Click here to download the PDF

• Recurrent Pregnancy Loss
• Unexplained Infertility
• Family History of Thrombosis
• Implantation Failure
• Poly Cystic Ovarian Syndrome
• Child with Cerebral Palsy
• High Risk Pregnancy Monitoring
• Pre-eclampsia

In pregnancy, any clotting pathology may be deleterious as fetal survival/growth depends on adequate circulation between the mother and the fetus. While women with thrombophilic defects can have normal pregnancies, Thrombophilia is considered to be a significant risk factor.

Primary Testing Panel

Antithrombin III Antigen

Antithrombin III is a potent inhibitor of multiple reactions in the coagulation cascade (factors XIa, IXa, Xa, and IIa, see figure) and is one of the primary natural anticoagulant proteins. This test determines the amount of antithrombin III in the patient’s plasma utilizing a nephelometric (light scatter) assay of antibody-induced turbidity. In type 1 antithrombin deficiency, the patients have an inadequate amount of antithrombin present in their circulation. In type 2 deficiency, the amount of antithrombin present is normal, but it does not function properly. Note: This test only identifies Type 1 deficiency.

Activated Partial Prothrombin Time (APTT)

APTT is a general screening clot/coagulation time test used primarily to evaluate abnormalities in the enzymes and co-factors of the intrinsic coagulation pathway necessary for prothrombin activation. APTT is especially sensitive to functional deficiencies of factors VIII, IX, XI and XII, Fletcher factor (prekalikrein) or Fitzgerald factor (high molecular weight kinniogen). APTT will be prolonged in coagulation defects (deficiencies) associated with these clotting factors. The presence of anti-coagulants in the blood will also result in a prolonged APTT, therefore patients on anti-coagulant therapy may receive false-positive results.

Factor V Leiden (APC Resistance, APCR)

Coagulation Test: This test measures the enhanced activity of factor V due to the Leiden mutation. This coagulation-based assay determines the APCR ratio using a DRVVT clotting test performed with and without activation of the endogenous Protein C by Southern Copperhead venom (SCV). The APCR ratios are calculated as DRVVT with SCV divided by DRVVT with saline. Low APCR ratios are indicative of the factor V Leiden mutation.

Background: Factor V is a pro-coagulatory protein in the common clotting pathway. In patients with a point mutation (G1691A) within the APC cleavage site in the factor V gene (factor V Leiden), APC can no longer cleave and inactivate factor V, resulting in prolonged coagulation events.

Hyper-Homocysteinemia (HHC)

Fasting Homocysteine: This test measures the amount of homocysteine in the patient’s serum utilizing HPLC. This test must be done after fasting, as fluctuations with diet are prevalent. Samples with elevated homocysteine levels may be reflexed to the MTHFR test to identify a putative cause for HHC.

Background: Homocysteine is a biochemical metabolite that can act as a pro-coagulatory protein. Among other functions, it activates the pro-coagulatory factor V, interferes with activation of the anti-coagulatory Protein C, suppresses expression of the anti-coagulatory thrombomodulin, and blocks the pro-fibrinolytic tissue plasminogen activator. Therefore, increased levels of homocysteine (hyper-homocysteinemia, HHC) represent a significant risk factor for T hrombophilia, and may indicate a deficiency in folate metabolism, which has been associated with neural tube defects.

Lupus Anticoagulant (LAC)

Lupus anticoagulant, an acquired defect, is a pro-coagulatory factor in the intrinsic clotting pathway. The LAC test (a modified Dilute Russell Viper Venom Test [DRVVT]) is a sensitive coagulation test used to specifically detect the presence of lupus anticoagulant in patient’s plasma. The presence of lupus anticoagulant in patient’s plasma is detected by a prolonged clotting/coagulation time.

Protein C Deficiency

Protein C Activity: This test measures the activity of the anti-coagulatory protein, Protein C, present in the patient’s plasma utilizing a clotting assay. The prolongation of clotting is proportional to the activity of Protein C in the plasma.

Background: Activated Protein C (APC) binds Protein S and inactivates coagulation factor V, limiting further coagulation. Protein C deficiency will lead to inadequate degradation of factor V and progression of hyper-coagulatory status. Protein C deficiency results from inadequate amounts of Protein C synthesis (type I) or synthesis of defective Protein C molecules (type II).

Protein S Deficiency

Protein S Activity: This test measures the activity of the anti-coagulatory protein, Protein S, present in the patient’s plasma utilizing a clotting assay. The prolongation of clotting is proportional to the activity of Protein S in the plasma.

Background: Similar to protein C deficiency, Protein S deficiency will lead to inadequate/lack of degradation of factor V and progression of hyper-coagulatory status. Protein S deficiency results from inadequate amounts of Protein S synthesis (type I), synthesis of defective Protein S molecules (type II), or inadequate amounts of the free form of Protein S, but normal total levels of protein S (type III) [Protein S exists in two forms – only free form can bind to APC and function as a co-factor].

