Coagulation Tests

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Coagulation Tests


Hemostasis has been described as "a process by which the body spontaneously stops bleeding and maintains blood in the fluid state within the vascular compartment." There are at least four major systems that are involved in this complex process: the vasculature system, the platelets, the fibrin-forming system, and the fibrin-lysing system. Hemostasis has also been further segregated into stages or steps. These are primary hemostasis, which is the interaction of the injured blood vessel and platelets; secondary hemostasis, which is referred to historically as the blood coagulation process; and tertiary hemostasis, which is the process of fibrinolysis (clot destruction). The first two stages are assessed by blood coagulation tests that evaluate platelets, circulating coagulation factors, and blood vessels.


Coagulation tests are performed to aid in the diagnosis of bleeding disorders, monitor the effectiveness of anticoagulant therapy, and confirm a patient's blood clotting status prior to surgery.


Before administering the test, the patient should be asked to list the medications he or she is taking, and whether or not he or she has recently experienced active bleeding, acute infection or illness, or undergone a blood transfusion, as these factors could adversely affect their coagulation test results. Some of the medications that can affect coagulation results include antacids, antibiotics, anticoagulants, antihistamines, aspirin, diuretics, nicotine, nonsteroidal anti-inflammatory drugs, oral contraceptives, steroids, tranquilizers, and vitamins C and K. If a patient is currently being medicated, the testing facility should be contacted to determine the acceptability of the blood sample. From the moment the blood specimen is drawn until the test is completed, the handling of the specimen is critical in coagulation studies. Assuming that the specimen is drawn correctly, the following procedures should be followed.

Collection of blood for coagulation tests

There is a generally held belief that the first tube in a case where multiple specimens are drawn should never be used for any hemostasis assay, because tissue thromboplastin from the initial venipuncture may affect coagulation test results. In addition, the tube(s) for coagulation testing should be filled before any tubes containing EDTA. If coagulation tests are the only studies ordered, a discard tube should be drawn before filling the blue-stoppered citrate tube used for the tests.

Anticoagulant for coagulation tests

The anticoagulant of choice for coagulation testing is sodium citrate, which reversibly chelates calcium. Evacuated blue-top tubes containing sodium citrate are available commercially with a 3.8% (129 mmol/L) or 3.2% (109 mmol/L) citrate concentration. Blue-stoppered tubes are manufactured to draw nine parts of whole blood to one part of liquid sodium citrate already present in the tube. Thus, when using an evacuated system, blood must be allowed to flow into the tube until it stops automatically. This provides for the 90% fill ratio required for coagulation testing. Ideally the plasma specimen is tested within four hours of collection. If this is not possible then the samples should be frozen until testing. Gross hemolysis is usually a criterion for sample rejection.


The first or primary event that stops bleeding from a very small wound is the formation of a platelet plug, which seals the hole in the vessel wall. This is followed by arteriolar vasoconstriction. The plug in turn is strengthened by fibrin strands. Exposure to collagen and subendothelial components is thought to be the trigger that causes the platelets to aggregate and form the primary plug. Aggregation has been proven to be dependent on the Von Willebrand factor and other plasma factors, such as ADP release from lysed red cells or platelets after exposure to collagen. A defect in one of these plasma factors, a qualitative defect in platelets, a reduced number of platelets (thrombocytopenia), or a defect in the blood vessel wall can result in failure of the primary hemostatic stage, causing spontaneous bleeding or purpura.

The blood clotting or coagulation system is a proteolytic cascade. Each enzyme of the pathway is present in plasma in an inactive (zymogen) form, which, when activated, releases the active factor from the precursor molecule. An active factor then "switches on" the next factor. The mechanism functions as a series of positive and negative feedback loops to effectively control the process. The main aim of this process is to produce thrombin, which can convert soluble fibrinogen into insoluble fibrin, thus forming the clot.

