Thrombophilia is a complex disease process, which clinically expresses as venous thrombosis. The presence of a genetic defect in one of the major contributing components (protein C [PC], protein S [PS], and antithrombin [AT]) to thrombophilia can be determined by clinical laboratory assays. However, understanding the limitations and problems associated with assays is paramount to an accurate analysis of the genetic status. This review will discuss the major analytical issues and provide recommendations for assaying PC, PS, and AT in plasma. Recommendations are also made about pre-analytical and postanalytical issues clinically affecting these assays.
Venous thromboembolism (VTE) is a complex disease (thrombophilia) that is caused by a number of genetic and acquired/environmental risk factors. Multiple risk factors with varying degrees of risk ‘potential’ acting together play a role in causing VTE . To date, three major genes have been identified as having significant relevance in thrombophilia expression: Protein C (PC), Protein S (PS), and Antithrombin (AT). These factors are routinely screened in the investigation for the cause of VTE.
In evaluating the contributions of PC, PS and AT, the only current practical assessment of these protein's genetic status is by laboratory assay of their plasma levels (Fig. 1). However, the laboratory phenotype (measured plasma level of the protein) is not always an accurate measure of the actual genotype . In addition, the laboratory phenotype does not always predict the potential for VTE formation (clinical phenotype) (Fig. 1) . However, if the plasma level of one of these proteins is accurately measured under the appropriate conditions, then an abnormal level is a good refection of the genotype .
All laboratory tests can be divided into three stages: pre-analytical, analytical, and postanalytical. Each stage has aspects that must be controlled for accurate results. This manuscript will discuss primarily the analytical stage of PC, PS, and AT assays, while summarizing the pre-analytical and postanalytical stages, and provide recommendations and references to reviews or documents for relevant details.
Patient variation deals with the pathological or pharmacological variables that can significantly affect the laboratory assays . The plasma levels of PC, PS, and AT in patients at the time of diagnosis of VTE may be consumed, thus leading to erroneously abnormal levels and an incorrect diagnosis [3, 4]. Other consumptive processes, liver disease, diabetes, and some renal disorders can also lead to a false diagnosis [3, 4]. Another main patient-related issue is measuring levels while using anticoagulants. Warfarin, heparin, and direct oral anticoagulants (DOAC) can cause significantly different values, again resulting in an erroneous diagnosis. Laboratory analysis of PC, PS, and AT for determination of a genetic deficiency should not be undertaken when pathological or pharmacological interferences might cause erroneous diagnosis to be made [3, 4]. Improper specimen or sample handling (obtaining and processing the specimen, transporting and storing the sample) can cause erroneous results and incorrect diagnosis (see detailed discussions in ref. #3–5). Routine assays (PT, PTT, and thrombin time) should be considered to rule out acquired abnormalities and anticoagulants before assaying for PC, PS, or AT.
Patient results are determined by comparison to a calibration curve of known concentrations. In the coagulation field, values should be based on an international standard (e.g., WHO) expressed as an international unit (IU); however, some laboratories report results as a percent of a reference population (adult mixed gender healthy population) [6, 7]. Currently PC, PS, and AT values are based on an international reference standards and are expressed as IU/mL.
The interpretation of results for PC, PS, and AT is made by a ‘comparative decision-making process’ . In this method, the patient's result is compared to the appropriate population-based reference interval [6, 7]. Reference intervals for AT, PC, and PS are established for a normal healthy adult population. Reference intervals for specific populations are available and should be used for specific groups. PC, PS, and AT reference intervals specific for neonatal and pediatric populations should be used for these age groups . All results are expressed in adult values and then the patient's result is compared to the specific reference interval of the appropriate age reference interval . Based on the PS method, adult gender-specific reference intervals may need to be established for PS; however, some laboratories have found these differences are not significant for reporting male- and female-specific reference intervals. The decision to use two gender-specific adult ranges should be decided by the Coagulation Laboratory Director [6, 7, 9, 10]. Reference intervals for AT, PC, and PS should not be established for pregnant patients, patients receiving anticoagulation, or patients with thrombotic or other pathologic abnormalities. The variations within these groups are too great and could lead to an incorrect diagnosis and inappropriate treatment .
