using a polymerase chain reaction (PCR) assay
coupled with detection systems to distinguish the
wild type and mutant alleles in clinical laboratories
is rapidly increasing. The assays include allele-specific restriction enzyme cleavage analysis, differential PCR priming with allele-specific primer, and
oligonucleotide ligation assays. 6 < 33-36
Protein C
Patients with an abnormal PC concentration or
function may have an increased risk for the development of venous thrombosis. The homozygote or
compound heterozygote with PC deficiency has a
marked tendency for venous thrombosis and purpura fulminans. Unless treated, patients die of massive disseminated intravascular coagulation. 1 ' 37 The
risk for venous thrombosis in the heterozygote is
variable, a n d severity may vary in certain p e d i grees. 3 8 The frequency of the homozygous condition is estimated to be 1 in 500,000, and the heterozygous condition may be found in 1 in 200 to
300 p e r s o n s , a l t h o u g h the frequency of the heterozygote with venous thrombotic disease has been
reported as 1 in 15,000.3-38
The gene for PC has been localized to chromosome 2 q l 3 - q l 4 . 4 0 ' 4 1 The gene spans 11 kilobases;
there are 9 exons that encode a 461-amino acid precursor protein. 42 Two messenger RNAs (mRNAs) are
formed because of an alternate p o l y a d e n y l a t i o n
site. 43 Protein C is a vitamin K-dependent glycoprotein that is s y n t h e s i z e d in the liver as a single
polypeptide with posttranslational P-hydroxylation,
y-carboxylation, and glycosylation. Studies on PC
have shown that there is a Pre-Pro sequence targeting the y-carboxylation and secretion. During processing, 42 amino acids are cleaved and 9 Gla (y-carboxyglutamic acid) residues are formed. The Gla
domain is necessary for the Ca + 2 and phospholipid
binding that is critical for the function of PC. The
mature protein is 62 kd, with 25% carbohydrate and
419 amino acids. Thrombin bound on thrombomodulin at the e n d o t h e l i a l cell level cleaves the PC
zymogen between Arg 169 and Leu 170 into the active
enzyme, APC. In the presence of PS and phospholipid, APC is an anticoagulant by enzymatically
cleaving factors Va and Villa. 44
Protein C deficiency is classified as type I or
type II. Type I, the most common form, involves a
AJCP-
concomitant decrease in activity and antigen and has
been associated with heterozygous and homozygous
deficiencies. A wide variety of mutations result in
type I deficiency; most of the mutations are of the
missense configuration. 45 The variable penetrance of
the thrombotic phenotype in some families with PC
deficiency may be related to genes other than those
for PC.
In type II, there is a greater loss of function than
antigen. 46 ' 47 Type II deficiency is subdivided into PC
deficiencies in which the PC can cleave small substrates in amidolytic assays (ie, involving activation
and the active site) and those in which the PC functional defect is only evident in coagulation assays
(ie, involving APC-PS and APC-phospholipid interactions). 46 Most mutations that affect only the anticoagulant assay have been found in exon III (which
codes for the Glu residues involved in Gla format i o n ) ; s e v e r a l h a v e b e e n f o u n d in e x o n IX. 4 5
Mutations in exon IX, which encodes the serine protease activity, have been associated with loss of
activity in amidolytic a n d coagulation assays. 4 5
Denaturing gradient gel electrophoresis has been
used to scan for a variety of mutations. 4 8
The elucidation of the genetic defects for diagnosing PC deficiency is important because there is
a large overlap between the values found in nonaffected a n d affected p e r s o n s w h e n a n t i g e n a n d
activity assays are used. D e t e r m i n i n g the DNA
defect(s) may be i m p o r t a n t in a n a l y z i n g family
studies w h e n the p a r e n t s of the patient seem to
have normal PC but the patient may have a type II
deficiency or be a d o u b l e h e t e r o z y g o t e . 4 9 Also,
testing for the genetic diagnosis may be performed
w h e n the activity a n d i m m u n o l o g i c a s s a y s are
affected by anticoagulant therapy or by factor cons u m p t i o n because of a recent major t h r o m b o t i c
episode. These studies also can be used to exclude
APC-R due to a factor V mutation.
Protein S
Recurrent venous thromboses may develop in
patients with abnormal PS activity; it has been estimated that up to 5% of patients with thrombophilia
h a v e PS deficiency. 5 0 ' 5 1 P r o t e i n S is a v i t a m i n
K - d e p e n d e n t n o n e n z y m a t i c cofactor of PC that
enhances the inactivation of factors Va and Villa.
