COAGULATION AND TRANSFUSION MEDICINE Review Article. A Laboratory Approach to the Evaluation

Transcription

1 COAGULATION AN TRANSFUSION MEICINE Review Article A Laboratory Approach to the Evaluation of Hereditary Hypercoagulability OROTHY M. ACOCK, M, 1-2 LOUIS FINK, M, 3 AN RICHAR A. MARLAR, Ph 2 ' 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 The incidence of venous thrombosis in the United States is estimated between 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 coagulation regulatory systems, vascular injury, and endothelial cell perturbation. The mechanism and cause of venous thrombosis involve numerous components of plasma proteins, blood cells, and blood vessels. 2 uring 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 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: grouped into a clinically related genetic disorder termed hereditary thrombotic disease (HT). 1,3,4 CONCEPT OF RISK FACTORS The concept of risk for the development of a thrombotic event is starting to gel into a hypothesis of a "two-hit" or "multihit" theory in which 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 HT 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. From the department of Pathology, Colorado Permanente Medical Multiple genetic risk factors with varying risk Group, Aurora, Colorado; the department of Pathology, University potentials of probably occur in more severely affected Colorado Health Sciences Center, enver, Colorado; the ^epartment individuals of and families. In addition, acquired factors (eg, effects of surgery, pregnancy, hormonal Pathology, Little Rock VA Medical Center, University of Arkansas School of Medicine, Little Rock, Arkansas; and the 4 Thrombosis Research Laboratory, Pathology and Laboratory Medicine Research Service, therapy, antiphospholipid syndrome, environmental, and nutritional factors) contribute to the over enver VA Medical Center, enver, Colorado. Manuscript received November 26, 1996; revision accepted all risk potential for each person. Risk factors March 5,1997. are independent factors that are synergistic in Address reprint requests to r Marlar: Laboratory Services nature and that increase the risk potential for the #113, enver Veterans Administration Medical Center, 1055 Clermont St, enver, CO development of venous thrombosis in the patient. ownloaded from by guest on 18 ecember

2 ACOCK ET AL 435 Evaluating Heredii \j Hypercoagulability 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 numerous factors (genetic and acquired) to produce a phenotype of venous thrombosis is familiar to geneticists and is termed multifactorial trait. 5 Multifactorial trait is the combined effect of multiple genes and possibly acquired factors to produce the observed phenotype. Because the concept of multifactorial trait relates to HT, we have termed the syndrome thrombotic threshold trait (R.A.M., January 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 and the acquired factors have not 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 HT or how best to evaluate for thrombotic threshold trait. KNOWN EFECTS ASSOCIATE WITH HT Knowledge of the interactions between the sequence and the functional deficiencies associated with certain mutations has facilitated mapping of the genes and domains for specific functions. Some mutations (polymorphisms) are apparently prevalent in the population and have normal or near-normal function. To accomplish mapping, 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 HT in widespread use in clinical laboratories is for the G»A mutation at base 1691 in the factor V Leiden gene, which is associated with activated protein C (APC) resistance. 6-9 uring the next several years, there undoubtedly will be increased genetic testing for HT. 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 the defect was elucidated by sequencing at least part of the affected gene. Factor V Uideu Hereditary resistance to APC-R has been described as an autosomal variable penetrance syndrome for thrombophilia and is the most common cause for inherited venous thrombosis in whites 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 Factor V has been mapped to chromosome lq21-25 and has 25 exons 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 12 ; 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 population The risk for patients carrying 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 Lejden ). 18 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. 21,22 Activated protein C resistance is approximately 10 times more common in the United States than the other known 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 factors for thrombophilia is not uncommon When various combinations occur, the affected patients may have an earlier onset of venous thromboses and a marked increase in the risk for venous thromboses. This disorder can be detected by genetic analysis or plasma-based screening. Genetic analysis has the advantage over plasma screening because the results can indicate the diagnosis of APC resistance when the patients are receiving anticoagulants or inhibitors of the ownloaded from by guest on 18 ecember 2018 Vol.1 No. 4

3 436 COAGULATION AN TRANSFUSION MEICINE Review activated partial thromboplastin time (APTT) test are present. 32 The availability of a rapid, direct, molecular examination of the mutation at the 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 < 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, and severity may vary in certain pedigrees. 38 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 persons, although the frequency of the heterozygote with venous thrombotic disease has been reported as 1 in 15, The gene for PC has been localized to chromosome 2ql3-ql4. 40 ' 41 The gene spans 11 kilobases; there are 9 exons that encode a 461-amino acid precursor protein. 42 Two messenger Rs (mrs) are formed because of an alternate polyadenylation site. 43 Protein C is a vitamin K-dependent glycoprotein that is synthesized 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. uring 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 endothelial 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 AJCPconcomitant 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 formation); several have been found in exon IX. 45 Mutations in exon IX, which encodes the serine protease activity, have been associated with loss of activity in amidolytic and coagulation assays. 45 enaturing gradient gel electrophoresis has been used to scan for a variety of mutations. 48 The elucidation of the genetic defects for diagnosing PC deficiency is important because there is a large overlap between the values found in nonaffected and affected persons when antigen and activity assays are used. etermining the defect(s) may be important in analyzing family studies when the parents of the patient seem to have normal PC but the patient may have a type II deficiency or be a double heterozygote. 49 Also, testing for the genetic diagnosis may be performed when the activity and immunologic assays are affected by anticoagulant therapy or by factor consumption because of a recent major thrombotic 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 have PS deficiency. 50 ' 51 Protein S is a vitamin K-dependent nonenzymatic 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. 52 ' 53 There is an overlap of the free and total PS levels between ownloaded from by guest on 18 ecember 2018 jer 1997

