Coagulation Studies

Coagulation studies are a group of hematologic laboratory studies that reflect the function of blood vessels, platelets, and coagulation factors, which all interact with one another to achieve hemostasis. Coagulation studies are usually ordered to evaluate patients with bleeding or hypercoagulation disorders.

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Overview

Definition

Coagulation studies are a group of hematologic studies that reflect the function of blood vessels, platelets, and coagulation factors, which all work in harmony to achieve hemostasis.

Uses of coagulation studies

  • Evaluation of abnormal bleeding or thrombosis
  • Preoperative testing
  • Management of anticoagulation therapy
  • Assist in resuscitation management during massive transfusions

Review: phases of the hemostatic process

The following is a summary of the hemostatic process:

  • Constriction of the blood vessel: limits blood flow to the area
  • Formation of the platelet plug: the initial, temporary plug formed via the following steps:
    • Adhesion: exposed von Willebrand factor (VWF) binds to the glycoprotein (Gp) Ib receptors on platelets
    • Aggregation: GpIIb/IIIa receptors on platelets bind fibrinogen
    • Secretion: Substances are released that stimulate further platelet activation and aggregation and initiate the coagulation cascade.
  • Activation of the coagulation cascade: forms a more stable fibrin clot
    • Extrinsic pathway: 
      • Primarily responsible for initiation of the cascade
      • Involves (in order): tissue factor, factor VII, and factor X
    • Intrinsic pathway: 
      • Primarily involved in amplification of the cascade
      • Can be directly activated by vessel injury
      • Involves (in order): factors XII, XI, IX, VIII, and X
    • Common pathway: 
      • The extrinsic and intrinsic pathways join together when factor X is activated to form the final common pathway.
      • Involves (in order): factors X, V, II (thrombin), I (fibrin), and XIII
  • Inhibition of clotting and fibrinolytic phase: 
    • Stops clotting and breaks down the clot once it is no longer necessary
    • Involves: 
      • Plasmin
      • Antithrombin
      • Proteins C and S

Formation of the temporary hemostatic plug:
The disrupted endothelial surface exposes von Willebrand Factor (vWF) to the passing blood. Platelets bind to the vWF via their GpIb receptors and are activated. Platelet activation triggers them to secrete adenosine diphosphate (ADP), which stimulates the expression of the GpIIb/IIIa receptors on the platelets. The GpIIb/IIIa receptors bind to fibrinogen, which is able to bind a platelet on each end, causing platelets to aggregate. As more platelets are bound to one another, the platelet plug is generated. As the coagulation cascade is activated, thrombin converts the weaker fibrinogen into the stronger fibrin, creating a much more stable clot.

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Overview of the coagulation cascade
a: activated form
PF3: platelet factor 3 (phospholipids)

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Coagulation Studies

The following studies can assist in diagnosing issues related to hemostasis.

Platelet assessments

Platelet counts:

  • Measure platelet number/concentration
  • Normal range: 150,000–450,000
  • ↑ in thrombocytosis, which can be caused by:
    • Acute blood loss/iron deficiency anemia
    • Metastatic cancer
    • Inflammatory conditions: 
      • Rheumatologic disorders
      • Inflammatory bowel disease
      • Kawasaki disease
      • Nephrotic syndrome
    • Infections
    • Trauma and burns
  • ↓ in thrombocytopenia:
    • Immune thrombocytopenic purpura
    • Thrombotic thrombocytopenic purpura
    • Gestational thrombocytopenia
    • HIV
    • Drug-induced thrombocytopenia

Bleeding time (BT):

  • Measures the time for bleeding to stop after a lancet incision
  • Indirect measure of platelet function
  • Normal range: 2–7 minutes
  • Prolonged in:
    • Thrombocytopenia
    • Disseminated intravascular coagulation (DIC)
    • Von Willebrand disease (VWD)
    • Bernard–Soulier disease
    • Glanzmann thrombasthenia
    • Renal failure
    • NSAID and/or aspirin use

Clotting times

These studies are used to assess the amount of time it takes plasma to clot when various substances are added.

  • PT: 
    • Measures the time it takes plasma to clot when exposed to tissue factor
    • Measures function of the extrinsic and common pathway
    • Normal range: approximately 11–13 sec
    • Prolonged in:
      • Warfarin therapy
      • Vitamin K deficiency
      • Factor deficiency: II, V, VII, or X
      • Liver disease 
      • DIC
  • INR:
    • A ratio comparing the patient’s PT to a reference PT set by the WHO
    • Measures function of the extrinsic and common pathways
    • Normal range: approximately 0.8–1.1
  • aPTT: 
    • Measures the time it takes plasma to clot when exposed to a negatively charged substance (which activates the intrinsic pathway):
      • Celite
      • Silica
    • Measures function of both the intrinsic and common pathways
    • Normal range: 25–40 sec
    • Prolonged in:
      • Heparin therapy 
      • Hemophilia disorders (abnormal factor VIII or IX)
      • Von Willebrand disease
      • Liver disease
      • DIC
  • TT: 
    • Time for plasma to clot after exposure to thrombin
    • Measures the final step in the common pathway: the conversion of fibrinogen to fibrin
    • Normal range: 14–19 sec
    • Prolonged in:
      • ↓ Fibrinogen
      • Use of anticoagulants that inhibit thrombin: heparin, direct thrombin inhibitors (e.g., Argatroban)
      • DIC
      • Liver disease

Assessing the coagulation cascade

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Coagulation factor assays

Coagulation factor assays primarily used to diagnose specific factor deficiencies. The tests are used to assess the concentration or activity level of individual factors.