Protein S exists in a free (active) form that normally accounts for 40% of the total PS and a fraction bound to C4b BP, inactive form. 5 2 ' 5 3 There is
an overlap of the free and total PS levels between
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ADCOCK ET AL
Evaluating Heredit
1/ Hypercoagulability
control subjects with normal levels and patients
with deficient levels. This overlap in plasma concentrations and physiologic fluctuation of the C4b
and PS levels can complicate the attempts to diagnose PS deficiency. 54
Protein S maps to chromosome 3 where there is
an active gene (PROS-1) and a pseudogene (PROS-2)
t h a t is n o t t r a n s c r i b e d . 5 5 The p r e s e n c e of the
p s e u d o g e n e ( w h i c h lacks exon 1 a n d h a s s t o p
codons within the gene) makes genetic analysis difficult. P r o t e i n S is s y n t h e s i z e d in h e p a t o c y t e s ,
endothelial cells, megakaryocytes, Leydig cells, and
osteoblasts. 53 The active gene is 80 kilobases with 15
exons that code for a 635-amino acid precursor protein. Exon I codes for a signal peptide, exon II codes
for a p r o p e p t i d e a n d the Gla d o m a i n s , exon III
codes for an aromatic domain, exon IV is a thrombin-sensitive domain, and exons V to VIII have epidermal growth factorlike domains. 5 3
Several approaches to identification of mutations
in PS have been used. Searching for mutations by
using reverse transcriptase-PCR mRNA elucidated
m u t a t i o n s in which the alteration was not in the
exons and when there was allelic exclusion of mutant
mRNA. 5 5 Recently, mutations have been found by
analyzing the PCR products obtained using primers
for all 15 exons that are not present in the pseudogene. This method includes examination of the exonintron boundaries. 56
Type I PS deficiency has a decreased level of
free PS and a decreased level of PS (APC cofactor)
activity. Most of the m u t a t i o n s associated w i t h
type I PS deficiency are located in the exons coding for the region of PS homologous to the steroid
h o r m o n e - b i n d i n g g l o b u l i n s . Most are m i s s e n s e
mutations, but there are changes causing premature termination or frame shifts. 57 Type Ha PS deficiency involves decreased free and total PS antigen
a n d d e c r e a s e d PS activity. Some p a t i e n t s w i t h
apparent type II PS deficiency probably have APCR rather than genetic mutation of PS. 2 9 A Ser 460 to
Pro m u t a t i o n i n v o l v i n g a T to C t r a n s i t i o n is
known as the Heerlen polymorphism and is found
in 18.8% of patients with PS deficiency and 0.8% of
h e a l t h y subjects. 5 8 Several o t h e r d e l e t i o n s a n d
mutations have been associated with the type Ila
PS deficiency phenotype. 3 Type III (formerly type
lib) PS deficiency is a qualitative defect in which
only the PS activity is decreased. Zoller et al suggested that type I and type III are phenotypic variants of the same genetic disease. 5 9 The differences
between the I and III phenotypes may be related to
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437
the relative concentration of the C4b binding protein (3+ isoform. 5 9
Antithrombin
III
Antithrombin III deficiency has been associated
with an increase in venous thromboses; the prevalence of AT deficiency in familial thrombophilia has
been reported as 2% to 4.2%. 50,51 The frequency of
AT deficiency in the general population is between
1:2,000 and 1:5,000.3 The database for AT mutations,
d e l e t i o n s , frame shifts, missense, and n o n s e n s e
mutations has been described. 3 , 6 0 Antithrombin III
maps to chromosome lq23-25 and has 7 exons spanning 13.5 kilobases with 10 Alu repeats. 61 A leader
sequence is encoded in exons I and II. The remaining polypeptide of 432 amino acids is encoded by
exons II through VI. The reactive site (Arg 393-Ser
394) is in exon VI; the heparin-binding site is in
exons I and III, in which mutations cause qualitative
defects in AT. 54,62 It is now feasible to use PCR to
identify AT mutations. Type I mutations consist of
frame-shift mutations, deletions, insertions, splicesite alterations, and premature terminations. In type
I mutations, one allele is often not expressed, leading to a 50% reduction in circulating AT. These heterozygotes usually have venous thromboses before
age 45 years. The type II, or qualitative, deficiencies
have mutations in the reactive site, mutations transforming AT into a thrombin cleavable substrate,
mutations preventing AT-protease interactions, and
m u t a t i o n s affecting h e p a r i n b i n d i n g . 6 0 The heterozygotes with reactive-site mutations are at similar risk for venous thrombosis as are persons with
type I deficiencies, and heterozygotes with mutations in the heparin-binding site are at less risk for
v e n o u s t h r o m b o s i s than are h e t e r o z y g o t e s with
reactive-site mutations. There are some mutations in
the reactive loop that result in a type II pleiotropic
effect in which expression of a nonfunctional prot e i n is d e c r e a s e d . 6 1 , 6 2 R e c e n t r e v i e w s list a n d
describe the types and sites of mutations associated
with quantitative and qualitative defects in AT. 54,60
Hyperhomocysteinemia
An elevated blood level of homocysteine (hyperhomocysteinemia) is a risk factor for the development of early atherosclerotic vascular disease and
venous thrombosis (See recent review by Guba, et
al. 63 ) Studies suggest that elevated levels of homocysteine should be included among the inherited
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COAGULATION AND TRANSFUSION MEDICINE
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disorders associated with venous thrombosis. 6 4 - 6 6
Den Heijer et al 6 6 recently reported a relationship
between increased homocysteine levels and venous
thrombosis that could not be attributed to other
well-described risk factors. This risk was greatly
increased when plasma homocysteine levels were
greater than 22 u m o l / L , suggesting that a threshold may exist above w h i c h h o m o c y s t e i n e has a
thrombogenic effect. Fermo et al 6 4 examined 107
consecutive patients younger than 45 years of age
with venous thrombosis without any known pred i s p o s i n g factors. Elevated homocysteine levels
were seen in 13%. In this study, 64 venous thrombosis w a s seen at a y o u n g e r age a n d there w a s a
higher rate of recurrence.
Hyperhomocysteinemia may be seen in a variety
of genetic and acquired conditions. An important
recently identified predisposing factor associated
with hyperhomocysteinemia is the inheritance of a
thermolabile variant form of the enzyme, methylene
t e t r a h y d r o f o l a t e r e d u c t a s e (MTHFR). 6 6 In the
homozygous state, the gene frequency varies among
different populations and is reported in 5% of the
general population and 12% to 15% of European,
Middle Eastern, and Japanese populations. Elevated
homocysteine levels may be seen in patients with the
thermolabile variant of MTHFR w h e n there is an
associated deficiency of folic acid.
EVALUATION OF SUSPECTED
HEREDITARY THROMBOTIC DISEASE:
SHOULD INDIVIDUALS BE EXAMINED
FOR HTD?