4 ACOCK ET AL 437 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) that is not transcribed. 55 The presence of the pseudogene (which lacks exon 1 and has stop codons within the gene) makes genetic analysis difficult. Protein S is synthesized in hepatocytes, 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 propeptide and the Gla domains, exon III codes for an aromatic domain, exon IV is a thrombin-sensitive domain, and exons V to VIII have epidermal growth factorlike domains. 53 Several approaches to identification of mutations in PS have been used. Searching for mutations by using reverse transcriptase-pcr mr elucidated mutations in which the alteration was not in the exons and when there was allelic exclusion of mutant mr. 55 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 mutations associated with type I PS deficiency are located in the exons coding for the region of PS homologous to the steroid hormone-binding globulins. Most are missense mutations, but there are changes causing premature termination or frame shifts. 57 Type Ha PS deficiency involves decreased free and total PS antigen and decreased PS activity. Some patients with apparent type II PS deficiency probably have APC- R rather than genetic mutation of PS. 29 A Ser 460 to Pro mutation involving a T to C transition is known as the Heerlen polymorphism and is found in 18.8% of patients with PS deficiency and 0.8% of healthy subjects. 58 Several other deletions and 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. 59 The differences between the I and III phenotypes may be related to the relative concentration of the C4b binding protein (3 + isoform. 59 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, The database for AT mutations, deletions, frame shifts, missense, and nonsense mutations has been described. 3,60 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 mutations affecting heparin binding. 60 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 venous thrombosis than are heterozygotes 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 protein is decreased. 61,62 Recent reviews list and 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 ownloaded from by guest on 18 ecember 2018 Vol.1 No. 4

5 438 COAGULATION AN TRANSFUSION MEICINE Review Article disorders associated with venous thrombosis en Heijer et al 66 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 umol/l, suggesting that a threshold may exist above which homocysteine has a thrombogenic effect. Fermo et al 64 examined 107 consecutive patients younger than 45 years of age with venous thrombosis without any known predisposing factors. Elevated homocysteine levels were seen in 13%. In this study, 64 venous thrombosis was seen at a younger age and there was 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 tetrahydrofolate reductase (MTHFR). 66 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 when there is an associated deficiency of folic acid. EVALUATION OF SUSPECTE HEREITARY THROMBOTIC ISEASE: SHOUL INIVIUALS BE EXAMINE FOR HT? Persons with HT are at greater risk for the development of venous thromboembolic complications than are persons without the associated deficiency. 1,3 ' 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 prophylactic therapy during 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 ' 67-69,71,72 Most people in the general population with genetic or laboratory-defined abnormalities who have HT will not suffer clinically apparent thrombotic disease Therefore, prescribing prophylactic anticoagulant therapy solely on the basis of having a defect is not justified. Asymptomatic persons with a deficiency, however, are at an increased risk of spontaneous venous thrombosis and thrombosis in high-risk situations (eg, trauma, surgery, prolonged immobilization, and pregnancy). 68,70,76 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. 68,77 Knowledge of the diagnosis and administration of prophylactic therapy have been demonstrated to decrease the incidence of venous thrombosis from 1.3/100 patient-years to 0.2/100 patient-years in persons with HT who are younger than 40 years. 67 Patients with a history of deep venous thrombosis have a higher risk of recurrent thrombosis regardless of whether an underlying deficiency is identified. 78,79 In those without HT, 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. 80 In patients with HT, 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. 68,72 Symptomatic patients with HT should accordingly be considered for long-term anticoagulant therapy. 67,69 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 with AT, PC, or PS deficiency who suffered thromboembolic disease, symptoms of postphlebitic syndrome developed in 50%. 69 Most of these patients believed that the symptoms negatively affected their quality of life and would have chosen to receive oral anticoagulant therapy before the first thrombotic episode. The knowledge of having HT allows the person to make rational decisions about therapy, as well as about contraceptive practices, obstetric care, and family planning through genetic counseling. 69,81-84 The desire to identify distinct deficiencies is enhanced as more specific forms of therapy are made accessible. Purified human AT concentrates and PC concentrates are now commercially available. 76,85,86 Forms of intervention other than ownloaded from by guest on 18 ecember 2018 AJCP Oc :tober 1997

6 ACOCK ET AL 439 Evaluating Heredit Hypercoagulability concentrates and anticoagulant agents may have a role in prophylaxis and therapy for some forms of HT. 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. 87,88 Vitamin supplementation therefore may affect the risk assessment in some persons with HT. 89 ' 90 Proper identification and provision of adequate vitamin supplementation may have a preventive effect. In some circumstances, laboratory evaluation for HT 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, whether documentation of HT in the patient's record affects insurance rates or the ability to obtain health insurance must be considered. LABORATORY EVALUATION The laboratory evaluation of HT 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 routine HT 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 HT 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. 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 approved with changes, and 10 were denied. More than 50% of the original requests for HT 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 HT 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 recommend HT workups 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. iscussing 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 ORER When the laboratory receives a request for a hypercoagulability workup, the necessary samples are drawn 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) ETA (purple-stoppered) tube or acid-citrate-dextrose (AC; yellow-stoppered) tube for whole blood and analysis. Most plasma-based studies are performed from derated blood. The citrate tube must be processed immediately, and the platelet-poor plasma divided into three aliquots, placed in plastic tubes, and frozen. The studies can be performed on a variety of whole blood samples (ETA or AC tubes). AC solution B may have greater ownloaded from by guest on 18 ecember 2018 Vol. No. 4