  • Clot-based assays use PT or aPTT testing calibrated for individual factors.
  • Chromogenic assays use cleavage of a chromogenic (colored) substrate to measure factor activity.
  • Antigenic assays: ELISAs are used to measure clotting factor levels.
  • Fibrinogen:
    • The precursor to fibrin
    • Abnormally low levels can increase bleeding risk.
    • Normal range: 200–400 mg/dL
    • Abnormal bleeding typically occurs at levels < 100 mg/dL.

Mixing studies

Mixing studies are used to further evaluate an unexplained prolongation of a clotting test, including a prolonged PT, aPTT, or TT.

  • Can determine whether an abnormal PT/aPTT is due to:
    • A factor deficiency OR
    • Presence of inhibitor: 
      • Autoantibody to a specific factor
      • Heparins
      • An elevated C-reactive protein
  • Measures clotting time of the patient’s plasma when serially diluted with normal plasma
  • The normal plasma:
    • Contains all the factors
    • Does not contain enough factors to overcome the presence of inhibitors
  • Results: The clotting time will either correct with the addition of normal plasma or fail to correct.
    • If clotting time (PT or aPTT) corrects:
      • Diagnosis: factor deficiency
      • Next step: Identify the specific factor using a coagulation factor assay.
    • If clotting time (PT or aPTT) fails to correct:
      • Diagnosis: presence of a factor inhibitor 
      • Next steps:
        • Consult with hematology.
        • Review medications for potential cause.
        • Determine the titer of antibody present using further serial dilutions.

Clinical Relevance: Hypocoagulable Conditions

Hypocoagulable conditions are a group of diseases that result in abnormal hemostasis. They manifest with minor bleeding (petechiae, mucocutaneous bleeding) or more severe internal bleeding (hemarthrosis, intracranial bleeding).

Disorders of formation of the platelet plug

Patients with these disorders will most likely have an increased bleeding time but normal PT and aPTT.

  • Glanzmann thrombasthenia: an autosomal recessive bleeding syndrome characterized by a deficiency of the GpIIb/IIIa receptor, resulting in a lack of platelet aggregation
  • Bernard–Soulier syndrome: an autosomal recessive bleeding syndrome characterized by deficiency of the GpIb receptor, resulting in failure of platelet adhesion: Bernard–Soulier syndrome can be diagnosed with a ristocetin assay. Ristocetin activates VWF to allow binding to the platelet GpIb receptor; however, in Bernard–Soulier syndrome, platelets will fail to adhere with the assay.
  • Thrombocytopenia: When platelet levels are low, formation of the platelet plug may be impaired. Thrombocytopenia is usually diagnosed with a CBC with platelet count and morphology assessment. Causes may include immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), HIV, drug-induced thrombocytopenia, or other bone marrow disorders (including malignancy).

Disorders of the coagulation cascade

Hemophilia: a rare blood clotting disorder in which the body lacks blood-clotting factors (factor VIII in hemophilia A and factor IX in hemophilia B). Affected individuals present with abnormal bleeding that occurred spontaneously or after minor trauma. Patients can bleed into joint spaces and can develop life-threatening internal bleeding. Coagulation studies typically reveal a prolonged aPTT due to the abnormalities in the intrinsic pathway, but the PT and platelet counts are normal. Mixing studies will correct, and the level of the specific factor activity in which the patient is deficient will be low.

Mixed disorders affecting both platelets and coagulation factors

  • Von Willebrand disease (VWD): the most commonly inherited disorder of hemostasis, caused by a qualitative or quantitative deficiency in VWF: The three primary types of VWD differ in severity, though all tend to present with bleeding abnormalities. Von Willebrand factor is required for both initial platelet adhesion, and it helps stabilize factor VIII in the intrinsic pathway. Testing shows a normal platelet count, but the aPTT may be prolonged (depending on the reduction of factor VIII activity). A VWF antigen test, specific functional assays, and genetic testing can aid in the diagnosis.
  • Disseminated intravascular coagulation: a serious medical condition in which the coagulation cascade is activated systemically, leading to multiple clots that can lead to permanent end-organ damage and in the process, coagulation factors are used up: Disseminated intravascular coagulation always has a secondary cause. Infections, burns, and malignancies are among the most common causes, but DIC can also occur during severe postpartum hemorrhage. Laboratory findings include thrombocytopenia, prolongation of the PT and aPTT, and elevation of D-dimer.
  • Cirrhosis: The liver is the primary site of synthesis for a majority of the clotting factors. In addition to impaired synthesis of clotting factors, cirrhosis also may independently result in thrombocytopenia due to splenic sequestration of platelets and decreased thrombopoietin production. Platelets themselves may also be dysfunctional. The platelet count may be low and the PT and aPTT may both be prolonged.

References

  1. Longo, D., Fauci, A., Kasper, D., Hauser, S., Jameson, J., Loscalzo, J. Harrison’s Manual of Medicine, 18th ed. McGraw-Hill Professional, 2012, pp 2159–2238.
  2. Zehnder, J. (2020).  Clinical use of coagulation tests. In Tirnauer, J.S. (Ed.), UpToDate. Retrieved March 8, 2021, from https://www.uptodate.com/contents/clinical-use-of-coagulation-tests
  3. Arnold, D.M. (2021). Diagnostic approach to the adult with unexplained thrombocytopenia. In Tirnauer, J.S. (Ed.), UpToDate. Retrieved March 30, 2021, from https://www.uptodate.com/contents/diagnostic-approach-to-the-adult-with-unexplained-thrombocytopenia

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