P e r s o n s w i t h H T D are at greater risk for the
development of venous thromboembolic complications than are persons without the associated deficiency. 1,3 ' 67-70 The advantages of properly classifying persons with increased thrombotic potential
include the following: (1) Persons without symptoms but with a deficiency can be educated about
the signs, symptoms, and risks of venous thrombosis. (2) Asymptomatic persons with a deficiency can
be given p r o p h y l a c t i c t h e r a p y d u r i n g high-risk
periods. (3) Symptomatic persons with a deficiency
can be offered the option for more intensive anticoagulant therapy. (4) More specific therapies can be
provided as available. 4 ' 6 7 - 6 9 , 7 1 , 7 2
Most p e o p l e in the g e n e r a l p o p u l a t i o n w i t h
genetic or laboratory-defined abnormalities w h o
h a v e H T D w i l l n o t suffer c l i n i c a l l y a p p a r e n t
t h r o m b o t i c d i s e a s e . 7 3 - 7 5 Therefore, p r e s c r i b i n g
p r o p h y l a c t i c a n t i c o a g u l a n t t h e r a p y s o l e l y on
t h e b a s i s of h a v i n g a d e f e c t is n o t j u s t i f i e d .
Asymptomatic persons with a deficiency, however,
are at an increased risk of s p o n t a n e o u s v e n o u s
thrombosis and thrombosis in high-risk situations
(eg, trauma, surgery, p r o l o n g e d immobilization,
and pregnancy). 6 8 , 7 0 , 7 6 A study of 161 normal and
heterozygote relatives of 24 symptomatic persons
with PC deficiency documented that manifestations
of thromboembolism developed by age 45 years in
50% of the heterozygotes and 10% of the normal
relatives. 70 Prophylactic administration of heparin
or oral anticoagulants during periods of risk prevents thrombosis in this population. 6 8 , 7 7 Knowledge
of the diagnosis and administration of prophylactic
therapy have been d e m o n s t r a t e d to decrease the
i n c i d e n c e of v e n o u s t h r o m b o s i s from 1.3/100
patient-years to 0.2/100 patient-years in persons
with HTD who are younger than 40 years. 6 7
Patients with a history of deep venous thrombosis h a v e a h i g h e r risk of r e c u r r e n t t h r o m b o s i s
regardless of whether an underlying deficiency is
i d e n t i f i e d . 7 8 , 7 9 In t h o s e w i t h o u t H T D , the risk
declines significantly at 3 to 6 months of oral anticoagulant therapy, which suggests that the anticoagulant therapy can be discontinued at that time. 8 0
In patients with HTD, however, the thrombotic risk
is persistent. In patients with hereditary deficiency
of AT, PC, or PS, development of venous thrombosis occurs at a rate of approximately 2% to 4% per
year. 6 8 , 7 2 Symptomatic patients with HTD should
accordingly be considered for long-term anticoagulant therapy. 6 7 , 6 9 The risks and benefits of longterm anticoagulation must be assessed on an individual basis, considering the potential thrombotic
and hemorrhagic complications. 1 In a cohort of 230
patients w i t h AT, PC, or PS deficiency w h o suffered thromboembolic disease, symptoms of postphlebitic syndrome developed in 50%. 6 9 Most of
these patients believed that the s y m p t o m s negatively affected their quality of life and would have
chosen to receive oral anticoagulant therapy before
the first thrombotic e p i s o d e . The k n o w l e d g e of
h a v i n g HTD allows the person to m a k e rational
decisions about therapy, as well as about contraceptive practices, obstetric care, and family planning through genetic counseling. 6 9 , 8 1 - 8 4
The desire to identify distinct deficiencies is
e n h a n c e d as more specific forms of t h e r a p y are
m a d e accessible. Purified h u m a n AT concentrates
and PC concentrates are now commercially availa b l e . 7 6 , 8 5 , 8 6 F o r m s of i n t e r v e n t i o n o t h e r t h a n
AJCP • O:tober
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ADCOCK ET AL
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concentrates and anticoagulant agents may have a
role in prophylaxis and therapy for some forms of
HTD. For example, in persons who are homozygous
for the thermolabile MTHFR polymorphism, nutritional factors have an important role in the development of hyperhomocysteinemia. Elevated plasma
homocysteine levels occur only when plasma folic
acid levels are below the median. 8 7 , 8 8 Vitamin supplementation therefore may affect the risk assessment in some persons with HTD. 89 ' 90 Proper identif i c a t i o n a n d p r o v i s i o n of a d e q u a t e v i t a m i n
supplementation may have a preventive effect.
In some circumstances, laboratory evaluation for
HTD is not cost-effective. If clinical management for
the patient and family would be unchanged by the
evaluation, then whether a need exists for the evaluation should be strongly considered. Furthermore,
w h e t h e r d o c u m e n t a t i o n of HTD in the p a t i e n t ' s
record affects insurance rates or the ability to obtain
health insurance must be considered.
LABORATORY EVALUATION
The laboratory evaluation of HTD is not only
complex, but also expensive. Plasma-based studies
cost approximately $30 to $75 per test at most referral centers, while the costs of PCR testing varies
substantially, from $75 to $250. A r o u t i n e HTD
workup that typically includes three to five different
tests can often cost $300 to $800. The complex nature
of the workup reflects the variety of therapeutic and
physiologic conditions that can substantially alter
assay results. Unless clinicians are aware of these
conditions and their impact, results may be misinterpreted, leading to inappropriately categorizing
patients as having a deficiency when they do not or
vice versa. Therefore, carefully selecting the patients
who should undergo testing, ordering the appropriate tests, suitably timing the testing, and evaluating
results accurately is important.
In our laboratories, we treat orders for special
coagulation tests as a request for a clinical pathology consultation. All orders for HTD are reviewed
before testing is initiated. Based on the clinical history, discussion with the clinician, or both, requests
are approved as ordered, approved with changes, or
denied. (No test requests are changed or denied
without the approval of the ordering provider.) This
system has been in place in each of our laboratories
for at least 6 years and has been successful not only
in reducing cost, but most importantly in improving
the quality of patient care.