7 440 COAGULATION AN TRANSFUSION MEICINE Article yield. Samples for genetic testing are generally stable for 3 days when stored at 4 C. Longer storage is associated with decreasing yield. Samples for genetic studies should not be frozen. After the specimens have been drawn, the order and the available history are reviewed. The designated laboratory consultant contacts the ordering provider and discusses the request (see "Evaluation of an Order"). Over time, many clinicians have become familiar with this system of test approval and frequently call before requesting special coagulation tests. In this instance, orders can be reviewed and approved in advance. Occasionally, the patients are directly referred to the laboratory specialist for assessment before the blood specimen is obtained. EVALUATION OF AN ORER In our evaluation of test requests, the following questions are routinely asked: (1) oes the patient's history justify an evaluation for HT? (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? oes the Patient's History an Evaluation for HT? Justify Not all patients who experience a venous thrombotic event require an HT workup. Many thrombotic events occur in association with acquired factors. In fact, the majority of patients who experience venous thrombosis do not have underlying thrombophilia. 74,91 Patients with a common acquired cause of thrombosis, eg, myeloproliferative disorders, prolonged immobilization, malignancy, anatomic defects, or high-risk operations, may not require an HT workup. 1 ' 92 If an unselected population of patients with 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. 71 If patients with venous thrombosis are screened, and if testing is performed only on those who are younger than 45 years, who have a positive family history, or who have recurrent 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 HT. 71 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 HT ultimately must be made on an individual basis. Clinical features strongly associated with underlying HT 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 HT 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 HT is not present. 67 ' 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 Leiden heterozygous, 28 years; and factor V Leiden homozygous, 18 years (R.A.M., June, 1996, unpublished data). These data are corroborated in other studies. 67,69 Only rarely do persons with HT experience the first thrombotic event before puberty. At 14 years of age, the risk increases sharply for unknown reasons. 4,69 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 criterion of young age of first venous thrombosis may not be as consistent a feature in patients with factor V Leiden or hyperhomocysteinemia. The thrombotic potential associated with these two disorders may be lower because these persons tend to be asymptomatic until they reach an advanced age. 51,87 ' 97 The incidence of first venous thrombosis in patients with factor V Leiden or hyperhomocysteinemia in older age groups is higher than in patients with PC, PS, and AT deficiencies. 15,51,66 This may be related in part to the increased risk of venous thrombosis associated ownloaded from by guest on 18 ecember 2018 AJCP- tober1997

8 ACOCK ET AL 441 Evaluating Heredit y Hypercoagulability with increasing age in the general population. 98 " 100 ahlback reported only a 30% risk of developing venous thrombosis by age 60 years for persons with APC-R. Furthermore, in a prospective study of healthy men with factor V Leiden mutation, the mean age of the first venous thrombosis was 63.2 years. 97 Rosendaal et al 101 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 Leiden 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 Leiden genotype. 101 For persons homozygous for factor V Leiden, the risk is 10 to 30 per 10,000 per year. 101 In patients with mild hyperhomocysteinemia, den Heijer demonstrated an increased risk for venous thrombosis with increasing age to 70 years. 102 In this study, the odds ratio for thrombosis for men and women was 2.4 for patients between the ages of 30 and 50 years, but the odds 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 predispose to clot formation. In the review by Pabinger 73 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 venous thrombosis, including first- and second-degree relatives, the prevalence of deficiency increased to 14.1%. 73 Heijboer et al 93 reported that the relative odds of having a deficiency are 2.7 when patients have venous thrombosis but no family history, and the relative odds are 8.8 in patients with thrombosis and one symptomatic first-degree relative. Other studies note a positive family history in up 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 member when venous thrombosis developed or other thrombotic risk factors. 51,78 Family history achieves 98% sensitivity for thrombophilia when at least two first-degree relatives of the propositus are symptomatic. 103 A negative family history does not exclude HT; many of these disorders have low penetrance and new mutations may occur. 74 Recurrent venous thrombotic disease In patients with HT, 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. 78 The incidence of recurrence is even higher when initial anticoagulant therapy is inadequate. 79 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 with factor V Leiden or hyperhomocysteinemia are few. In 180 patients with recurrent venous thrombosis, den Heijer found hyperhomocysteinemia in 25%. 102 In patients with hyperhomocysteinemia, recurrent venous thrombosis was seen in 75%. 65 In one small study, 21 patients with factor V Leiden (5 homozygous and 16 heterozygous) were compared with an age- and sex-matched control group with venous thrombosis but without deficiency. No statistical difference was found in recurrence rates between the heterozygous and control groups. The homozygous group showed a trend toward a higher rate of recurrence, although the sample was too small to draw a conclusion. 104 Venous thrombosis involving unusual sites The most frequent clinical event in symptomatic patients with HT is deep leg vein or pelvic vein thrombosis. 69,73 In almost 50% of patients, this is associated with pulmonary embolus. Thrombosis involving unusual sites, such as mesenteric, portal, splenic, renal, cerebral, or retinal veins, also can be seen in patients with HT. 105 Mesenteric venous thrombosis seems to be the most commonly recognized unusual site for thrombosis in HT because it occurs in 4% to 10% of patients. 73 The other sites of thrombosis are reported in 3% or less of patients with HT. Retinal vein thrombosis is rarely reported in inherited thrombophilia. As more clinicians from varied specialties become increasingly familiar with HT, the reported incidence of thrombosis involving unusual sites may change. Thrombosis involving unusual sites is often listed as a clinical indication of HT, but whether the incidence of thrombosis in unusual sites increases in HT cannot be determined. Spontaneous venous thrombosis Idiopathic or spontaneous venous thrombosis is often cited as a clinical feature of HT. 4,67,70,94 In a 1956 Swedish ownloaded from by guest on 18 ecember 2018 Vol.1 No. 4