There are three phases of coagulation, the intrinsic and extrinsic (tissue factor) pathways that provide alternative routes for the generation of factor X and the final common pathway that results in thrombin formation. The distinction between the intrinsic and extrinsic pathways is important for understanding the laboratory tests of coagulation. It is not relevant for the real-life process of blood clotting in the body, which may involve both pathways to different extents. Experts are unclear as to which pathway is involved in venous thromboembolic diseases such as deep vein thrombosis and pulmonary embolism.

Bleeding disorders

According to the cascade theory, each coagulation factor is converted to its active form by the preceding factor in a series of biochemical chain reactions. If there is a deficiency of any one of the factors, coagulation cannot proceed at a normal rate, initiation of the next reaction is delayed, and the time required for clotting is prolonged, resulting in bleeding from injured vessels for a longer time. Bleeding disorders can be either acquired or inherited (congenital). Von Willebrand's disease is the most common hereditary coagulation disorder; it results in problems with platelet quality and can cause mild to moderately severe bleeding. Inherited disorders also include hemophilia A and B, which are associated with a decrease in factor VIII or IX activity, respectively. Because the liver produces many of the coagulation factors, diseases that affect the liver may be associated with clotting abnormalities. Disseminated intravascular coagulation (DIC), anticoagulant therapy, and thrombocytopenia also increase bleeding tendencies.

There are several tests available to aid in the diagnosis of bleeding disorders and monitor anticoagulation therapy, and the most common are listed below.

Platelet count

Platelets are disk-shaped structures formed by the detachment of cytoplasm from megakaryocytes. They aid in the coagulation process by attaching or adhering to the walls of injured blood vessels, where they stick together to form the initial platelet plug. A low platelet count may occur in patients with AIDS, viral infections, lymphoma, and lupus erythematosus, or in patients taking certain drugs, most notably quinine and quinidine. Decreased platelet production is also a cause of thrombocytopenia, and may be due to aplastic anemia, leukemia, lymphoma, or bone marrow fibrosis.

When collecting a specimen for a platelet count, EDTA is the anticoagulant of choice. The test is most commonly performed on an automated instrument which employs impedence measurement to count platelets, red blood cells, and white blood cells. Impedence counting is also known as the Coulter principle. According to Beckman Coulter, a major manufacturer of cell counters, the methodology works as follows: "A small opening (aperture) between electrodes is the sensing zone through which suspended particles pass. In the sensing zone each particle displaces its own volume of electrolyte. Volume displaced is measured as a voltage pulse; the height of each pulse being proportional to the volume of the particle. Several thousand particles per second are individually counted and sized with great accuracy." Automated cell counters also report the Mean Platelet Volume (MPV) or Platelet Distribution Width (PDW), two indices used to determine the relative size of the platelets being counted. Since a variety of factors can affect results (e.g., fibrin clots in EDTA blood, platelet clumping in capillary samples), a platelet estimate can also be performed to verify count accuracy. This is done by counting the platelets seen on a blood film by microscopic examination. Each platelet seen per field is estimated to be equivalent to 20,000 platelets/μL. This means 10 platelets per field would correspond to a platelet count of 200,000/μL. When results are questionable, manual platelet counts may be performed using light or phase contrast microscopy. The procedure is as follows: the sample is first treated with 1% ammonium oxalate counting fluid, then charged into a Neubauer hemacytometer chamber. The chamber is placed in a moist Petri dish for 10 minutes to allow the cells to settle. The chamber is then placed on the microscope platform and the platelets in the 25 small center squares of the chamber grid are systematically counted. The number obtained is multiplied by 1,000 to give the platelet count per microliter of blood.

