Secondary Hemostasis

• Secondary Hemostasis


  Formation of irreversible clot (Fibrin)
• Coagulable cascade


  Coagulation Pathway and Coagulation Factors
• Coagulation pathways and Activators

  1. Extrinsic: Activated by tissue thromboplastin

  2. Intrinsic: Activated by Collagen

  3. Common: both intrinsic and extrinsic by formation of prothrombinase complex
• Coagulation factors


  • Synthesize in Liver except III and IV
  • Circulating in its inactive/zymogen form except III and IV
  • All coagulation factors, including PK and HMWK, active forms are in Serine Proteases except: Factor 1 (Fibrinogen), Factor V and VIII (as cofactors), Factor XIII (Transglutaminase/Transaminase) for fibrin plug stabilization
• Coagulation factors


  • I: Fibrinogen
  • II: Prothrombin
  • III: Tissue Factor
  • IV: Calcium
  • V: Proaccelerin
  • VII: Proconvertin
  • VIII: Antihemophilic Factor
  • IX: Plasma Thromboplastin Component
  • X: Stuart-Prower Factor
  • XI: Plasma Thromboplastin Antecedent
  • XII: Hageman Factor
  • XIII: Fibrin-Stabilizing Factor
  • Prekalikrein (PK)
  • High Molecular Weight Kininogen
• Factor VIII subtypes circulate in blood bound to vWF
• Factor VIII subtypes


  • Factor VIII/Factor VIIIC/ Factor VIII:C
  • Factor VIII: Ag
  • Factor VIIIR:RCo (Restocetin Cofactor)
  • Factor VIII: vWF
• Factor VIII: Ag


  • Refers to antigenic properties
  • Measured by immunoassays
• Factor VIIIR:RCo (Restocetin Cofactor)


  Refers to portion responsible for platelet aggregation in the presence of restocetin
• Factor VIII: vWF


  • Termed as Von Willebrand Factor
  • Required for normal platelet adhesion
• vWF: Ag (Previously: Factor VIIIR: Ag)


  • Antigenic portion of the Von Willebrand Factor
  • Measured by immunoassays
• GROUP OF CLOTTING FACTORS - According to the Coagulation Pathway

  • Intrinsic Coagulation Pathway
  • Extrinsic Coagulation Pathway
  • Common Coagulation Pathway
• GROUP OF CLOTTING FACTORS - According to Properties

  • Fibrinogen Group
  • Prothrombin Group
  • Contact group
• GROUP OF CLOTTING FACTORS - Vitamin K independent(Prothrombin Group)

  • Calcium Dependent
  • Vitamin K dependent
• GROUP OF CLOTTING FACTORS - Calcium Dependent


  • Vitamin K Independent
  • Calcium Independent
• OTHERS


  • Extrinsic Tenase
  • Intrinsic Tenase
  • Prothrombinae group
  • Labile Factor
  • Cold temperate Factors
• REES ECKER COUNTING


  Improved Neubauer Counting Chamber
• REES ECKER DILUTING FLUID COMPONENTS

  • BRILLIANT CRESYL BLUE
  • SODIUM CITRATE
  • DISTILLED WATER
  • 1% AMMONIUM OXALATE
• METHODS


  1. By automated hematology analyzers

  2. Direct smear also gives information about platelets’ size, shape, and clumping

  3. Direct count from the peripheral blood smears
• THE MANUAL METHOD OF PLATELETS COUNT

  1. Take 20 µL of blood

  2. Add 1.8 mL of 1% ammonium oxalate

  3. Leave for 15 minutes for complete lysis of RBCs

  4. Mount the Neubauer chamber

  5. Leave the chamber for 15 minutes in high humidity

  6. Count the large central square labeled as P
• FORMULA: When a 1:20 dilution is used, the four large squares on side yield count 21, 34, 10, 28, 29. On the other side yielded 11, 23, 24, 29, 30. What is the leucocyte count?
• EVALUATION OF SECONDARY HEMOSTASIS: Error in both pathway, Error in Extrinsic Pathway, Error in Intrinsic pathway, Specimen consideration and processing, Problem in Common pathway, Problem in Intrinsic Pathway, Problem in Extrinsic pathway
• Leucocyte count is yielded as 11, 23, 24, 29, 30
• Evaluation of Secondary Hemostasis

  1. Error in both pathway

  2. Error in Extrinsic Pathway

  3. Error in Intrinsic Pathway

  4. Specimen consideration and processing

  5. Problem in Common Pathway

  6. Problem in Intrinsic Pathway

  7. Problem in Extrinsic Pathway
• Sample for Coagulation


  1. PPP (Platelet Poor Plasma)

  2. Must be processed within 1 hr at room temperature 18-24hrs

  3. PT: 24 hrs

  4. APTT: 4 hrs
• APTT (Activated Partial Thromboplastin Time)


  • To monitor Intrinsic and Common pathway
  • To monitor Warfarin (Coumadin) therapy
  • Affected by circulating Anticoagulant
• PT (Prothrombin time)


  • To monitor Extrinsic and Common pathway
  • To monitor Heparin therapy
• Normal values for APTT: 35-45 seconds
• Normal values for PT: 10-14 seconds
• Reagents for Coagulation tests

  • Platelet substitute (Phospholipids)
  • Negative Charge Activator (Kaolin, Ellargic acid, Celites, Silica)
  • 0.025M CaCl2
• Materials for Coagulation tests


  • Controls
  • Test tubes (12x75 mm)
  • Pipets
  • 27C water bath
• Procedure for APTT test

  Add PPP to APTT reagent, incubate, add CaCl2, record clotting time
• Procedure for PT test

  Warm plasma and control, add PT reagent, incubate, record clotting time
• Reporting for APTT is in seconds to the nearest tenth along with the reference value
• Reporting for PT can be in several ways: Px time with the control time, Px time with the reference range, Prothrombin ratio, INR (International Normalized Ratio)
• INR (International Normalized Ratio) is a standardized way of reporting PT to minimize differences in results due to reagent-instrument combinations
• PT ratio of Px under Oral Anticoagulants: NV 1, 1-2.5 under warfarin
• ISI (International Sensitivity Index) is performed to determine the sensitivity of reagent
• Common tests for evaluation of Secondary Hemostasis include APTT and PT
• Therapeutic Anticoagulants include Heparin and Warfarin