9 442 COAGULATION AN TRANSFUSION MEICINE Review Article study of 303 persons with thrombosis, spontaneous thrombosis was seen in 17.7%. 98 In patients with HT, 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. 108 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 consultant, the clinical pathologist, is responsible for providing the physician with pertinent up-to-date information and advice for testing. 93,111 The consultant also must discourage the use of studies that add little, if any, clinical information. To encourage cost-effective and clinically appropriate testing, we developed a series of sequential test panels for the HT workup. 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 HT 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 HT 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 V Leiden Protein C activity Protein S activity Panel 2 Methylene tetrahydrofolate reductase by Plasma homocysteine and red blood cell folate ownloaded from by guest on 18 ecember 2018 Panel 3 Antithrombin III Plasminogen activity Thrombin time/fibrinogen activity Panel 4 Research assays HT = hereditary thrombotic disease. AJCP October 1997

10 ACOCK ET AL 443 Evaluating Hereditary Hypercoagulability when there is a personal or family history of early venous thrombosis 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 depends on diagnosis. These panels are continuously reviewed and updated 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 made 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 HT testing. In our approval scheme, a common reason a test request is "approved with changes" is that only a limited number of tests have been ordered. The request typically does not include assays that account for the greatest cause of hypercoagulability (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 while receiving oral contraceptive agents. However, the incidence of AT deficiency is quite low. There is, moreover, a strong association between the use of oral contraceptives, factor V Leiden, 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 during treatment with oral contraceptives. 113 Hereditary deficiency of antithrombin accounts for only 1% of patients with HT, while deficiencies of PC and PS together account for 18% (R.A.M., September 1995, unpublished data). Evaluations that do not include APC-R will miss deficiencies in 17% to 50% of patients with HT 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 HT 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 V Lddcn by polymerase chain reaction Panel 2 Methylene tetrahydrofolate reductase by 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 ownloaded from by guest on 18 ecember 2018 Panel 3 Antithrombin III Plasminogen activity Thrombin time/fibrinogen activity Panel 4 Research assays HT = hereditary thrombotic disease. Vol. 108 No. 4

11 444 COAGULATION AN TRANSFUSION MEICINE Revieiv Article Another example in which the test order may not be inclusive enough is when 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 appropriate 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 not use added factor V-deficient plasma. 117 In these instances, alternative test methods must be used. In all requests for HT workups, we use our laboratory information system to determine whether a recent prothrombin time result, if available, indicates that the patient 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 run. If the result is abnormal, 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, 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 postpartum state. uring 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 normal 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 up 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-dependent 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. 120 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%. 17,37 In patients with PS deficiency receiving oral anticoagulant therapy, the plasma total and free antigen levels range from 3 to 10 ug/ml and 0 to 5 ug/ml, 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. 127 Plasma levels of AT decrease in patients receiving continuous intravenous heparin therapy. 118,128 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 APC- R. 129 The baseline APTT must be within normal limits for proper test performance. The presence of a lupus anticoagulant, factor deficiency, oral anticoagulant therapy, or heparin therapy will ownloaded from by guest on 18 ecember 2018 AJCP Oc tober1997

12 ACOCK ET AL 445 Evaluating Hereditary Hypercoagulability TABLE 3. CONITIONS ASSOCIATE WITH ALTERE PLASMA CONCENTRATIONS OF TURALLY OCCURRING ANTICOAGULANTS Condition Physiologic Age Adult Child Infant (<6 mo) Pregnancy Pathologic Acute thrombosis Postoperative state isseminated intravascular coagulation Hepatic disease, severe Nephrotic syndrome Inflammatory state Vitamin K deficiency iabetes mellitus Therapeutic Oral anticoagulants Heparin L-asparaginase Estrogen or oral contraceptives = not affected; = decreased; I = increased. Antithrombin increase the APC/APTT ratio and cause false-negative results. The use of added factor V-deficient plasma can eliminate many of these preanalytic variables. Short APTTs can be seen in patients with increased acute-phase reactants and may cause false-positive results. In general, we do not recommend the evaluation of AT, PC, and PS during anticoagulant therapy (R.A.M., June 1995, unpublished data). 129 ' 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 antigen with vitamin K-dependent antigen 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 overestimation of the protein 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 used during testing. Another consideration is examination of family members with a history of venous thrombosis but who are not receiving anticoagulant therapy. I Protein C I I or I I Protein S or or I orl We also do not recommend evaluations of hypercoagulability in patients immediately after a thrombotic event. Some clinicians, aware that heparin and oral anticoagulant therapy will interfere with assay results, order HT 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 the immediate postthrombotic period, 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 ownloaded from by guest on 18 ecember 2018 Vol. 108 No. 4