Protein C Assays
Protein C is a vitamin K-dependent serine protease proenzyme that is converted to activated protein C (APC) by the thrombin and thrombomodulin complex. Upon activation, APC forms a complex with protein S on a phospholipid surface to rapidly inactivate factors Va and VIIIa. Clinically, heterozygous deficiencies of PC lead to increased risk of VTE , whereas homozygous PC deficiency results in significant purpura fulminans, DIC, and death in the newborn . The prevalence of heterozygous PC deficiency in the general population is approximately 1 in 300; however, the prevalence of VTE symptomatic PC deficiency is 1 in 3000–5000 . The mechanism causing the different asymptomatic and symptomatic patients is unknown. Physiologically, plasma levels of PC are significantly lower for neonates and children, and must be taken into account when comparing results to the adult reference interval . Age-specific ranges for newborns and children up to the age of 13 years must be used when assessing these patients .
The PC molecule consists of five functional domains. Genetic defects can occur in the different domains, creating difficulty in accurately diagnosing PC deficiency by laboratory assay [13, 14]. Quantitative defects (Type I) are the most common (75–80%) and are relatively easy to diagnose . Qualitative defects in the activation site and the active site (Type IIa) are also relatively easy to detect [13-15]. Difficultly ascertaining defects in the PC substrate, surface or cofactor binding (Type IIb) are problematic and dependent on the type of assay used [13-15]. Of the 20–25% of PC deficiencies that are qualitative defects, the majority (24.5%) are Type IIa and the remaining Type IIb PC deficiencies constitute about 0.5–1.0% of all deficiencies. There appears to be no difference in clinical expression of phenotype (thrombosis) between the Type I and Type II defects of PC [13, 15].
PC activity can be measured in plasma by either a clotting-based assay or chromogenic (amidolytic)-based assay (Table 1) . In commercial assays, both clotting and chromogenic types of assays use a snake venom activator to convert the plasma PC to APC . The chromogenic assay is based on the addition of chromogenic substrate to the generated APC in the diluted plasma sample [15, 17]. The formation of chromogenic color is proportional to the amount of APC (hence PC) in the plasma sample . This assay only analyzes two of the functional domains of PC: activation and active site (Type IIa) [13, 17]. Defects in cofactor binding, surface binding, and other substrate binding regions (Type IIb) are not detected (Table 1) . An additional issue associated with chromogenic assays is potential overestimation of PC activity because of the lack of specificity of the chromogenic substrate . If a plasma sample is partially activated, PC levels may appear artificially increased. These increased levels can be detected if a chromogenic blank is used for comparison. Underestimation is a possibility with samples with significant hemolysis, lipemia, and icterus [4, 5]. A major concern of chromogenic PC assays is the overestimation of the physiological levels of PC for patients on vitamin K antagonist (VKA). Vitamin K induces a carboxy PC molecule that is nonfunctional in vivo but is measured with the chromogenic assay. The chromogenic assay is not a measure of all functional domains of PC . PC levels may be approximately twice the level of the clot-based assay .
Table 1. Detection of qualitative and quantitative deficiencies by laboratory-testing methods
Deficiency type detected
May or may not detect this type of deficiency without modification of the method.
The clotting-based assays utilize mainly the PTT, but assays using the PT or RVVT have been developed. The PTT-based assays are not influenced by the PTT activator, but are or can be by the phospholipid source and concentration (rare defects) [14, 17]. In addition, increased levels of factor VIII and factor VLeiden underestimate, and increased levels of PS can overestimate the PC value in plasma [14, 17]. These influences can be eliminated by dilution of the plasma with PC-deficient plasma; however, this modification reduces the lower limit of PC detection and increases assay cost .
PC antigenic assays determine the presence of PC molecules, but not its function or activity. The role of the PC antigen assay is to distinguish between Type I and Type II deficiencies when a defect in PC function is found (Table 1) [13, 15]. Commercially available and laboratory-developed tests for PC antigen include ELISA or radial immunodiffusion (RID) methodologies . Comparison of the ratio of PC activity and PC antigen values can differentiate Type I from Type II .
Protein S Assays
Unlike the other vitamin K-dependent plasma proteins, PS is not a serine protease, but functions as a cofactor in the protein C system . Cofactor PS enhances APC inactivation of factors V and VIII . Measurement of PS is complicated further by the fact that a PS is partitioned in plasma between free functional PS and the portion bound to the complement protein, C4b-binding protein (C4b BP) . In the healthy individual, about 40% is free and functions as the cofactor of APC [10, 11].