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439
Over a 9-month period, 75 requests for hypercoagulability workups were made for outpatients. Of
these 75 requests, 35 orders were approved without
change, 30 were a p p r o v e d with changes, and 10
were denied. More than 50% of the original requests
for HTD workups for outpatients were considered
inappropriate. Orders were approved with changes
most commonly because only a limited, incomplete
battery of tests was ordered or the tests ordered
were not appropriate for the patient's drug regimen
or physiologic condition (see "Evaluation of an
Order"). Orders may be denied for a variety of reasons, for example; we do not perform HTD assays
as a preoperative evaluation to determine potential
thrombotic risk in patients without a personal or
family history of thrombosis. In addition, orders
may be denied when patients experience the first
thrombotic event when they are older than 70 years
and have an underlying acquired condition, such as
malignancy, associated with thrombosis. We also do
not r e c o m m e n d HTD w o r k u p s for patients with
acute disseminated coagulation or who are in the
immediate postthrombotic period. The cost savings
associated with this type of program can be substantial. We saved an estimated $7,000 in outpatient
workups during a 9-month period.
Overall, our "approval" program has been well
accepted by most clinicians; they welcome consultation about the timing of testing and a discussion of
which tests are indicated. Discussing workups with
the clinicians has a second distinct advantage—it
gives them a contact person in the special coagulation laboratory to answer questions about test interpretation or therapy.
PROCESSING AN ORDER
W h e n the l a b o r a t o r y receives a r e q u e s t for a
hypercoagulability workup, the necessary samples
are d r a w n and stabilized. Regardless of the tests
ordered, at least two vacuum-type tubes of blood
should be drawn: (1) citrate (blue-stoppered) tube
for plasma testing and (2) EDTA (purple-stoppered)
tube or acid-citrate-dextrose (ACD; yellow-stoppered) tube for whole blood a n d DNA analysis.
Most plasma-based studies are performed from derated blood. The citrate tube m u s t be processed
immediately, and the platelet-poor plasma divided
into three aliquots, placed in plastic tubes, and
frozen. The DNA studies can be performed on a
variety of w h o l e blood s a m p l e s (EDTA or ACD
tubes). ACD solution B may have greater DNA
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COAGULATION AND TRANSFUSION MEDICINE
Article
yield. Samples for genetic testing are generally stable for 3 days when stored at 4°C. Longer storage is
associated with decreasing DNA yield. Samples for
genetic studies should not be frozen.
After the specimens have been drawn, the order
and the available history are r e v i e w e d . The
d e s i g n a t e d l a b o r a t o r y c o n s u l t a n t c o n t a c t s the
ordering provider and discusses the request (see
"Evaluation of an Order"). Over time, many clinicians have become familiar with this system of test
a p p r o v a l a n d frequently call before r e q u e s t i n g
special coagulation tests. In this instance, orders
c a n b e r e v i e w e d a n d a p p r o v e d in a d v a n c e .
Occasionally, the patients are directly referred to
the laboratory specialist for assessment before the
blood specimen is obtained.
EVALUATION OF AN ORDER
In our evaluation of test requests, the following
questions are routinely asked: (1) Does the patient's
history justify an evaluation for HTD? (2) Are the
tests requested inclusive enough to properly evaluate the patient's condition? (3) Will underlying therapeutic, pathologic, or physiologic conditions interfere with the interpretation of test results?
Does the Patient's History
an Evaluation for HTD?
Justify
Not all patients who experience a venous thrombotic event require an HTD workup. Many thrombotic events occur in association with acquired factors. In fact, the majority of patients who experience
venous thrombosis do not have underlying thromb o p h i l i a . 7 4 , 9 1 Patients w i t h a c o m m o n a c q u i r e d
cause of thrombosis, eg, myeloproliferative disorders, prolonged immobilization, malignancy,
anatomic defects, or high-risk operations, may not
require an HTD workup. 1 ' 9 2
If an u n s e l e c t e d p o p u l a t i o n of p a t i e n t s w i t h
venous thrombosis is assessed, the frequency of AT,
PC, or PS deficiency is 7% to 8%. 82,93 Given this low
prevalence in the general population and low mortality rate of untreated undiagnosed patients, it is
more cost-effective to assess patients that meet certain criteria. 7 1 If patients with venous thrombosis
are screened, and if testing is performed only on
those who are younger than 45 years, who have a
p o s i t i v e family history, or w h o h a v e r e c u r r e n t
venous thrombotic events, up to 17% of patients will
be determined as having AT, PC, or PS deficiency. 67
This percentage may rise to approximately 40% with
the inclusion of APC-R testing.
To be cost-effective, therefore, screening should
be limited to patients with evidence-based clinical
features of underlying HTD. 7 1 However, if strict criteria are used to screen patients, then the diagnosis
will be missed in a small percentage. The decision to
pursue the evaluation for HTD ultimately must be
m a d e on an i n d i v i d u a l b a s i s . Clinical features
strongly associated with underlying HTD include
the following: (1) venous thromboembolic disease
before 45 years of age, (2) family history of venous
thrombosis, and (3) recurrent venous thrombotic
disease. Evaluation for HTD also should be considered for patients in whom spontaneous thrombosis,
coumarin-induced skin necrosis, or venous thrombosis involving unusual sites (eg, mesenteric vein or
cerebral vein) develops.