13 446 COAGULATION AN TRANSFUSION MEICINE Review Article or activation; approximately 50% of patients had normal levels of factors VII and IX. Certainly orders for PC and PS assays need not be ordered for immediate performance. Assays that are based on analysis are not influenced by physiologic and therapeutic conditions because genetic makeup does not change. Therefore, PCR assays for factor V Leiden and MTHFR and other genetic sequences can be performed regardless of the patient's condition and medication regimen. SUMMARY The goal of laboratory testing for HT is to predict and ultimately prevent venous thrombosis and its complications. A rational approach to prophylactic therapy must be multifaceted and consider nutritional aspects (eg, folate or pyridoxine supplementation), environmental risks (eg, surgery), hormonal and other drug treatments, and the type, dose, and duration of anticoagulant therapy. The thrombotic threshold trait concept integrating acquired and genetic risk factors for venous thrombosis for their relative importance and interactions must be used to develop plans for quality and cost-effective diagnoses and treatments. Knowledge of acquired and genetic risk factors is increasing rapidly. With the evolution of these concepts, the laboratory must remain flexible. We advocate that each request for HT testing be treated as a request for a clinical pathology consultation. Given this, the laboratory must adapt its own approach that evolves based on current research and test availability with the goal of providing cost-effective and quality evaluations. In this endeavor, we recommend that the laboratory align itself or develop an expert (internal or external) who will assist in tailoring the workup with these goals in mind. Acknowledgments: We thank the personnel in the Special Coagulation Laboratories at the enver (Colo) VA Medical Center and the Little Rock (Ark) VA Medical Center for their help in the development of a cost-effective sequential testing scheme. This work was supported in part by a VA Merit Review grant (R.A.M., enver, Colo). REFERENCES 1. Hirsh J, Hoak J. Management of deep vein thrombosis and pulmonary embolism. Circulation. 1996;93: van den Belt AGM, Prins MH, Huisman MV, Hirsh J. Familial thrombophilia: a review analysis. Clin Appl Thromb Hemost, 1996;2: estefano V, Finazzi G, Marmucci PM. Inherited thrombophilia: pathogenesis, clinical syndromes, and management. Blood. 1996;87: 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 and Clinical Practice. Philadelphia, Pa: JB Lippincott; 1994: Lewis R. Multifactorial traits. In: Human Genetics: Concepts and Applications. ubuque, Iowa: WC Brown Publishers; 1994: Bertina RM, Koeleman BPC, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994;369: 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: Voorberg J, Roelse J, Koopman R, et al. Association of idiopathic thromboembolism with single point mutation at Arg 506 of factor V. Lancet. 1994;343: Zoller B, Svensson PJ, He X, ahlback B. Identification of the same factor V gene mutation in 47 out of 50 thrombosisprone families with inherited resistance to activated protein C. / Clin Invest. 1994;94: Griffin JH, Evatt B, Wideman C, Fernandez JA. Anticoagulant protein C pathway defective in majority of thrombophilia patients. Blood. 1993;82: Rodgers GM. Activated protein C resistance and inherited thrombosis. Am ] Clin Pathol. 1995;103: Kalafatis M, Haley PE, Lu, Bertina RM, Long GL, Mann KG. Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay. Blood. 1996;87: Wang H, Riddell C, Guinto ER, MacGillivray RTA. Localization of the gene encoding human factor V to chromosome lq Genomics. 1988;2: Ortel TL, Kane WH, Keller FG. Factor V in molecular basis of thrombosis and hemostasis. In: High KA, Roberts HR, eds. Molecular Basis of Thrombosis and Hemostasis. New York, NY ekker; 1995: Svensson PJ, ahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med. 1994;330: Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C Leiden thrombophilia study. Lancet. 1993;342: Voelkerding KV. Resistance to activated protein C and a novel factor V gene mutation. Clin Lab Med. 1996;16: Sun X, Evatt B, Griffin JH. Blood coagulation factor V a abnormally associated with resistance to activated protein C in venous thrombophilia. Blood. 1994;83: ahlback B. Inherited thrombophilia: resistance to activated protein C as a pathogenic factor of venous thrombomodulin. Blood. 1995;85: ahlback B, Hillarp A, Rose S, Zoller B. Resistant to activated protein C, the FV:Q 506 allele, and venous thrombosis. Ann Hematol. 1996;72: Rees C, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet. 1995;346: Ko YL, Hsu TS, Wu SM, et al. The G1691A mutation of the coagulation factor V gene (factor V Leiden ) is rare in Chinese: an analysis of 618 individuals. Hum Genet. 1996;98: ownloaded from by guest on 18 ecember 2018 AJCP October 1997