Clinically, heterozygous deficiencies of PS lead to increased risk for VTE. The overall prevalence of heterozygous PS deficiency in the general population is approximately 1 in 300–400, but the prevalence of VTE symptomatic PS deficiency is significantly less at 1 in 3000–6000 . As in PC deficiency, the mechanism for the difference between asymptomatic and symptomatic patients is unknown . In pregnancy, birth control hormone use, inflamation and other acquired conditions, plasma levels of functional PS are significantly lower . This must be taken into account when comparing the reference intervals . PS determination should not be performed on women who are pregnant or receiving birth control hormones or hormone replacement medications [19, 20]. In addition, patients receiving VKA should also not be tested for PS. Heterozygous defects of PS can be subdivided into three types: two quantitative defects (Type I and Type III ) which are the most common (80% and 15%, respectively) and qualitative defects (Type II) with about 5% incidence [10, 11]. Difficultly diagnosing the different types is problematic and dependent on the type of assay used . There appears to be no difference in clinical phenotype (VTE) between the different PS types [10, 11].
PS levels are difficult to ascertain as PS is a cofactor for the APC pathway and can be bound to C4b BP [10, 18]. The PS activity assay is determined using a clotting-based assay to measure the cofactor activity. The unbound (free) portion of PS is a surrogate measure of PS activity and can be determined with a free PS antigen assay [10, 15]. The third clinical assay for PS is an antigen assay measuring both free and bound PS antigen (termed total PS antigen) . The PS activity assay can be PT- or RVVT–based, but most commonly it is PTT-based [10, 15]. PS activity assay can detect both of the quantitative PS deficiencies and the Type II dysfunctional molecules (Table 1) [10, 15]. The amount of PS activity is proportional to the clotting value. Increased levels of factor VIII and factor VLeiden can underestimate the level of PS in plasma [10, 15, 18]. Dilution of the plasma with PS-deficient plasma can eliminate these variables . Lupus anticoagulant can either over- or underestimate the PS levels in the PTT-based assays. The most troubling aspect of the PS activity assay is the sporadic decreased levels in normal patients, reverting to normal when repeated [10, 15, 19]. The cause of this phenomenon is unknown. It creates a high percent of abnormal results when using the activity assay as the primary screening test [10, 19]. The PS activity assay is not recommended for screening or initial assessment of PS levels [10, 15, 19].
PS antigen assays determine the presence of PS molecules in the plasma, but not PS function [10, 15]. Using free PS antigen as a surrogate assessment of the PS activity, the majority of PS deficiencies (Types I and III) can be diagnosed (Table 1) [10, 15, 18, 19]. Free PS antigen is usually determined by latex particle turbidimetric methods or ELISA . Clinically available free PS antigen assays utilize different methodologies [10, 15]. A common method employs a monoclonal antibody recognizing only the free form of PS and not the bound form. The second method is much more complicated as it uses PEG to precipitate the bound PS fraction leaving only the free PS in solution. The PEG method has numerous technical issues. Other unique methodologies have been developed and used in the clinical laboratory. Neither of these antigenic methods for free PS antigen will detect Type II defects [10, 15].
The measurement of total PS antigen is performed using the same methods as free PS antigen but the detection antibody recognizes both bound and free forms of PS [10, 15]. The clinical value of determining the total PS antigen is in debate (Table 1) [10, 15]. Many laboratories do not perform this assay as it adds cost, but very little clinical information for treatment of the PS-deficient patient. Commercial assays and laboratory-developed tests have been developed for PS antigen, including ELISA or latex particle turbidimetric assay [10, 15, 18]. These activity and antigenic assays are used to distinguish between Type I and III or Type II defects (Table 1) [10, 18]. The ratios of PS activity, free and total PS antigen values are compared to differentiate the types (Table 1) [10, 15, 18].
AT, a serine protease inhibitor, regulates the coagulation enzymes, mainly factor Xa and thrombin . The activity of AT is significantly increased by heparin . Clinically, patients with a heterozygous deficiency of AT manifest with VTE, but are fairly uncommon in the general population (1 in 5000) [21, 22]. Acquired deficiencies of AT are associated with hypercoagulable or consumptive states . Genetic defects of AT occur in different functional domains making some defects difficult to accurately diagnose [21, 22]. Quantitative defects (Type I) or defects in the reactive site of AT (Type IIa) are the easiest to determine as they do not react with the enzyme in the assay (Table 1) . In contrast, defects in the heparin-binding site (Type IIb or Type II–HBS) and pleiotropic defects (Type IIc) may be more diagnostically problematic (Table 1) [15, 21, 22].
In the evaluation of a thrombophilic individual, a chromogenic (amidolytic) assay should be the initial or screening assay [15, 21, 22]. Upon determination and confirmation of a decreased AT activity level, an AT antigenic assay will differentiate between Type I and Type II (Table 1) . For nonclinical purposes, Type II deficiencies can be further subtyped using specialized assays [15, 22].
