Hemostasis and Diagnostic Testing

Hemostasis

I. Introduction to Hemostasis

  • Hemostasis refers to the physiological process that prevents and stops bleeding.

  • It is critical in surgical procedures and managing bleeding disorders.

II. Diagnostic Testing for Hemostasis

A. Blood Collection

  • Volume Requirements:

    • 2.7 mL whole blood and 0.3 mL Na3C6H5O7.2H2O (3.2% sodium citrate, 0.109M).

    • Ratio: 1 part sodium citrate to 9 parts whole blood.

  • Collection Tubes Required:

    1. Blood cultures

    2. Plain serum glass tubes

    3. Sodium citrate tubes (light blue top)

    4. Gel separator/plain serum plastic tubes

    5. Heparin tubes

    6. EDTA tubes

    7. Glucose preservative tubes

  • Order of Collection Importance: Proper order minimizes contamination and ensures accurate test results.

B. Adjusting Anticoagulant for Polycythemia Vera (PV) Patients

  • PV patients often have hematocrit levels >55%.

  • Need for Adjustments:

    • Before collection, adjust anticoagulant to prevent false prolongation of results due to decreased plasma volume.

  • Adjustment Formula:
    C=(1.85imes10−3)(100−H)VC = (1.85 imes 10^{-3}) (100 - H)V

    • Where:

      • C = volume of sodium citrate in mL

      • V = volume of whole blood collected in mL

      • H = hematocrit (%)

  • Example Calculation:

    • For 3 mL of whole blood with Hct of 65%:
      C=(1.85imes10−3)(100−65)imes3.0C = (1.85 imes 10^{-3}) (100 - 65) imes 3.0
      C=(1.85imes10−3)(35)imes3.0C = (1.85 imes 10^{-3}) (35) imes 3.0
      C = 0.19 ext{ mL of 3.2% sodium citrate}

  • Implications of Imbalance: Additional red cells decrease plasma volume affecting calcium levels and potentially interfere with coagulation tests (e.g., PT and APTT) due to excess citrate binding to calcium reagents.

C. Potential Errors in Blood Collection

  1. Short Draw

    • Results prolongation due to anticoagulant excess neutralizing test reagents.

  2. Clot in Specimen

    • Results in unusable samples.

  3. Traumatic Venipuncture

    • Can falsely shorten results.

  4. Visible Hemolysis

    • Results are unreliable due to hemolysis effect.

  5. Lipemia

    • Can falsely prolong results.

D. Hemolysis Effect

  • Hemolysis releases ADP from red cell stroma, which activates platelets and results in shorter clotting times.

E. Storage and Testing Conditions

  • Test Storage Conditions:

    • PT: 2-4°C or 18-24°C for 24 hours.

    • APTT without heparin: 2-4°C or 18-24°C for 4 hours.

    • APTT for heparin therapy: separate plasma within 1 hour and test within 4 hours.

F. Processing Specimens

  • Platelet Poor Plasma (PPP): Centrifuge sodium citrate specimens at 1500 x g for 15 min.

  • Platelet Rich Plasma (PRP): Centrifuge sodium citrate specimens at 50 x g for 30 min.

  • Normal Platelet Counts:

    1. Platelet count <10,000/µL for PPP.

    2. Platelet count 200,000 to 300,000/µL for PRP.

    3. Tests: PT, APTT, fibrinogen assays, factor assays, platelet aggregometry.

G. Bleeding Time (BT)

  • Introduction: Initiated in the 1930s to predict risk of intraoperative hemorrhage.

  • Method: Automated lancet produces a 5mm long, 1mm deep incision on the forearm; blood absorption measured every 30 seconds at constant pressure (40 mmHg).

  • Normal Range: 2-9 minutes.

  • Disease Associations:

    • vWD and Bernard-Soulier syndrome lead to prolonged bleeding times (BT).

H. PFA-100 Assay

  • Measures platelet function (in vitro).

  • Requirements: Platelet count must be >100,000/µL.

  • Test Procedure:

    • Test cartridges contain collagen/EPI or collagen/ADP for stimulating platelet aggregation.

    • Citrated whole blood is aspirated through a membrane; closure time (CT) indicates platelet function.

  • Normal Values for Closure Time:

    • Collagen/EPI: 78-199 seconds.

    • Collagen/ADP: 55-137 seconds.

  • Disease Associations: Prolonged closure time seen in vWD, Bernard-Soulier syndrome, Glanzmann’s Thrombasthenia, Gray Platelet Syndrome.

I. TEG (Thromboelastography)

  • History: Developed in 1948 by Hellmut Hartert and became widely accepted in the U.S. in the 1980s.

  • Purpose: Assesses patient's coagulopathy status.

  • Measurement Method: Captures metrics between a rotating cup and a torsion pin using optical systems, cameras, and a heating unit.

J. TEG Components and Measurements

  • Reagents Used in TEG:

    • Kaolin for conventional TEG.

    • Heparinase for patients on heparin.

    • Tissue factor + kaolin for rapid TEG.

  • TEG Parameters:

    1. R: Time to initial clot formation.

    2. K: Time to reach a predetermined clot strength.

    3. MA (Maximum Amplitude): Clot strength indication.

    4. α (Alpha Angle): Relation to fibrinogen concentration.

    5. LY30: Fibrinolysis percentage after 30 minutes.

  • Treatment Recommendations Based on TEG Analysis:

    • Decreased R time: administer fresh frozen plasma.

    • Increased K time or decreased α angle: administer cryoprecipitate.

    • Decreased MA: administer platelets.

    • Increased LY30: administer tranexamic acid.

K. TEG Tracing Interpretation

  • Key Interpretation Foresees Hemostasis and Coagulation Problems:

    • Normal R Time: ~7 minutes.

    • elevated K: indicates deficient fibrinogen.

    • Normal MA: for adequate platelet function.