14 23. Mandel H, Brenner B, Berant M, et al. Coexistence of hereditary homocystinuria and factor V Leiden -effect on thrombosis. N Engl] Med. 1996;334: vanboven HH, Reitsma PH, Rosendaal FR, et al. Factor V Leiden (FV R506Q) in families with inherited antithrombin deficiency. Thromb Haemost. 1996;75: Simioni P, Scudeller A, Radossi P, et al. 'Pseudo homozygous' activated protein C resistance due to double heterozygous factor V defects (factor V Lcidcn mutation and type I quantitative factor V defect) associated with thrombosis: report of two cases belonging to two unrelated kindreds. Thromb Haemost. 1996;75: Zoller B, He X, ahlback B. Homozygous APC-resistance combined with inherited type I protein S deficiency in a young boy with severe thrombotic disease. Thromb Haemost. 1995;73: Beauchamp NJ, aly ME, Cooper PC, Makris M, Preston FE, Peake IR. Molecular basis of protein S deficiency in three families also showing independent inheritance of factor v Leiden- Bhod -1996;88: Zehnder JL, Jain M. Recurrent thrombosis due to compound heterozygosity for factor V Leiden and factor V deficiency. Blood Coagul Fibrinolysis. 1996;7: Koeleman BP, van Rumpt, Hamulyak K, Reitsma PH. Bertina RM. Factor V, eiden: an additional risk factor for thrombosis in protein S deficient families?. Thromb Haemost. 1995; 74: Bloemenkamp KWM, Rosendaal FR, Helmerhorst FM, Buller HR, Vandenbroucke J P. Enhancement by factor V Leiden mutation of risk of deep-vein thrombosis associated with oral contraceptives containing third-generation progestagen. Lancet. 1995;346: Hirsch R, Mikkola KM, Marks PW, et al. Pulmonary embolism and deep venous thrombosis during pregnancy or oral contraceptive use: prevalence of factor V Leiden. Am Heart J. 1996;131: Florell SR, Rodger GM. Utilization of testing for activated protein C resistance in a reference laboratory. Am ] Clin Pathol. 1996;106: Greengard JS, Xu X, Gandrille S, Griffin J. Alternative PCR method for diagnosis of mutation causing activated protein C resistant Gln 506 -factor V. Thromb Res. 1995;80: Kirschbaum NE, Foster PA. The polymerase chain reaction with sequence specific primers for the detection of the factor V mutation associated with protein C resistance. Thromb Haemost. 1995;74: Zotz RB, Maruhn B, Scharf RE. Mutation in the gene coding for coagulation factor V and resistance to activated protein C: detection of the genetic mutation by oligonucleotide ligation assay using a semiautomated system. Thromb Haemost. 1996;76: Benson JM, Phillips J, Holloway BP, Evatt BL, Hooper WC. Oligonucleotide ligation assay for detection of the factor V mutation (Arg 506 "'Gln) causing protein C resistance. Thromb Res. 1966;83: Marlar RA, Mastovich S. Hereditary protein C deficiency: a review of the genetics, clinical presentation, diagnosis and treatment. Blood Coagul Fibrinolysis. 1990;1: Tomczak JA, Ando RA, Sobel HG, Bovill EG, George LL. Genetic analysis of a large kindred exhibiting type I protein C deficiency and associated thrombosis. Thromb Res. 1994;74: ACOCK ET AL 447 Evaluating Hereditary Hypercoagulability 39. Rocchi M, Roncuzzi L, Santamaria R, Archidiacono N, ente L, Romeo G. Mapping through somatic cell hybrids and c probes of protein C to chromosome 2, factor X to chromosome 13, and alpha 1-acid glycoprotein to chromosome 9. Hum Genet. 1986;74: Kato A, Miura O, Sumi Y, Aoki N. Assignment of the human protein C gene (PROC) to chromosome region 2ql4-q21 by in situ hybridization. Cytogenet Cell Genet. 1988;47: Patracchini P, Aiello V, Palazzi P, Calzolari E, Bernardi F. Sublocalization of the human protein C gene on chromosome 2ql3-ql4. Hum Genet. 1989;81: Foster C, avie EW. Characterization of a c coding for human protein C. Proc Natl Acad Sci USA. 1984;81: Beckmann RJ, Schmidt RJ, Santerre RF, Plutzky J, Crabtree GR, Long GL. The structure and evolution of a 461 amino acid human protein C precursor and its messenger R, based upon the sequence of cloned human liver cs. Nucleic Acids Res. 1985;13: Suzuki K. Protein C. In: High KA, Roberts HR, eds. Molecular Basis of Thrombosis and Hcmostasis. New York, NY: ekker; 1995:392^ Reitsma PH, Bernardi F, oig RG, et al. Protein C deficiency: a database of mutations: 1995 update. Thromb Haemost. 1995;73: Greengard JS, Fisher CL, Villoutreix B, Griffin JH. Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: patterns revealed by three-dimensional molecular modelling of mutations of the protease domain. Proteins Struct Fund Genet. 1994;18: Aiach M, Gandrille S, Emmerich J. A review of mutations causing deficiencies of antithrombin, protein C and protein S. Thromb Haemost. 1995;74: Gandrille S, Goossens M, Aiach M. Scanning method to establish the molecular basis of protein C deficiencies. Hum Mutat. 1994;4: Ido M, Ohiwa M, Hayashi T, et al. A compound heterozygous protein C deficiency with a single nucleotide G deletion encoding Gly 381 and an amino acid substitution of Lys for Gla 26. Thromb Haemost. 1993;70: Comp PC. Congenital and acquired hypercoagulable states. In: Hull R, Pineo GF, eds. isorders of Thrombosis. Philadelphia, Pa: WB Saunders; 1996: Bertina RM, Reitsma PH, Rosendaal FR, Vandenbroucke JP. Resistance to activated protein C and factor V Lciden as risk factors for venous thrombosis. Thromb Haemost. 1995;74:449^ ahlback B. Protein S and C4b-binding protein: components involved in the regulation of the protein C anticoagulant system. Thromb Haemost. 1991;66: Suzuki K. Protein S. In: High KA, Roberts HR, eds. Molecular Basis of Thrombosis and Hemostasis. New York, NY: ekker; 1995: Aiach M, Gandrille S, Emmerich J. A review of mutations causing deficiencies of antithrombin, protein C and protein S. Thromb Haemost. 1995;74: Formstone CJ, Wacey Al, Berg L-P, et al. etection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy. Blood. 1995;86: Gandrille S, Borgel, Eschwege-Gufflet V, et al. Identification of 15 different candidate causal point mutations and three polymorphisms in 19 patients with protein S deficiency using a scanning method for the analysis of the protein S active gene. Blood. 1995;85: ownloaded from by guest on 18 ecember 2018 Vol. 108 No. 4