Chromogenic assays for AT are accurate, precise, reproducible, and automated. The AT assay is based on either factor Xa or thrombin as the enzymatic source [21, 22]. The enzyme is incubated for a standard time interval with diluted plasma and heparin. After the chromogenic substrate is added, the color generated is proportional to the residual enzyme. Hence, the amount of AT in the sample is inversely proportional to color generated by the residual enzyme. Either human or bovine factor Xa is the enzymatic source in most commercial assays. However, thrombin from either bovine or human sources is also used. It should be noted that overestimation of AT activity due to heparin cofactor II (a second AT-like molecule found in plasma) is observed when human thrombin is the basis of the AT assay [15, 21-23].
Difficulties in the diagnosis of Type IIb deficiencies or Type IIc (pleiotropic) AT defects may arise when factor Xa is the enzymatic source (Table 1) [21, 22, 24]. Either reducing the enzyme or shortening the incubation time can help to determine these types of unique deficiencies [22, 24]. Oddly enough, bovine thrombin used as the enzymatic source may be able to detect these rare disorders better than human thrombin or factor Xa (Table 1) [22, 24]. There is no single assay configuration (enzyme, dilution, and chromogenic substrate) that can identify all AT Type II defects (Table 1) [15, 21, 25]. The rarely performed ‘progressive’ AT assay in which the diluted plasma is incubated with the enzyme without heparin can determine only Type I and Type IIa [21, 25, 26].
AT antigenic assay determines the presence of AT molecule, but not AT activity [15, 21, 26]. This assay is used to determine Type I or Type II defects (Table 1). A range of assay methods for AT antigen is available including ELISA, latex particle turbidimetric assay, or RID [15, 21, 26]. The ratio of activity and antigen values is compared to differentiate Type I from Type II.
Patient variability, pre-analytical variables, and postanalytical issues may cause an erroneous diagnosis. These problematic sources must be kept to a minimum to ensure accurate results.
Laboratory diagnosis of decreased or abnormal levels of PC, PS, or AT does not always reflect a genetic deficiency. Decreased levels also do not confirm clinical phenotype expression (VTE). Repeat abnormal values in 4–8 weeks to confirm a deficiency.
Not all commercial kits or laboratory-developed tests measure the plasma protein in the same manner. Normal or abnormal values on the same sample or patient may be generated with different kits or assay methods.
Testing at incorrect times (immediate post-thrombosis or on anticoagulation therapy) may result in inaccurate results.
Consider performing PT and PTT assays to rule out acquired abnormalities or artifacts that may impact true PC, PS, or AT values. However, this additional assays increase the overall cost of the assays.
Testing for PC, PS, or AT deficiencies in the presence of anticoagulants may result in inaccurate results.
Testing for PC, PS, or AT deficiencies in the presence of acquired abnormalities or conditions (such as lupus anticoagulant, DIC, or surgery) may result in inaccurate results.
Comparison of patient's results to the correct reference interval will result in the correct interpretation.
Protein C testing
To determine the presence of PC deficiency, use either a chromogenic or clot-based assay (Table 2). The chromogenic PC assay is recommended for cost savings.
Table 2. Recommended tests for protein C, protein S, and antithrombin in the thrombophilia panel
Component to determine
Primary screening assay
Reflex if abnormal
AT activity – factor Xa-based
PC activity – chromogenic or clotting-based
Free PS antigen
PS activity (total PS antigen)
If the PC activity is abnormal, then a PC antigen assay is performed to determine Type I or Type II (Table 2).
Patient's age must be taken into account for final diagnosis. Pediatric and newborn reference intervals are lower than adult ranges.
PC assays should not be performed when patient is on vitamin K antagonists (VKA) or DOAC.
Protein S testing
The initial or screening assay should be a free PS antigen assay (Table 2).
The initial or screening assay should not be a PS activity assay.
If the free PS antigen assay is abnormal, then PS activity and total PS antigen assays should be performed to determine the deficiency type (Type I, Type II, or Type III) (Table 2).
For the determination of PS deficiency, compare the patient's value to age-appropriate reference interval and gender-specific reference intervals depending on the PS assay.
PS assays should not be performed during pregnancy, hormone therapy, vitamin K antagonist (VKA), or DOAC therapy.
Screening for AT deficiency, a chromogenic assay using factor Xa or thrombin and heparin is recommended (Table 2).
If the activity assay is abnormal, then an AT antigen assay is performed. The ratio confirms Type I or Type II deficiency (Table 2).
If Type II deficiency, further specialized testing can be performed to determine the Type II subclass (reactive site, heparin-binding site, or pleiotropic).