Venous thrombotic disease before 45 years of age—
In the presence of a hereditary deficiency, the first
venous thrombosis typically occurs in younger persons than if HTD is not present. 67 ' 92 - 94-96 The mean
ages of the first venous thrombotic event are similar
for the known deficiencies and are as follows: AT,
21 years; PC, 24 years; PS, 26 years; factor V L e i d e n
heterozygous, 28 years; and factor V L e i d e n homozygous, 18 years (R.A.M., June, 1996, u n p u b l i s h e d
data). These data are corroborated in other studies. 6 7 , 6 9 Only rarely do persons with HTD experience the first thrombotic event before puberty. At
14 y e a r s of a g e , the risk i n c r e a s e s s h a r p l y for
unknown reasons. 4 , 6 9
Venous thrombosis develops between the ages of
15 and 40 years in more than 50% of persons with AT,
PC, or PS deficiency, while up to 85% experience a
thrombotic event by 50 years of age. 67,95 ' 96 If venous
thrombosis occurs for the first time after the age of 45
years, the probability of a deficiency of PC, PS, or AT
is extremely low. 82
The c r i t e r i o n of y o u n g a g e of first v e n o u s
thrombosis may not be as consistent a feature in
patients with factor V L e i d e n or hyperhomocysteinemia. The t h r o m b o t i c p o t e n t i a l a s s o c i a t e d w i t h
these two disorders may be lower because these
persons tend to be asymptomatic until they reach
an a d v a n c e d a g e . 5 1 , 8 7 ' 9 7 The i n c i d e n c e of first
venous thrombosis in patients with factor V L e i d e n
or hyperhomocysteinemia in older age groups is
higher than in patients with PC, PS, and AT deficiencies. 1 5 , 5 1 , 6 6 This may be related in part to the
increased risk of v e n o u s t h r o m b o s i s associated
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Evaluating Heredit
y Hypercoagulability
w i t h i n c r e a s i n g a g e in t h e g e n e r a l p o p u l a tion. 9 8 " 1 0 0 Dahlback reported only a 30% risk of
developing venous thrombosis by age 60 years for
persons with APC-R. Furthermore, in a prospective s t u d y of h e a l t h y m e n w i t h factor V L e i d e n
mutation, the mean age of the first venous thrombosis w a s 63.2 y e a r s . 9 7 R o s e n d a a l et a l 1 0 1 estimated that the risk of venous thrombosis in persons younger than 30 years is 1 per 10,000 per year
with a normal genotype and 6 per 10,000 per year
for heterozygous factor V L e i d e n carriers. For persons older than 50 years, the risk is 2 per 10,000
with a normal genotype and 15 per 10,000 with a
heterozygous factor V L e i d e n genotype. 1 0 1 For persons homozygous for factor V L e i d e n , the risk is 10
to 30 per 10,000 per year. 101
In patients with mild h y p e r h o m o c y s t e i n e m i a ,
d e n Heijer d e m o n s t r a t e d an i n c r e a s e d risk for
v e n o u s t h r o m b o s i s w i t h i n c r e a s i n g age to 70
years. 1 0 2 In this study, the odds ratio for thrombosis
for men and women was 2.4 for patients between
the ages of 30 a n d 50 years, b u t the o d d s ratio
increased to 5.5 for patients between the ages of 50
and 70 years.
Family history of venous thrombotic
disease—A
positive family history of venous thrombosis may
be a predictor of inherited abnormalities that pred i s p o s e to clot f o r m a t i o n . In t h e r e v i e w by
Pabinger 7 3 of 680 consecutive patients with venous
thrombosis, the prevalence of AT, PC, or PS deficiency was 7.1%. When patients were screened for
a positive family history of v e n o u s t h r o m b o s i s ,
including first- and second-degree relatives, the
p r e v a l e n c e of deficiency i n c r e a s e d to 1 4 . 1 % . 7 3
Heijboer et al 9 3 reported that the relative odds of
h a v i n g a deficiency are 2.7 w h e n p a t i e n t s h a v e
venous thrombosis but no family history, and the
relative o d d s are 8.8 in patients with thrombosis
and one symptomatic first-degree relative. Other
studies note a positive family history in u p to 63%
of patients, but these studies typically did not characterize whether this represented first- or seconddegree relatives, and they did not consider the age
of the family m e m b e r w h e n v e n o u s t h r o m b o s i s
d e v e l o p e d or other thrombotic risk factors. 5 1 , 7 8
Family history achieves 98% sensitivity for thrombophilia when at least two first-degree relatives of
the propositus are symptomatic. 1 0 3
A negative family history does not exclude HTD;
many of these disorders have low penetrance and
new mutations may occur.74
Vol.1
441
Recurrent venous thrombotic disease—In patients
with HTD, the risk for recurrent venous thrombosis is greater than in those without an underlying
deficiency. 67,71,94 In patients without a deficiency,
the risk of recurrent deep venous thrombosis is 6%
to 10% after one thrombotic episode and 25% after
multiple episodes. 7 8 The incidence of recurrence is
even higher when initial anticoagulant therapy is
inadequate. 7 9 Recurrent venous thrombotic disease
is seen in 60% to 80% of persons with AT, PC, or
PS deficiency. 64,67,69,82
Studies evaluating recurrence rates in patients
w i t h factor V L e i d e n or hyperhomocysteinemia are
few. In 180 patients with recurrent venous thrombosis, d e n Heijer found h y p e r h o m o c y s t e i n e m i a in
25%. 1 0 2 In patients with hyperhomocysteinemia,
recurrent venous thrombosis was seen in 75%. 65 In
one small study, 21 patients with factor V L e i d e n (5
homozygous and 16 heterozygous) were compared
with an age- and sex-matched control group with
venous thrombosis but without deficiency. No statistical difference w a s f o u n d in r e c u r r e n c e r a t e s
between the heterozygous and control groups. The
homozygous group showed a trend toward a higher
rate of recurrence, a l t h o u g h the sample w a s too
small to draw a conclusion. 104
Venous thrombosis involving unusual
sites—The
m o s t f r e q u e n t c l i n i c a l e v e n t in s y m p t o m a t i c
patients with HTD is deep leg vein or pelvic vein
thrombosis. 6 9 , 7 3 In almost 50% of patients, this is
associated with p u l m o n a r y embolus. Thrombosis
involving unusual sites, such as mesenteric, portal,
splenic, renal, cerebral, or retinal veins, also can be
seen in patients with HTD. 1 0 5 Mesenteric venous
thrombosis seems to be the most commonly recognized unusual site for thrombosis in HTD because it
occurs in 4% to 10% of patients. 7 3 The other sites of
thrombosis are reported in 3% or less of patients
w i t h H T D . R e t i n a l v e i n t h r o m b o s i s is r a r e l y
reported in inherited thrombophilia. As more clinicians from varied specialties become increasingly
f a m i l i a r w i t h H T D , t h e r e p o r t e d i n c i d e n c e of
thrombosis involving u n u s u a l sites may change.