Thrombin time

This test is used to screen for abnormalities in the conversion of fibrinogen to fibrin. These may be caused by qualitative or quantitative abnormalities of fibrinogen or by inhibitors, such as heparin or fibrin/fibrinogen split products. The principle of the thrombin time test is that the exogenous addition of thrombin to plasma converts fibrinogen to fibrin and bypasses both the intrinsic and extrinsic pathways. The time it takes for the patient's plasma to clot on addition of thrombin, referred to as the thrombin time, is a function of fibrinogen concentration. The test procedure involves adding one part of a known concentration of thrombin to one part of the patient's citrated plasma and the clotting time is recorded. A reference range should be established by each testing facility, but a range of 14.0 to 20.0 seconds is considered normal.

Prothrombin time (PT)

The PT measures the function of the extrinsic and common pathways of the coagulation cascade. A reagent containing tissue thromboplastin (factor III) of rabbit brain origin and calcium is commonly used as an activating substance. Factor III (thromboplastin) initiates the extrinsic pathway by forming a complex with factor VII. Calcium is needed to replace the plasma calcium that was chelated by the citrate in the blood collection tube. The PT is timed from the moment that the reagent is added to the plasma until the sample clots. As the factor VII-tissue thromboplastin complex activates factor X, the coagulation cascade proceeds into the common pathway. After formation of thrombin, fibrin will form at a normal rate only if factors involved in the extrinsic pathway (VII) and common pathway (X, V, II, I) are present in adequate amounts. If the PT is normal, then the sample contains adequate levels of factors VII, X, V, II, and I.

The PT test is used to (1) screen for hereditary or acquired factor deficiencies in the extrinsic/common pathway (i.e., VII, X, V, II, I), (2) screen for specific factor or non-specific inhibitors (such as the lupus anticoagulant), (3) monitor anticoagulant therapy with vitamin K antagonists such as Coumadin and warfarin, and (4) assess the effect of vitamin K deficiency, which is an index of liver damage in patients with chronic liver disease.

The results of the PT depend on the source and preparation of the thromboplastin reagent used, the instrumentation selected to perform the test, and the handling of the patient's sample. The PT may be reported as the clotting time in seconds or as the INR (international normalized ratio). The INR is preferred to the clotting time measured in seconds because different thromboplastin reagents have different sensitivities to warfarin-induced changes in levels of clotting factors. Since the potency of different commercially prepared calcium-thromboplasin reagents varies, the International Sensitivity Index (ISI) was developed to describe the relative strength of each reagent. This index is a measure of the sensitivity of the thromboplastin used in PT assays. The higher the ISI, the less sensitive the thromboplastin. The INR is calculated by dividing the patient PT value by the established mean PT value of the normal population in each testing facility. This value is then raised to the power of the ISI. Use of the INR eliminates the interlaboratory variation seen with prothrombin times measured in seconds.

Activated partial thromboplastin time (aPTT)

The aPTT measures the function of the intrinsic and common pathways of the coagulation cascade. To begin the test, a reagent containing a contact activator (cephalin) and a phospholipid substitute for platelet factor III is incubated with the patient's platelet-poor plasma. During this incubation period, the intrinsic pathway is initiated by conversion of XII to XIIa, which then converts XI to XIa. The cascade does not proceed any further, since the subsequent conversion of IX to IXa requires calcium ions. At this point, calcium is added as a second reagent. After recalcification, fibrin will form at a normal rate only if the factors involved in the intrinsic pathway (prekallikrein, high molecular weight kininogen [HMWK], XII, XI, IX, VIII) and common pathway (V, X, II, I) are present in adequate amounts. This rate of clotting measures the overall coagulant activity. The term "partial" in aPTT is used because platelet substitutes (the phospholipid emulsion) are not capable of activating the extrinsic pathway. This requires "complete" thromboplastin (tissue factor). Thus, the aPTT bypasses the extrinsic pathway and is unaffected by any abnormalities of factor VII. The aPTT is used to (1) screen for hereditary or acquired deficiencies of factors in the intrinsic/common pathway (i.e., all factors except VII and XIII), (2) screen for specific factor or non-specific inhibitors (such as the lupus anticoagulant), and (3) monitor anticoagulant therapy with parenteral heparin. Intrinsic coagulation disorders are apparent when the aPTT is prolonged and the prothrombin time is normal. For screening purposes, the aPTT is usually done in combination with other tests, such as the prothrombin time, which evaluates the factors of the extrinsic pathway. The combination of tests narrows the list of possible missing or defective factors. Results of the aPTT are prolonged by factor VIII deficiency (Hemophilia A) and factor IX deficiency (Hemophilia B). When the aPTT test is being used to monitor the effect of heparin, the test is done before the first dose of heparin and then as necessary to monitor therapeutic dosage. The activated clotting test (ACT) may be used in place of the aPTT to monitor heparin therapy. This test is performed by adding kaolin, (a clay that activates factor XII), or other clotting activator to the patient's plasma and measuring the time required for clot formation.

