BIO230 Class 01/30

Hematology and Coagulation

  • Endothelial Cells:

    • They line the entire vascular system and act as a physical barrier between blood and subendothelial collagen.

    • Hemostatic Properties: Under normal conditions, they maintain an anticoagulant environment by producing nitric oxide (NONO) and prostacyclin (PGI2PGI_2) to inhibit platelet activation.

    • Procoagulant Transition: Upon injury, they express Tissue Factor (Factor III) and von Willebrand Factor (vWF) to initiate clotting and platelet adhesion.

  • Protein C and Protein S Pathway:

    • Activation: Protein C is a zymogen converted to activated protein C (APC) by thrombin. This reaction is significantly accelerated (approx. 1000-fold) when thrombin is bound to thrombomodulin on the endothelial surface.

    • Cofactor Requirement: Protein S serves as a crucial non-enzymatic cofactor for APC. Both Protein C and S are Vitamin K-dependent proteins.

    • Role of Activated Protein C (APC):

    • Proteolytically inactivates activated Factor V (Va) and activated Factor VIII (VIIIa).

    • This negative feedback loop is essential to prevent the runaway expansion of the fibrin clot.

Coagulation Mechanism

  • The Cascade Model:

    • Intrinsic Pathway: Initiated by contact with negatively charged surfaces (Contact Phase). Involves Factors XII, XI, IX, and VIII.

    • Extrinsic Pathway: Initiated by vascular injury and the release of Tissue Factor (TF). Involves Factor VII.

    • Common Pathway: Where both pathways converge at the activation of Factor X. Involves Factors X, V, II (Prothrombin), and I (Fibrinogen).

  • Complex Formation:

    • Tenase Complex: Consists of Factors IXa, VIIIa, calcium, and phospholipids; activates Factor X.

    • Prothrombinase Complex: Consists of Factors Xa, Va, calcium, and phospholipids; converts Prothrombin (II) to Thrombin (IIa).

Therapeutic Anticoagulant Therapy

  • Classes of Anticoagulants:

    • Heparin (Unfractionated Heparin - UFH):

    • Mechanism: Binds to Antithrombin III (ATIII), causing a conformational change that increases ATIII's affinity for Thrombin and Factor Xa by 1000 times.

    • Monitoring: Measured via the Activated Partial Thromboplastin Time (APTT).

    • Clinical Use: Immediate anticoagulation for DVT, Pulmonary Embolism (PE), or Acute Coronary Syndrome.

    • Low Molecular Weight Heparin (LMWH):

    • e.g., Enoxaparin. More predictable than UFH and primarily inhibits Factor Xa.

    • Warfarin (Coumadin):

    • Mechanism: Inhibits Vitamin K Epoxide Reductase (VKOR), preventing the recycling of Vitamin K. This leads to the production of non-functional Factors II, VII, IX, X, and Proteins C and S (PARNAs).

    • Monitoring: Prothrombin Time (PT) and the International Normalized Ratio (INR). Target INR is typically 2.0−3.02.0 - 3.0.

    • Note: Initial bridge therapy with heparin is required because Protein C has a shorter half-life than the procoagulant factors, potentially causing a transient prothrombotic state.

Coagulation Tests and Assays

  • Routine Screening:

    • APTT (Activated Partial Thromboplastin Time): Screens the intrinsic and common pathways. Reference range is usually 25−3525 - 35 seconds.

    • PT (Prothrombin Time): Screens the extrinsic and common pathways. Sensitive to Factor VII deficiencies.

  • Mixing Studies:

    • Performed when a patient has an unexplained prolonged PT or APTT.

    • Correction: Clotting time normalizes within 10%10\% of the normal range, indicating a factor deficiency.

    • No Correction: Clotting time remains prolonged, indicating the presence of a circulating inhibitor (e.g., Lupus Anticoagulant or an antibody against Factor VIII).

  • D-Dimer Assay:

    • A specific marker for cross-linked fibrin degradation. It requires the action of Thrombin (to form the clot), Factor XIII (to cross-link it), and Plasmin (to break it down).

    • Clinical Utility: High negative predictive value; a negative D-Dimer can rule out DVT/PE in low-risk patients.

Practical Considerations in Specimen Collection

  • The Blue Top Tube: Uses 3.2%3.2\% Sodium Citrate. The ratio must be exactly 9:19:1 (blood to anticoagulant).

  • Pre-analytical Errors:

    • Under-filling: Excess citrate binds case-level calcium in the test reagent, causing falsely prolonged clotting times.

    • Polycythemia: If Hematocrit is >55\%, the plasma volume is reduced relative to the citrate, requiring a manual adjustment of the anticoagulant volume.

  • Platelet Function Analysis:

    • Closure Time (PFA-100): Assesses platelet adhesion and aggregation under high shear stress.

    • Light Transmission Aggregometry (LTA): Gold standard; uses agonists like ADP, Collagen, Epinephrine, and Arachidonic Acid to observe aggregation curves.

Point of Care Testing (POCT)

  • Essential for monitoring patients on bypass during surgery or bedside monitoring of Warfarin/Heparin.

  • Requires strict adherence to Quality Control (QC) to ensure results align with central laboratory standards.