Thrombosis involving unusual sites is often listed
as a clinical indication of HTD, but w h e t h e r the
incidence of thrombosis in unusual sites increases
in HTD cannot be determined.
Spontaneous
venous thrombosis—Idiopathic
or
spontaneous venous thrombosis is often cited as a
clinical feature of HTD. 4 , 6 7 , 7 0 , 9 4 In a 1956 Swedish
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COAGULATION AND TRANSFUSION MEDICINE
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study of 303 persons with thrombosis, spontaneous
thrombosis was seen in 17.7%. 98 In patients with
HTD, thrombosis develops in approximately 50%
without an identifiable provocation.4<67<94 In patients
whose first venous thrombotic episode is spontaneous, recurrent thrombotic episodes were unprovoked in 72%. 69 Based on a review of the literature
from 1965 to 1992, the calculated risk of spontaneous thrombosis in patients with AT, PC, or PS
deficiency is 0.6% to 1.6% per year. 80
Patients with coumarin-induced skin necrosis—Skin
necrosis is a rare complication of coumarin therapy,
with an incidence of 0.01% to 1%. 106 ' 107 This complication, which is more common in females, typically
develops 3 to 6 days after the initiation of oral anticoagulant therapy. 1 0 8 Clinically the lesions occur
most commonly over the breasts, thighs, buttocks, or
legs. Skin necrosis manifests first with pain, then
petechiae, followed by sharply demarcated regions
of ecchymoses, and, finally, gangrenous necrosis.
Approximately one third of patients with coumarininduced skin necrosis prove to have hereditary PC
deficiency. 109 Coumarin-induced skin necrosis also
has been reported in patients with PS deficiency and
antiphospholipid antibodies. 106,110
Are the Tests Requested Inclusive Enough To Properly
Evaluate the Patient's Condition?
As a c o n s u l t a n t , t h e clinical p a t h o l o g i s t , is
responsible for providing the physician with pertin e n t u p - t o - d a t e i n f o r m a t i o n a n d a d v i c e for
testing. 9 3 , 1 1 1 The consultant also must discourage
the use of studies that a d d little, if any, clinical
information. To encourage cost-effective and clinically appropriate testing, we developed a series of
s e q u e n t i a l t e s t p a n e l s for t h e H T D w o r k u p .
Patients are examined in a serial fashion, beginning
with a review of the common acquired causes of
hypercoagulability (Table 1, panel 0). 91 In panel 1,
which is our basic hypercoagulability workup, the
more common and well-established causes of HTD
are evaluated (see Table 1). A similar sequential
test panel is available for patients receiving oral
anticoagulant therapy (Table 2). Most evaluations
include only panels 0 and 1. Panel 2 (MTHFR and
plasma homocysteine) is generally recommended
TABLE 1. A COST-EFFECTIVE APPROACH TO THE EVALUATION OF HTD USING A SEQUENTIAL SERIES OF TEST PANELS
Panel 0
Exclude common acquired causes of hypercoagulability such as malignancy, prolonged immobilization, high-risk surgery, and anatomic
abnormality.
Activated partial thromboplastin time to evaluate for lupus anticoagulant
Antiphospholipid antibody by enzyme-linked immunosorbent assay
Panel 1
Activated protein C resistance/factor VLeiden
Protein C activity
Protein S activity
Panel 2
Methylene tetrahydrofolate reductase by DNA
Plasma homocysteine and red blood cell folate
Panel 3
Antithrombin III
Plasminogen activity
Thrombin time/fibrinogen activity
Panel 4
Research assays
HTD = hereditary thrombotic disease.
AJCP • October 1997
ADCOCK ET AL
Evaluating Hereditary Hypercoagulability
when there is a personal or family history of early
v e n o u s t h r o m b o s i s and atherosclerosis. Panel 3
includes more esoteric tests with a low prevalence
and is recommended only in special circumstances
(eg, when other test results are negative or a high
suspicion of a deficiency exists) or when therapy
d e p e n d s on diagnosis. These panels are continuously reviewed and u p d a t e d by the coagulation
specialists based on current research. Offering tests
as panels provides an efficient method to ensure
that evaluations include all of the most appropriate
assays rather than relying on practicing physicians
to keep abreast of newer tests as they are m a d e
available and to order the appropriate tests individually. The panel approach also enables other
laboratorians to assist in the clinical pathology special coagulation consultation practice because they
can refer to the recommended established panels of
HTD testing.
In our approval scheme, a common reason a test
request is "approved with changes" is that only a
limited n u m b e r of tests have been ordered. The
request typically does not include assays that
account for the greatest cause of hypercoagulability
443
(eg, patients with APC-R). For example, some clinicians still believe that AT is the only test needed for
a young woman who experiences venous thrombosis w h i l e r e c e i v i n g o r a l c o n t r a c e p t i v e a g e n t s .
