Hemostasis Reviewer

Here's your comprehensive exam reviewer for Hemostasis (MLS 417):

HEMOSTASIS — Complete Exam Reviewer

PART 1: WHAT IS HEMOSTASIS?

Definition: The physiological process that keeps blood fluid under normal conditions and forms a clot when a vessel is injured. (From Greek: haima = blood + stasis = stopping)

Four Components:

• Blood vessels

• Platelets

• Coagulation factors

• Fibrinolysis components

PART 2: FOUR STAGES OF HEMOSTASIS

Stage 1 — Vasoconstriction (Vascular Spasm)

• Immediate narrowing of the blood vessel

• Three purposes: reduce blood flow, reduce blood loss, enhance platelet adhesion

Stage 2 — Primary Hemostasis

• Main player: Platelets

• Three platelet stages: Adhesion → Activation → Aggregation

• Key receptor: GPIb (GPIb/IX/V) → binds von Willebrand factor (vWF) → platelet adhesion to exposed collagen

• Binding to vWF triggers platelet activation → shape changes from discoid → round with pseudopods

• ATP released → recruits more platelets

• PDGF released → promotes healing

• Fibrinogen binds activated platelet via GPIIb/IIIa → platelet aggregation

• End product: Unstable Platelet Plug

Stage 3 — Secondary Hemostasis (Coagulation Cascade)

• Goal: Form fibrin to stabilize the platelet plug → becomes fibrin clot

• Three pathways: Intrinsic, Extrinsic, Common

Stage 4 — Fibrinolysis

• Clot dissolution; plasminogen → plasmin (via TPA/UPA activators)

• Plasmin degrades fibrin → produces D-dimer, X, Y, D, E fragments

PART 3: SPECIMEN COLLECTION & PROCESSING

General Considerations (Prevent Premature Activation)

• ONE HIT rule — redirected needles activate coagulation; discontinue and repeat

• Coagulation tests are the most sensitive tests in hematology

Needle Size

• Small bore needles → more likely to cause hemolysis

• Evacuated tubes/syringes must be silicone coated

Anticoagulants

Specimen Handling

PART 4: TESTS FOR PRIMARY HEMOSTASIS

1. Bleeding Time (BT)

• Measures the time for a standard wound to stop bleeding (platelet plug formation)

• Detects: platelet number/function abnormalities, Factor VIII:vWF deficiency (screening only), vessel wall abnormalities

Interpretation Table:

2. Capillary Resistance (Fragility) Test

• Detects capillary structural weakness or thrombocytopenia

• Abnormal in: hereditary telangiectasia, hemophilia, Vitamin K deficiency

Grading:

3. Clot Retraction Time

• McFarlane Technique

• Procedure: 5 mL whole blood incubated at 37°C for 1 hour; clot shrinks from tube walls

• NV: 44-67% (formula: Vol. of serum / Total vol. of whole blood × 100)

• Factors: number of contractible platelets, presence of calcium and ATP, normal fibrinogen concentration

• Normal clot: firm, easily retractable

• No retraction = no fibrin clot; Weak clot = factor/platelet deficiency

4. Platelet Count

• NV: 150,000-450,000/µL

5. Platelet Aggregation

• Principle: aggregation agents added to PRP → platelets aggregate → sample becomes clearer → more light transmitted → measured by aggregometer

• Aggregation agents: Epinephrine, Collagen, ADP, Ristocetin

• Normal: clear supernatant, high light transmission

• Abnormal: turbid supernatant, low light transmittance

6. Platelet Adhesiveness — Glass Bead Test

• Tests: vWF and GPIb receptor on platelets

• Procedure: 1st sample (EDTA — baseline platelet count) vs 2nd sample (through glass bead column)

• Formula: PA = [(PC₁ - PC₂) / PC₁] × 100; NV: ≥70%

• Normal: platelets adhere to beads with vWF; platelet count in bead system ≈ EDTA count

• Abnormal: no adhesion; low platelet count; deficiency in vWF or GPIb

PART 5: COAGULATION SYSTEM

Coagulation Factors (MEMORIZE)

• Zymogen = inactive enzyme precursor → becomes active enzyme

• Cofactor = accelerates enzymatic reaction

Pathways

• Contact factors (XII, XI, HMWK, Prekallikrein): no significant role in physiologic coagulation; deficiencies prolong aPTT