Plasminogen Activator Inhibitor (PAI-1)

PAI-1 Activity: This test measures the binding activity of PAI-1 in the patient’s plasma utilizing an ELISA-based assay that uses tPA as a capture protein to capture active PAI-1, which is then detected with antibodies. This test will not detect PAI-1 in its inactive form.

Background: PAI-1 is an anti-fibrinolytic protein, which when overexpressed results in thrombophilia as it inhibits the natural breakdown of clots after the blood vessels are repaired. PAI-1 disease is caused by defects in the PAI-1 gene transcription promoter region. Certain genotypes (4G/4G and 4G/5G) are associated with increased plasma levels of PAI-1 protein.

Prothrombin Mutation G20210A (PTG20210A)

This test measures the increased expression of prothrombin, which is a pro-coagulatory protein in the common clotting pathway. PTG20210A is a mutation in the prothrombin gene promoter that results in higher levels of expression. PTG20210A is diagnosed using a PCR/DNA ligation assay. This molecular test detects the PTG20210A mutation with extremely high sensitivity and specificity and determines the patient’s homozygous or heterozygous status.

Additional Testing Panel

Hyper-Homocysteinemia (HHC)

MTHFR C677T Mutation: One of the most common genetic defects of homocysteine metabolism is a mutation in the enzyme methylene-tetrahydrofolate reductase (MTHFR). The most abundant MTHFR mutation is a nucleotide transition from C-to-T at position 677 (C677T), which results in a non-functional enzyme. This mutation is detected using a PCR/DNA ligation assay. This molecular test detects the C677T mutation with extremely high sensitivity and specificity. While this test determines the patient’s homozygous or heterozygous status, only homozygous mutations result in HHC and can result in pregnancy complications (prevalence of homozygosity in women with obstetric complications is up to 22%).

Background: Homocysteine is a biochemical metabolite that can act as a pro-coagulatory protein. Among other functions, it activates the pro-coagulatory factor V, interferes with activation of the anti-coagulatory Protein C, suppresses expression of the anti-coagulatory thrombomodulin, and blocks the pro-fibrinolytic tissue plasminogen activator. Therefore, increased levels of homocysteine (hyper-homocysteinemia, HHC) represent a significant risk factor for thrombophilia, and may indicate a deficiency in folate metabolism, which has been associated with neural tube defects.

Plasminogen Activator Inhibitor (PAI-1)

PAI-1 antigen: This test compliments the PAI-1 activity test by measuring the amount of PAI-1, both active and inactive, in the patient’s plasma. PAI-1 antigen is quantitated with an ELISA assay.

Background: PAI-1 is an anti-fibrinolytic protein, which when overexpressed results in thrombophilia as it inhibits the natural breakdown of clots after the blood vessels are repaired. PAI-1 disease is caused by defects in the PAI-1 gene transcription promoter region. Certain genotypes (4G/4G and 4G/5G) are associated with increased plasma levels of PAI-1 protein.

Protein C Deficiency

Protein C Antigen: This test compliments the activity test by measuring the amount of Protein C present in the patient’s plasma with an ELISA assay. The results will determine whether the decreased activity is due to a type I or type II deficiency.

Background: Activated Protein C (APC) binds Protein S and inactivates coagulation factor V, limiting further coagulation. Protein C deficiency will lead to inadequate degradation of factor V and progression of hyper-coagulatory status. Protein C deficiency results from inadequate amounts of Protein C synthesis (type I) or synthesis of defective Protein C molecules (type II).

Protein S Deficiency

Protein S Antigen: This test compliments the activity test by measuring the amount of Protein S present in the patient’s plasma with an ELISA assay. Free Protein S is measured on all samples tested. Total Protein S can be measured upon request. Determining both Total and Free Protein S will identify which type of deficiency the patient has.

Background: Similar to protein C deficiency, Protein S deficiency will lead to inadequate/lack of degradation of factor V and progression of hyper-coagulatory status. Protein S deficiency results from inadequate amounts of Protein S synthesis (type I), synthesis of defective Protein S molecules (type II), or inadequate amounts of the free form of Protein S, but normal total levels of protein S (type III) [Protein S exists in two forms – only free form can bind to APC and function as a co-factor].

Factor V Leiden (APC Resistance, APCR)

G1691A Mutation Test: This test is used as a confirmation test for the coagulation test or for those cases where the results from the coagulation test are inconclusive (ratios of 1.5-2.0). This test detects the presence of the single nucleotide point mutation at the site 1691 in the factor V gene that results in a G?A transition using a PCR/DNA ligation assay. This molecular test detects the Factor V Leiden mutation with extremely high sensitivity and specificity and determines the patient’s homozygous or heterozygous status.