15 448 COAGULATION AN TRANSFUSION MEICINE Review Article 57. Gomez E, Poort SR, Bertina RM, Reitsma PH. Identification of eight point mutations in protein S deficiency type I: analysis of 15 pedigrees. Tltromb Haemost. 1995;73: uchemin J, Gandrille S, Borgel, et al. The Ser 460 to Pro substitution of the protein S a (PROS1) gene is a frequent mutation associated with free protein S (type Ha) deficiency. Blood. 1995;86: Zoller B, de Frutos PG, ahlback B. Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type II deficiencies to be phenotypic variants of the same genetic disease. Blood. 1995;85: Lane A, Olds RJ, Boisclair M, et al. Antithrombin III mutation database: first update for the thrombin and its inhibitors. Thromb Haemost. 1993;70: Olds RJ, Lane A, Chowdhury V, estefano V, Leone G, Thein SL. Complete nucleotide sequence of the antithrombin gene: evidence for homologous recombination causing thrombophilia. Biochemistry. 1993;32: Sheffield WB, Wu YI, Blajchman MA. Antithrombin structure and function. In: High KA, Roberts HR, eds. Molecular Basis of Thrombosis and Hemostasis. New York, NY: ekker; 1995: Guba SC, Fink LM, Fonseca V. Hyperhomocysteinemia: an emerging and important factor for thromboembolic and cardiovascular disease. Am ] Clin Pathol. 1996;106: Fermo I, 'Angelo SV, Paroni R, Mazzola G, Caroli G, 'Angelo A. Prevalence of moderate hyperhomocysteinemia in patients with early-onset venous and arterial occlusive disease. Ann Intern Med. 1995;123: Falcon CR, Cattaneo M, Panzeri, Martinelli I, Mannucci PM. High prevalence of hyperhomocysteinemia in patients with juvenile venous thrombosis. Arterioscler Thromb. 1994; 14: en Heijer M, Koster T, Blom HJ, et al. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med. 1996;334: e Stefano Y, Leone G, Mastrangelo S, et al. Clinical manifestations and management of inherited thrombophilia: retrospective analysis and follow-up after diagnosis of 238 patients with congenital deficiency of antithrombin III, protein C, protein S. Thromb Haemost. 1994;72: Pabinger I, Kyrle PA, Heistinger M, Eichinger S, Wittman E, Lechner K. The risk of thromboembolism in asymptomatic patients with protein C and protein S deficiency: a prospective cohort study. Thromb Haemost. 1994;71:441^ Pabinger I, Schneider B. Thrombotic risk in hereditary antithrombin III, protein C, or protein S deficiency: a cooperative, retrospective study. Arterioscler Thromb Vase Biol. 1996;16: Allaart CF, Port RP, Rosendaal FR, Reitsma PH, Bertina RM, Brief E. Increased risk of venous thrombosis in carriers of hereditary protein C defect. Lancet. 1993;341: Nachman RL, Silverstein R. Hypercoagulable states. Ann Intern Med. 1993;119: Bauer KA. Management of patients with hereditary defects predisposing to thrombosis including pregnant women. Thromb Haemost. 1995;74: Pabinger I. Hereditary thrombophilia. Biomed Prog. 1994;7: The British Committee for Standards in Haematology. Guidelines in the investigation and management of thrombophilia. / Clin Pathol. 1990;43: Miletich JP, Prescott SM, White R, Majerus PW, Bovill EG. Inherited predisposition to thrombosis. Cell. 1993;72: Vinazzer H. Clinical use of antithrombin concentrates. Vox Sang. 1987;53: Conard J, Horellou MH, Van reden P, Lecompte M. Samama M. Thrombosis and pregnancy in congenital deficiencies of AT III, protein C or protein S: study of 78 women. Thromb Haemost. 1990;63: Salzman EW, Hirsh J. The epidemiology, pathogenesis and natural history of venous thrombosis. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Philadelphia, Pa: JB Lippincott; 1994: Hull R, elmore T, Genton E, et al. Warfarin sodium versus low-dose heparin in the treatment of venous thrombosis. N Engl J Med. 1979;302: Sarasin FP, Bounameaux H. uration of oral anticoagulant therapy after proximal deep vein thrombosis: a decision analysis. Thromb Haemost. 1994;71: Alving BM, Comp PC. Recent advances in understanding clotting and evaluating patients with recurrent thrombosis. Am J Obstet Gynecol. 1992;167: Pabinger I, Brucker S, Kyrle PA, et al. Hereditary deficiency of antithrombin III, protein C and protein S: prevalence in patients with a history of venous thrombosis and criteria for rational patient screening. Blood Coagul Fibrinolysis. 1992;3: Malm J, Laurell M, ahlback B. Changes in the plasma levels of vitamin K-dependent proteins C and S and c4b-binding protein during pregnancy and oral contraceptives. Br J Haematol. 1988;68: Sanson BJ, Friederich PW, Simioni P, et al. The risk of abortion and stillbirth in antithrombin-, protein C-, and protein S- deficient women. Thromb Haemost. 1996;76: Schwartz RS, Bauer KA, Rosenberg R, Kavanaugh EJ, avies C Bogdanoff A. Clinical experience with antithrombin III concentrate in treatment of congenital and acquired deficiency of antithrombin. Am J Med. 1989;87(suppl 3B):53S-60S. 86. Marlar RA, Adcock M. Protein C replacement therapy as a treatment modality for homozygous protein C deficiency. AdvAppl Biotechnol. 1990;11: Motulsky AG. Nutritional ecogenetics: homocysteine-related arteriosclerotic vascular disease, neural tube defects, and folic acid. Am J Hum Genet. 1996;58: Selhub J, Jacques PF, Wilson PW, Rush, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA. 1993;270: Franken G, Boers GH], Blom HJ, Trijbels FJM, Kloppenborg PWC. Treatment of mild hyperhomocysteinemia in vascular disease patients. Arterioscler Thromb. 1994;14: Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. JAMA. 1995;274: Awidi AS, Abu-Khalaf A, Herzallah U, et al. Hereditary thrombophilia among 217 consecutive patients with thromboembolic disease in Jordan. Am J Hematol. 1993;44: Rodgers GM, Changler WL. Laboratory and clinical aspects of inherited thrombotic disorders. Am J Hematol. 1992;41: Heijboer H, Brandjes PM, Buller HR, Sturk A, Cate JW. eficiencies of coagulation-inhibiting and fibrinolytic proteins in outpatients with deep-vein thrombosis. N Engl J Med. 1990;323: ownloaded from by guest on 18 ecember 2018 AJCP October 1997

