Study type: Blood collected in a completely filled blue-top [3.2% sodium citrate] tube; related body system: Circulatory/Hematopoietic system. If the patient’s hematocrit exceeds 55%, the volume of citrate in the collection tube must be adjusted. The collection tube should be completely filled. Important note: When multiple specimens are drawn, the blue-top tube should be collected after sterile (i.e., blood culture) tubes. Otherwise, when using a standard vacutainer system, the blue-top tube is the first tube collected. When a butterfly is used, due to the added tubing, an extra red-top discard tube should be collected before the blue-top tube to ensure complete filling of the blue-top tube. Promptly transport the specimen to the laboratory for processing and analysis. The recommendation for processed and unprocessed samples stored in unopened tubes is that testing should be completed within 1 to 4 hr of collection.

NOTE: It has been documented that clot based detection methods for coagulation factor assays produce unreliable results when performed on specimens of patients being treated with direct oral anticoagulants (DOACs) (e.g. apixaban, betrixaban, dabigatran, edoxaban, etexilate, rivaroxaban) as these drugs interfere with clot based or chromogenic coagulation studies. In general, DOACs prolong the results of many clot based assays by decreasing either thrombin or factor Xa activity; some clot based assays are unaffected because there is no involvement of thrombin or factor Xa. DOACs prolong clotting assays to varying degrees based on a number of factors that include:

• the clotting assay being performed
• type of reagents used in the clotting assay
• DOAC present in the test sample
• concentration of DOAC present in the test sample

The Process of Hemostasis

Hemostasis involves three components: blood vessel walls, platelets, and plasma coagulation proteins. Primary hemostasis has three major stages involving platelet adhesion, platelet activation, and platelet aggregation.

• Platelet adhesion is initiated by exposure of the endothelium as a result of damage to blood vessels. Exposed TF-bearing cells trigger the simultaneous binding of von Willebrand factor to exposed collagen and circulating platelets.
• Activated platelets release a number of procoagulant factors, including thromboxane, a very potent platelet activator, from storage granules. These factors enter the circulation and activate other platelets, and the cycle continues.
• The activated platelets aggregate at the site of vessel injury, and at this stage of hemostasis, the glycoprotein IIb/IIIa receptors on the activated platelets bind fibrinogen, causing the platelets to stick together and form a plug.

Simultaneously, the coagulation process or secondary hemostasis occurs. In secondary hemostasis, the coagulation proteins respond to blood vessel injury in an overlapping chain of events. The contact activation and TF pathways (also known as the intrinsic and extrinsic pathways, respectively) of secondary hemostasis are a series of reactions involving the substrate protein fibrinogen, the coagulation factors (also known as enzyme precursors or zymogens), nonenzymatic cofactors (Ca²⁺), and phospholipids. The factors were assigned Roman numerals in the order of their discovery, not their place in the coagulation sequence. Factor VI was originally thought to be a separate clotting factor. It was subsequently proved to be the same as a modified form of factor Va, and therefore, the number is no longer used.

There is a balance in health between the prothrombotic or clot formation process and the antithrombotic or clot disintegration process. The antithrombotic process includes tissue factor pathway inhibitor (TFPI), antithrombin, protein C, and fibrinolysis.

The coagulation factors are formed in the liver. They can be divided into three groups based on their common properties:

1. The contact group is activated in vitro by a surface such as glass and is activated in vivo by collagen. The contact group includes factor XI, factor XII, prekallikrein, and high-molecular-weight kininogen.
2. The prothrombin or vitamin K– dependent group includes factors II, VII, IX, and X.
3. The fibrinogen group includes factors I, V, VIII, and XIII. They are the most labile of the factors and are consumed during the coagulation process. The factors listed in the table are the ones most commonly measured.

For many years, our understanding of the process of coagulation has been explained by the traditional model (the coagulation cascade), comprised of the intrinsic and extrinsic pathways. While this model is still valid, it is used more often to identify the role of different coagulation factors in the clotting process than to explain the actual process of coagulation.