Bleeding time test

The bleeding time (BT) is defined as the time between making a small incision through a skin capillary and the moment the bleeding stops. It is an in vivo measurement of platelet participation in small blood vessel hemostasis, and is one of the simplest and best tests of overall platelet function. This test is used to determine how well platelets interact with the blood vessel wall to form a blood clot. The test is usually performed on patients who have a history of prolonged bleeding after cuts, or who have a family history of bleeding disorders, or as a preoperative test to determine a patient's likely bleeding response during and after surgery. There are four methods used: the Ivy method, template, modified template, and Duke method. With all methods the skin is cut in an area void of visible veins. The time from when the incision is made until all bleeding has stopped is measured and recorded as the bleeding time. Every 30 seconds, filter paper or a paper towel is used to draw off the blood. The test is finished when bleeding has stopped completely.


The fibrinogen test aids in the diagnosis of suspected clotting or bleeding disorders caused by fibrinogen abnormalities which include the absence of fibrinogen, low fibrinogen concentration, or functionally abnormal fibrinogen. Reduced fibrinogen levels can be found in liver disease, prostate cancer, lung disease, bone marrow lesions, malnutrition, and certain bleeding disorders. Obstetric complications or trauma may also cause low levels. Patients who have received multiple blood transfusions may exhibit low fibrinogen levels because banked blood does not contain fibrinogen. Fibrinogen levels are also decreased in approximately 50% of patients with disseminated intravascular coagulation (DIC). In this condition the coagulation process is triggered by malignancy, severe injury, sepsis (and other conditions) and continues unabated, causing systemic clots to form until coagulation factors and platelets are depleted. This process is followed by internal hemorrhage. The fibrinogen test is performed by adding thrombin to specific dilutions of the patient's plasma and measuring the amount of time it takes for the sample to clot.

Anticoagulation therapy with low molecular weight heparin (LMWH)

The anticoagulating effects of LMWH (heparin of smaller chain length) are easier to predict than standard heparin, and LMWH is associated with a lower risk of excessive bleeding and heparin induced thrombocytopenia. However, the effects of LMWH cannot be measured using the aPTT or activated clotting test (ACT). LMWH therapy is monitored by the antifactor Xa assay. The antifactor Xa activity of heparin has been considered important because it is relatively unaffected by the molecular weight of heparin and therefore less influenced by the effect of potent heparin antagonists which can be released from platelets. The anti-factor Xa assay measures the anti-Xa effect of LMWH whereby heparin in a citrated plasma sample combines with antithrombin, forming a complex that inhibits an excess of purified factor Xa. Measurement of the clotting activity of residual factor Xa is done by the addition of phospholipids and calcium in the presence of a substrate plasma. This substrate plasma brings to the assay an excess of AT-III and other coagulation factors, thus eliminating interferences by the factors that are already present in the plasma being tested.


Other than potential bruising at the puncture site, and/or dizziness, there are no complications associated with this test.


The absolute numbers that are considered normal vary from one laboratory to another. Any results reported should come with information regarding the testing facility's normal range. The values listed below are representative of normal values. The patient's physician is the best person to consult about a specific test level.