16 94. Levy PJ, Gonzales FM, Rush S, Haynes JL. Hypercoagulable states as an evolving risk for spontaneous venous and arterial thrombosis. I Am Coll Surg 1994;178: Bovill EG, Bauer KA, ickerman J, et al. The clinical spectrum of heterozygous protein C deficiency in a large New England kindred. Blood. 1989;73: emers C, Ginsberg JS, Hirsh J, Henderson P, Blajchman MA. Thrombosis in antithrombin-iii-deficient persons: report of a large kindred and literature review. Ann Intern Med. 1992:116: Ridker PM, Hennekens CH, Lindpaintner K, Stampfer MJ, Eisenberg PR. Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N Engl ] Med. 1995;332: Gjores JE. The incidence of venous thrombosis and its sequelae in certain districts of Sweden. Acta Chir Scandl. 956;206: Zoller B, Svensson PJ, He X, ahlback B. Identification of the same factor V gene mutation in 47 out of 50 thrombosisprone families with inherited resistance to activated protein C. / Clin Invest. 1994; 94: Anderson FA Jr, Wheeler HB, Goldberg RJ, et al. A populationbased perspective of the hospital incidence and case-fatality rates of deep vein thrombosis and pulmonary embolism: the Worcester VT study. Arch Intern Med. 1991;151: Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in patients homozygous for factor V Leiden (APC-resistance). Blood. 1995;85: den Heijer M, Blom HJ, Gerrits WJ, et al. Is hyperhomocystaeinemia a risk factor for recurrent thrombosis? Lancet. 1995;345: Tosetto A, Frezzato M, Rodeghierro F. Family history and inherited thrombophilia. Br } Haematol. 1995;89: Rintelen C, Pabinger I, Knobl P, Lechner K, Mannhalter C. Probability of recurrence of thrombosis in patients with and without factor V Leiden. Thromb Haemost. 1996;76: Kitchens CS. Thrombophilia and thrombosis in unusual sites. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Philadelphia, Pa: JB Lippincott; 1994: Comp PC. Elrod JP, Karenski S. Warfarin-induced skin necrosis. Semin Thromb Hemost. 1990;16: Adcock M, Brozna J, Marlar RA. Proposed classification and pathological mechanisms of purpura fulminans and skin necrosis. Semin Thromb Hemost. 1990;16: Broekmans AW, Bertina RM, Loeliger EA, Hofman V, Klingman HG. Protein C and the development of skin necrosis during anticoagulant therapy. Thromb Haemost. 1983;49: Broekmans AW, Teepe RGC, van der Meer FJM, Briet E, Bertina RM. Protein C (PC) and coumarin-induced skin necrosis. Thromb Res. 1987;6(suppl): Friedman K, Marlar RA, Houston JG, Montgomery RM. Warfarin-induced skin necrosis in a patient with protein S deficiency. Blood. 1985;68(suppl l):333a. ACOCK ET AL 449 Evaluating Heredit Hypercoagulability 111. Koepke JA. Testing for hereditary hypercoagulability: activated protein C resistance. Am j Clin Pathol. 1996;106: Vandenbroucke JP, Koster T, Briet E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oralcontraceptive users who are carriers of factor V Leiden mutation. Lancet. 1994;344: Hellgren M, Svensson PJ, ahlback B. Resistance to activated protein C as a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Am f Obstet Gynecol. 1995;173: Florell SR, Rodgers GM. Utilization of testing for activated protein C resistance in a reference laboratory. Am / Clin Pathol. 1996;106: Marlar RA, Adcock M. Clinical evaluation of protein C: a comparative review of antigenic and functional assays. Hum Pathol. 1989;20: Berdeaux H, Abshire TC, Marlar RA. ysfunctional protein C deficiency (type II): a report of 11 cases in 3 American families and review of the literature. Am ] Clin Pathol. 1993;99: Tosetto A, Rodeghiero F. iagnosis of APC resistance in patients on oral anticoagulant therapy. Thromb Haemost. 1995;73: Hathaway WE. Clinical aspects of antithrombin III deficiency. Semin Hematol. 1991;28: Walker FJ. Protein S and thrombotic disease. Proc Soc Exp Biol Med. 1992;200: Miletich JP. Laboratory diagnosis of protein C deficiency. Semin Hemost Thromb. 1990;16: Marlar RA. Protein C in thromboembolic disease. Semin Hemost Thromb. 1985;11: Comp PC, Thurnau GR, Welsh J, Esmon CT. Functional and immunologic protein S levels are decreased during pregnancy. Blood. 1986;68: Weenink G, Treffers PE, Hahle LH, Ten Cate JW. Antithrombin III in normal pregnancy. Thromb Res. 1982;26: Marlar RA, Montgomery R, Broekmans A. iagnosis and treatment of homozygous protein C-deficient children. / Pediatr. 1989;114: Fourrier F, Chopin C, Goudemand J, et al. Septic shock, multiple organ failure, and disseminated intravascular coagulation: compared patterns of antithrombin III, protein C, and protein S deficiencies. Chest. 1992;101: Vigano-'Angelo S, 'Angelo A, Kaufman CE, Sholer C, Esmon CT, Comp PC. Protein S deficiency occurs in the nephrotic syndrome. Ann Intern Med. 1987;107: Marcinial E, Farley CH, esimone PA. Familial thrombosis due to antithrombin deficiency. Blood. 1974;43: Rao AK, Guzzo J, Niewiarowski S, ay HJ. Antithrombin III levels during heparin therapy. Thromb Res. 1981;24: Nichols WL, Heit JA. Activated protein C resistance and thrombosis. Mayo Clin Proc. 1996;71: Pabinger I, Kyrle PA, Speiser W, Stoffels U, Jung M, Lechner K. iagnosis of protein C deficiency on patients on oral anticoagulant treatment: comparison of three different functional protein C assays. Thromb Haemost. 1990;63: ownloaded from by guest on 18 ecember 2018 Vol. 108 No. 4