Background: Factor V is a pro-coagulatory protein in the common clotting pathway. In patients with a point mutation (G1691A) within the APC cleavage site in the factor V gene (factor V Leiden), APC can no longer cleave and inactivate factor V, resulting in prolonged coagulation events.

Related Tests

Anti-phospholipid antibodies (APA)

Anti-phospholipids are also a significant risk factor for thrombophilic events and recurrent pregnancy loss, and they are often associated with and can enhance/amplify the other thrombophilic defects addressed in this panel. Please consult our literature/flyer on the APA Test.

Expected Ranges for Repromedix Performed Tests

* Clotting time ratio ** +/- and +/+ represent heterozygosity and homozygosity
for mutation, respectively; -\- is wild type (normal)

Overview

Blood coagulation is a vital process that prevents excessive blood loss when a blood vessel gets damaged. A series (cascade) of events happen at the site of vessel injury, ultimately resulting in the formation of a platelet plug and a mesh of cross-linked fibrin referred to as a blood clot or thrombus. The clotting process is initiated by factors such as collagen, tissue factor, and phospholipids. The deposition and cross-linking of fibrin is mediated by the protein-based coagulation cascade, which involves the sequential activation of coagulation factors (depicted above in blue). Mechanisms that restrict excessive or prolonged clotting processes at the site of vessel injury are necessary to maintain normal blood flow and prevent hyper-coagulatory events. While thrombin is a crucial factor in the pro-coagulatory cascade, its generation also leads to the controlled activation of the anti-clotting, negative feedback loop that includes Protein C, Protein S, and thrombomodulin (depicted in red/yellow). Additionally, antithrombin provides a second method by which the coagulation cascade is regulated. Finally, the maintenance of proper blood flow requires the removal of blood clots, fibrinolysis, after the damaged blood vessel has been repaired. This process is mediated by plasmin, which is regulated by plasminogen activator and PAI-1 (depicted above in green). While outside of the coagulation/fibrinolysis cascade, the metabolite homocysteine (in orange) promotes thrombophilia by affecting the function of multiple proteins. A proper balance between the pro- and anti-coagulatory proteins/functions is required in order to maintain hemostasis, which prevents excessive bleeding or clotting. Defects in any part of this coagulation network can shift the balance and result in a pathologic state. These defects can be either inherited (genetic defects) or acquired (autoimmunity).

Clinical Significance

Thrombosis is a pathologic process in which platelet aggregation and fibrin clot formation occur in intact blood vessels and is often associated with heart disease, neuro-vascular complications, pulmonary embolism and edema, inflammation, and necrosis. Excessive coagulatory events lead to disruption in blood supply to the fetus or thrombosis in the placenta and can result in many complications: implantation failure, recurrent pregnancy loss (RPL), fetal death, fetal growth retardation (IUGR), pre-eclampsia and stillbirths. While prevalences vary depending on the exclusion criteria utilized by specific researchers, studies have shown that 40-66% of unexplained RPL are associated with thrombophilic defects, both inherited and acquired. These defects result in excessive clotting by increasing pro-coagulatory agents (LAC, APL, prothrombin, homocysteine), decreasing anti-coagulatory proteins (antithrombin and Proteins C and S), producing a pro-coagulatory factor that can’t be deactivated (factor V Leiden), or increasing anti-fibrinolytic proteins (PAI-1). In addition, increased homocysteine results in reduced flexibility of blood vessels, increasing susceptibility to thrombotic events. Having any one of these defects represents a significant risk factor; however, multiple defects often put a patient at a higher risk of pregnancy complications, with an odds ratio of 2-8 typically shown for each the defect or combination of defects

Literature Support

Follow-Up Therapies

1. Therapeutic regimens of heparin, warfarin, aspirin and low molecular weight heparin have proven effective for some situations. Those who have had multiple thromboembolic episodes or are at high risk for further episodes need to be investigated for multiple defects/deficiencies and given additional medications. Consult with a hematologist about the use of anti-coagulation medication may be appropriate. The efficacy of anti-coagulant treatment of thrombophilia on pregnancy outcome has been addressed in a clinical trial published by Grandone et al [Fert. & Ster. 78(2):371, 2002].
2. Homocysteinemia could be treated by supplementation with folic acid, Vitamin B6 and B12.

Testing Services Available

Primary Test Panel – Repromedix offers an economical, broad ranged, and comprehensive screen test panel for women who may have Thrombophilia disorders resulting in pregnancy complications

Additional Test Panel - Abnormal results from the primary tests may be reflexed to additional and advanced testing that confirms diagnosis at the molecular and antigenic levels. The physician will have the option of ordering these tests in advance or requesting reflex testing automatically.

*** Sample integrity is vitally important. Please call Client Services (781-937-8893) for detailed shipping instructions and supplies.

86-88 Cummings Park, Woburn, MA 01801
Telephone: (800) 667-8893 / (781) 937-8893
FAX: (781) 937-8895
Web: www.repromedix.com
lab@repromedix.com