The cellular-based model portrays a more dynamic concept than the traditional linear cascade model. It includes four overlapping phases in the formation of thrombin: initiation, amplification, propagation, and termination. It is now known that the TF pathway is the primary pathway for the initiation of blood coagulation. TF-bearing cells (e.g., endothelial cells, smooth muscle cells, monocytes) can be induced to express TF and are the primary initiators of the coagulation cascade either by contact activation or trauma.

The contact activation pathway is more related to inflammation, and although it plays an important role in the body’s reaction to damaged endothelial surfaces, a deficiency in factor XII does not result in development of a bleeding disorder, which demonstrates the minor role of the intrinsic pathway in the process of blood coagulation. Substances such as endotoxins, tumor necrosis factor alpha, and lipoproteins can also stimulate expression of TF. TF, in combination with factor VII and calcium, forms a complex that then activates factors IX and X in the initiation phase. Activated factor X in the presence of factor II (prothrombin) leads to the formation of thrombin. Tissue factor pathway inhibitor (TFPI) quickly inactivates this stage of the pathway so that limited or trace amounts of thrombin are produced, which results in the activation of factors VIII and V. Activated factor IX, assisted by activated factors V and VIII, initiates amplification and propagation of thrombin in the process. Thrombin activates factor XIII and begins converting fibrinogen into fibrin monomers, which spontaneously polymerize and then become cross-linked into a stable clot by activated factor XIII.

Qualitative and quantitative factor deficiencies can affect the function of the coagulation pathways. Factor V and factor II (prothrombin) mutations are examples of qualitative deficiencies and are the most common inherited predisposing factors for blood clots. Hemophilia A is an inherited deficiency of factor VIII and occurs at a prevalence of about 1 in 5,000 to 10,000 male births. Hemophilia B is an inherited deficiency of factor IX and occurs at a prevalence of about 1 in about 20,000 to 34,000 male births. Hemophilia A and B are inherited in an X-linked recessive pattern, a genetic disorder passed from a mother to male children. Genetic testing is available for inherited mutations associated with inherited coagulopathies. The tests are performed on samples of whole blood. Counseling and informed written consent are generally required for genetic testing.

Knowledge of genetics assists in identifying those who may benefit from additional education, risk assessment, and counseling. Genetics is the study and identification of genes, genetic mutations, and inheritance. For example, genetics provides some insight into the likelihood of inheriting a medical condition such as hemophilia. Some conditions are the result of mutations involving a single gene, whereas other conditions may involve multiple genes and/or multiple chromosomes.

The factor V Leiden mutation in the F5 gene is an inherited autosomal genetic disorder that results in thrombophilia and the development of blood clots. Severity of the disorder depends on whether a single F5 mutation is inherited from one parent or two mutations are inherited, one from each parent. The risk of forming abnormal blood clots is also increased in people who have multiple mutations in the F5 gene or mutations in a different gene that also codes for products involved in the coagulation process, including factor V Leiden. Variants to factor II can also affect the coagulation process and increase the risk of developing blood clots. Mutation testing for factors II and V can be performed on blood or buccal swabs. Testing should be considered for patients with a personal or family health history of venous thrombosis, thrombosis at an age of less than 50 years, thrombosis during pregnancy, or taking medications known to increase risk of thrombosis.

Genomic studies evaluate the interaction of groups of genes. The combined activity or combined expression of groups of genes allows assumptions or predictions to be made. As an example, genomic studies measure the levels of activity in multiple genes to predict how they and other factors influence the prolonged development of abnormal blood clots. Other factors that may contribute to the development of blood clots in people with the factor V Leiden mutation include obesity, tissue injury (e.g., from surgery or trauma), smoking, pregnancy, exposure to hormones (e.g., oral contraceptives or hormone replacement therapy), and advancing age. Further information regarding inheritance of genes can be found in the study titled “Genetic Testing.”

The PT/INR measures the function of the tissue factor pathway of coagulation and is used to monitor patients receiving warfarin or coumarin-derivative anticoagulant therapy. The aPTT measures the function of the contact activation pathway of coagulation and is used to monitor patients receiving heparin anticoagulant therapy.