  • Platelets: A normal platelet count ranges between 150,000 and 400,000/μL. Platelet counts under 50,000/μL put a patient at risk for severe bleeding, while counts below 30,000/μL are considered critical.
  • Thrombin time: Reference values for the thrombin time are 14 to 15 seconds or within five seconds of the control.
  • Prothrombin time: PT results are reported in seconds and/or the International Normalized Ratio (INR). The normal range for the prothrombin time reported in seconds is between 11 and 13 seconds. Therapeutic levels for patient receiving Coumadin therapy generally are between 1.5 and 2-fold normal, or in terms of the INR, between 2.0 and 3.0.
  • Activated partial thromboplastin time: The normal range is between 20 and 36 seconds. If a patient is being anticoagulated with heparin, a result approximately 1.5 to 2.5 times the normal control value is usually a therapeutic goal.
  • Bleeding time: A normal bleeding time for the Ivy method is less than five minutes from the time of the incision until all bleeding from the wound has stopped. Some texts extend the normal range to eight minutes. The normal value for the template method is eight minutes or less, while for the modified template method, up to 10 minutes is considered normal. Normal for the Duke method is three minutes or less.
  • Fibrinogen: Normal reference values for fibrinogen are 200 mg/dL-400 mg/dL for adults and 125 mg/dL-300 mg/dL for newborns.


Anticoagulant— A substance that suppresses or counteracts coagulation of the blood.

Coagulation— The process of blood clotting.

Coagulation cascade— The sequence of biochemical activities, involving clotting factors, that stops bleeding by forming a clot.

Coagulation factors— Substances in the blood that act in sequence to stop bleeding by forming a clot.

Coumadin— An anticoagulant taken to prevent blood clots.

Fibrin— The insoluble protein formed by the action of thrombin on fibrinogen and stabilized by the action of factor XIIIa. Fibrin forms strands that add bulk to a forming blood clot to hold it in place and help "plug" an injured blood vessel wall.

Fibrinogen— A type of blood protein called a globulin that interacts with thrombin to form fibrin.

Fibrinolytic (thrombolytic) therapy— The intravenous administration of a drug to break up a blood clot.

Hemostasis— The process the body uses to stop the flow of blood when the vascular system is damaged.

Heparin— A medication that prevents blood clots by enhancing the activity of antithrombin. The heparin-antithrombin complex inhibits the activity of factor Xa, thrombin, and other clotting factors.

International normalized ratio (INR)— A measurement system that standardizes the prothrombin time to help monitor anticoagulant activity for clients receiving warfarin (Coumadin) therapy. It is recommended by the World Health Organization (WHO) for more consistent reporting of prothrombin time results, as it eliminates variation in PT results between laboratories caused by differences in the sensitivity of thromboplastin reagents.

Partial thromboplastin time— A test that measures the function of the clotting factors of the intrinsic pathway.

Platelet— An irregularly shaped cell-like particle in the blood that is an important part of blood clotting. Platelets are activated when an injury causes a blood vessel to break. They change shape from round to spiny, "sticking" to the broken vessel wall and to each other to begin the clotting process.

Prothrombin— A protein in blood plasma that is converted to thrombin during the clotting process.

Thrombin— An enzyme in blood plasma that converts fibrinogen to fibrin during the last stage of the clotting process.

Thromboplastin— A tissue substance that initiates the intrinsic coagulation pathway.

Thrombus— A solid mass of blood constituents (blood clot) formed in the heart or vessels.

Health care team roles

Coagulation tests are ordered and interpreted by physicians (in some cases pharmacists). The samples may be collected by a nurse, physician assistant, phlebotomist, or technician. Testing is performed by a clinical laboratory scientist CLS(NCA)/medical technologist MT(ASCP) or by a clinical laboratory technician CLT(NCA)/medical laboratory technician MLT(ASCP).



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