However, the incidence of AT deficiency is quite
low. T h e r e is, m o r e o v e r , a s t r o n g a s s o c i a t i o n
b e t w e e n the u s e of oral c o n t r a c e p t i v e s , factor
V L e i d e n , and the development of thrombosis. In fact,
the risk of thrombosis is 35 times greater in those
homozygous for the factor V mutation who use oral
contraceptives. 112 Activated protein C resistance is
seen in approximately 30% of women with thromboembolic complications d u r i n g t r e a t m e n t with
oral contraceptives. 113
Hereditary deficiency of antithrombin accounts
for only 1% of patients with HTD, while deficiencies
of PC and PS together account for 18% (R.A.M.,
September 1995, u n p u b l i s h e d data). Evaluations
that do not include APC-R will miss deficiencies in
17% to 50% of patients with HTD. 15 - 16 - 101 In reviewing laboratory testing practices for inherited thrombosis, Florell and Rodgers 114 found that AT, PC, and
PS assays were ordered at a sixfold greater rate than
were requests for APC-R.
TABLE 2. A COST-EFFECTIVE APPROACH TO THE EVALUATION OF HTD USING A SEQUENTIAL SERIES OF TEST PANELS
IN PATIENTS RECEIVING ORAL ANTICOAGULANT THERAPY
Panel 0
Exclude common acquired causes of hypercoagulability such as malignancy, prolonged immobilization, high-risk surgery, and anatomic
abnormality.
Antiphospholipid antibody by enzyme-linked immunosorbent assay
Panel 1
Factor VLddcn by polymerase chain reaction
Panel 2
Methylene tetrahydrofolate reductase by DNA
Plasma homocysteine and red blood cell folate
Protein C antigen/ factor VII or factor X antigen
Protein S antigen/ factor VII or factor X antigen
Panel 3
Antithrombin III
Plasminogen activity
Thrombin time/fibrinogen activity
Panel 4
Research assays
HTD = hereditary thrombotic disease.
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COAGULATION AND TRANSFUSION MEDICINE
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Another example in which the test order may not
be inclusive e n o u g h is w h e n PC and PS antigen
assays are ordered rather than activity assays in
patients not receiving oral anticoagulant therapy.
Activity assays identify quantitative and qualitative
deficiencies and are therefore better screening tests. 115
The true incidence of type II PC and PS deficiencies is
unknown, but the deficiencies seem to be a more common cause of venous thrombosis than previously recognized. 116 Type II PC and PS deficiencies would be
missed with antigen assays. 115,116
The PC and PS activity assays, however, are not
always the most a p p r o p r i a t e tests to order, and,
therefore, we cannot routinely default to these. In
patients receiving oral anticoagulant therapy, PC
and PS activity assays are not accurate, and results
are invalid (see the next section). Oral anticoagulant
therapy also interferes with APTT-based plasma
assays for APC-R that do n o t use a d d e d factor
V-deficient plasma. 117 In these instances, alternative
test methods must be used. In all requests for HTD
workups, we use our laboratory information system
to determine whether a recent p r o t h r o m b i n time
result, if available, indicates that the p a t i e n t is
receiving oral anticoagulant therapy. If this information is not readily available, a prothrombin time is
performed before the more costly special coagulation tests are r u n . If the result is a b n o r m a l , the
ordering physician is contacted and the more appropriate evaluation discussed.
Will Underlying Therapeutic, Pathologic, or
Physiologic Conditions Interfere With the
Interpretation of Test Results?
A host of physiologic states, pathologic conditions, and drugs may affect plasma levels of AT, PC,
and PS (Table 3). 118,119 Some of these also may interfere with APTT-based APC-R assays. We discuss
only the more common interfering factors. For a
more extensive discussion of these variables, excellent reviews are available. 4,118-121
Tests must be performed when circumstances or
drugs will not interfere with results, or data must be
interpreted with these interferences in mind. One of
the greatest cost inefficiencies in the special coagulation laboratory is the performance or interpretation of
tests under inappropriate conditions. In our laboratories, one of the most common reasons for denial or
approval with changes of test requests is that assays
are ordered when physiologic conditions or therapies
will interfere with the interpretation of results.
Physiologic states associated with variant AT, PC,
and PS plasma concentrations include the newborn
period, childhood, pregnancy, and the early postp a r t u m state. During pregnancy, there may be a
slight decrease in AT levels or an increase in PC levels but a significant decrease in PS activity to 38% of
n o r m a l compared with average levels of 98% in
nonpregnant control subjects. 82,84,123,124
Age also must be considered when interpreting
values. Levels of PC are substantially reduced at
birth and do not achieve adult levels until 12 to 14
years of age. 37,124 The levels of PS in newborns and
children u p to 10 years of age may be lower than
adult levels. 37 If possible, the diagnosis of PS or PC
deficiency should not be attempted until the later
teenage years or beyond.
Pathologic conditions associated with a decreased
concentration of AT, PC, and PS include the immediate postthrombotic period, the postoperative state,
disseminated intravascular coagulation, and severe
hepatic disease. 37,121,125 Nephrotic syndrome is associated with decreased AT and PS levels, while PC levels
may be increased. 126
A variety of therapeutic drugs may affect levels
of the naturally occurring anticoagulants (see Table
3). Because PC and PS are vitamin K - d e p e n d e n t
proteins, levels are decreased in patients receiving
vitamin K antagonists, such as oral anticoagulants.
Given an average intensity of anticoagulation, antigen levels are typically reduced by 50% and activity
levels to an even greater degree. 1 2 0 In patients with
PC deficiency who are receiving oral anticoagulant
therapy, plasma antigen levels range from 25% to
40%, while clotting activity is 1% to 25%. 1 7 , 3 7 In
patients with PS deficiency receiving oral anticoagulant therapy, the plasma total and free antigen levels range from 3 to 10 u g / m L and 0 to 5 u g / m L ,
respectively, while the activity is in the 1% to 25%
range (R.A.M., January 1995, unpublished data). In
a small percentage of patients with AT deficiency,
AT levels are increased secondary to warfarin therapy. 1 2 7 Plasma levels of AT decrease in p a t i e n t s
receiving c o n t i n u o u s i n t r a v e n o u s h e p a r i n therapy. 1 1 8 , 1 2 8 Heparin therapy will not affect plasma
levels of PC and PS but will interfere with APTTbased activity assays.