• Thrombin converts fibrinogen → fibrin (initial clot = unstable, held by H bonds)

• Factor XIII stabilizes clot by forming covalent cross-links → strong, stable fibrin clot

• Tenase complexes are called "Tenase" because they activate Factor X

Fibrinolysis

• TPA and UPA convert plasminogen → plasmin

• Plasmin degrades fibrin → X, Y, D, E fragments + D-dimer

• Fibrin degradation products (FDPs): inhibit hemostasis, prevent platelet activation, inhibit fibrin polymerization, indicate plasmin activity

• D-dimer: specific to fibrin; marker of thrombosis AND fibrinolysis

PART 6: TESTS FOR SECONDARY HEMOSTASIS

1. Clotting Time (CT)

• Measures free fibrin formation

• Combined with BT: CT/BT (CT = secondary; BT = primary)

Lee and White Method: NV: 7-15 mins

1. Incubate 3 tubes at 37°C

2. Add 1 mL blood per tube

3. Start timer when blood enters tube 1

4. Gently tilt tube 3 every 30 seconds

5. After clotting in tube 3 → observe tube 2 then tube 1

2. Prothrombin Time (PT)

• Measures extrinsic + common pathway factors

• Monitors: coumarin/coumadin/warfarin therapy

• Warfarin inhibits Vitamin K-dependent factors: II, VII, IX, X (mnemonic: 1972)

• Patients on warfarin → prolonged PT

• NV: 10-13 seconds

INR (International Normalized Ratio):

• Standardized PT reporting

• Formula: INR = (Patient PT / Mean Normal PT)^ISI

• NV: 2.0-3.0

3. aPTT (Activated Partial Thromboplastin Time)

• Measures intrinsic + common pathway factors

• Monitors heparin therapy

• NV: <35 seconds

• "Partial" = reagents must be added before clotting occurs

• Common reagent: kaolin (contact activator; mimics collagen)

• Components: Activators (contact factors), Phospholipid (platelet substitute), CaCl₂ (cofactor)

4. Stypven/Russell Viper Venom Time (RVVT)

• Measures common pathway; directly activates Factor X

• Bypasses intrinsic and extrinsic pathways

• Detects deficiencies of: Fibrinogen, Prothrombin, Factors V and X

• Cannot detect Factor VII deficiencies

• Reagent: East Indian Viper (Vipera russelli)

• NV: 6-10 seconds

5. Thrombin Time (TT)

• Detects fibrinogen deficiency and heparin inhibition

• Prolonged in: fibrinogen deficiency, thrombolytic agents, FDPs, heparin presence

• NV: 10-14 seconds

6. Reptilase Time

• Detects fibrinogen deficiency

• Reagent: Bothrops atrox snake venom → converts fibrinogen to fibrin

• UNAFFECTED BY HEPARIN → better than thrombin time when heparin is suspected

• Prolonged in: fibrinogen deficiency, thrombolytic agents, FDPs (NOT heparin)

• NV: 10-15 seconds

7. Duckerts/5M Urea Solubility Test

• Detects Factor XIII deficiency

• Reagents: 5M Urea (H-bond disruptor), 1% monochloroacetic acid, 2% acetic acid

• Normal result: insoluble clot (FXIII provides covalent bonds)

• FXIII deficiency → clot has only H-bonds → dissolves in urea

8. Substitution Studies

• Detects factor deficiencies by mixing correction reagents with patient sample then measuring PT and aPTT

• Reagents: Adsorbed Plasma (contains V, VIII) and Serum (contains VII, IX, X, XI)

QUICK REFERENCE: TEST SUMMARY

HIGH-YIELD MNEMONICS & PEARLS

• Warfarin inhibits factors 1972 → I (no), IX, VII, X, II

• Blue top = sodium citrate = coagulation tests (9:1 ratio — walang labis, walang kulang)

• GPIb → adhesion (binds vWF); GPIIbIIIa → aggregation (binds fibrinogen)

• Reptilase > Thrombin time when heparin is in the picture

• D-dimer = specific marker of both thrombosis AND fibrinolysis

• Unstable platelet plug (primary) → stabilized by fibrin (secondary) = fibrin clot

• Factor XIII = fibrin stabilizing factor (covalent cross-links; dissolves in urea if deficient)

• Kaolin mimics collagen as a contact activator in aPTT

• RVVT directly activates Factor X — bypasses both intrinsic and extrinsic