A number of conditions interfere with the performance and interpretation of testing for APCR. 129 The baseline APTT m u s t be within n o r m a l
limits for proper test performance. The presence of
a lupus anticoagulant, factor deficiency, oral antic o a g u l a n t t h e r a p y , or h e p a r i n t h e r a p y w i l l
AJCP • Otober1997
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ADCOCK ET AL
Evaluating Hereditary Hypercoagulability
445
TABLE 3. CONDITIONS ASSOCIATED WITH ALTERED PLASMA CONCENTRATIONS
OF NATURALLY OCCURRING ANTICOAGULANTS
Condition
Physiologic
Age
Adult
Child
Infant (<6 mo)
Pregnancy
Pathologic
Acute thrombosis
Postoperative state
Disseminated intravascular coagulation
Hepatic disease, severe
Nephrotic syndrome
Inflammatory state
Vitamin K deficiency
Diabetes mellitus
Therapeutic
Oral anticoagulants
Heparin
L-asparaginase
Estrogen or oral contraceptives
Antithrombin
Protein C
NA
NA
D
D
NA
D
D
I
NA
DorNA
D
D
D
D
D
NA
NA
NA
D
D
D
D
I
NA
D
Dor I
D
D
D
D
D
D
D
Dor I
I
D
D
D
D
D
NA
D
I
NAorl
D
D
Protein S
D
D
NA = not affected; D = decreased; I = increased.
increase the APC/APTT ratio and cause false-negative results. The use of added factor V-deficient
plasma can eliminate m a n y of these preanalytic
v a r i a b l e s . Short APTTs can be seen in p a t i e n t s
with increased acute-phase reactants and may
cause false-positive results.
In general, we do not recommend the evaluation
of AT, PC, and PS d u r i n g anticoagulant therapy
(R.A.M., June 1995, u n p u b l i s h e d data). 1 2 9 ' Some
advocate the use of PC and PS antigen assays, comparing these levels to other vitamin K-dependent
factors, such as factor IX or X.119 Ratios using PC or
PS a n t i g e n w i t h v i t a m i n K - d e p e n d e n t a n t i g e n
assays are not reliable for a variety of reasons. The
antigenic assays cannot detect defects in the activity
of the protein or type II deficiencies. Furthermore,
the antigen assays may detect carboxylated and
noncarboxylated forms of the protein, giving an
o v e r e s t i m a t i o n of the p r o t e i n level. Finally, the
diagnostic accuracy of this method has not been
validated. If evaluation of these proteins is essential, we recommend that coumarin therapy be discontinued for 2 weeks and that heparin therapy be
u s e d d u r i n g t e s t i n g . A n o t h e r c o n s i d e r a t i o n is
examination of family members with a history of
venous thrombosis but who are not receiving anticoagulant therapy.
We also do not recommend evaluations of hypercoagulability in patients immediately after a thrombotic
event. Some clinicians, aware that heparin and oral
a n t i c o a g u l a n t t h e r a p y will interfere w i t h assay
results, order HTD assessments when the patient is
first examined but before therapy is initiated. While
normal protein levels strongly suggest that no congenital deficiency exists, protein concentrations may
be decreased because of coagulation factor consumption. We repeatedly have seen patients inappropriately characterized as having an AT or PS deficiency
because the person interpreting the assay results or
reviewing the chart was unaware of the inappropriate
timing of the workup.
In general, AT, PC, and PS assays are best suited
to the outpatient setting and have little role for the
hospitalized patient. Most hospitalized patients are
in t h e i m m e d i a t e p o s t t h r o m b o t i c p e r i o d , are
receiving anticoagulant therapy, or have serious illnesses that may interfere with testing. Miletich 120
reported decreased plasma PC levels in hospitalized patients associated with a variety of serious
illnesses. Of hospitalized patients, 12% had PC levels below the lower limit of the reference range.
These patients did not demonstrate an increased
incidence of venous thrombosis, and no relationship was evident to global suppression of synthesis
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COAGULATION AND TRANSFUSION MEDICINE
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3. DeStefano V, Finazzi G, Marmucci PM. Inherited thrombophilia:
pathogenesis, clinical syndromes, and management. Blood.
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4. Hirsh J, Prins MH, Samama M. An approach to the thrombophilic patient. In: Colman RW, Hirsh J, Marder VJ,
Salzman EW, eds. Hemostasis and Thrombosis: Basic Principle
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5. Lewis R. Multifactorial traits. In: Human Genetics: Concepts and
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1994:93-109.
6. Bertina RM, Koeleman BPC, Koster T, et al. Mutation in blood
coagulation factor V associated with resistance to activated
protein C. Nature. 1994;369:64-67.
SUMMARY
7. Greengard JS, Xi S, Xiao X, Fernandez JA, Griffin JH, Evatt B.
Activated protein C resistance caused by Arg 506Gln mutation in factor V. Lancet. 1994;343:1361-1362.
The goal of laboratory testing for HTD is to predict
8. Voorberg J, Roelse J, Koopman R, et al. Association of idiopathic
and ultimately prevent venous thrombosis and its
thromboembolism with single point mutation at Arg506 of
complications. A rational approach to prophylactic
factor V. Lancet. 1994;343:1536.
therapy must be multifaceted and consider nutri9. Zoller B, Svensson PJ, He X, Dahlback B. Identification of the
tional aspects (eg, folate or pyridoxine supplementasame factor V gene mutation in 47 out of 50 thrombosisprone families with inherited resistance to activated protein
tion), environmental risks (eg, surgery), hormonal
C. / Clin Invest. 1994;94:2521-2524.
and other drug treatments, and the type, dose, and
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Griffin
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development of a cost-effective sequential testing scheme. This
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