MLHP 106 Hematology and Transfusion Science - Lesson 4 NOTES & Flashcards

HEMOSTASIS

Hemostasis is the process that stops bleeding and involves the coordinated action of four systems working together: blood vessels, platelets, coagulation factors, and fibrinolysis (clot breakdown). The body forms a stable fibrin clot to halt bleeding and then remodels and dissolves the clot once healing occurs. The fibrin clot is also called a thrombus or hemostatic plug. Hemostasis encompasses two main components: (1) blood clot formation (coagulation) and (2) blood clot breakdown (fibrinolysis).

PRIMARY AND SECONDARY HEMOSTASIS

Hemostasis is traditionally divided into two parts. Primary hemostasis involves the vascular response and platelets. It includes vasoconstriction of damaged vessels and platelet adhesion and aggregation to form a platelet plug. Secondary hemostasis involves coagulation factors that form a stable fibrin clot. Fibrinolysis then dissolves the clot when healing occurs.

In more detail:

  • Primary Hemostasis involves: Blood vessels (vascular constriction) and Platelets (adhesion and aggregation).

  • Secondary Hemostasis involves: Coagulation factors (I–XIII), vitamin K–dependent factors, calcium, and the coagulation cascade leading to fibrin formation.

  • Fibrinolysis involves plasminogen activation to plasmin, which degrades fibrin.

It is important to prevent tissue fluid from entering collection tubes because tissue factor exposure can activate coagulation and alter test results.

ABNORMAL AND NORMAL HEMOSTASIS; DRUGS AND MONITORING

Abnormal hemostasis ranges from easy bruising to severe hemorrhage and can include unwanted clotting events such as thrombosis or embolism. Anticoagulant drugs (e.g., warfarin, heparin, aspirin) are used to prevent intravascular clotting and are monitored in the laboratory to ensure therapeutic effects while minimizing bleeding risk.

  • Warfarin (Coumadin) inhibits vitamin K–dependent coagulation factors (vitamin K antagonist).

  • Heparin inhibits thrombin and factor Xa.

  • Aspirin inhibits platelet aggregation.

The laboratory monitoring of anticoagulant therapy is essential to maintain balance between bleeding and clotting risk.

ROLE OF BLOOD VESSELS AND TISSUE FACTOR; ROLE OF PLATELETS

Role of Blood Vessels (PRIMARY HEMOSTASIS):

  • Vasoconstriction reduces blood flow to the damaged area.

  • When endothelium is damaged, collagen is exposed and tissue factor is released. Collagen helps activate platelets; tissue factor is a major trigger for the coagulation cascade (secondary hemostasis).

  • Tissue factor exposure is a key driver of coagulation; avoid contamination of blood samples with tissue fluids.

Role of Platelets (PRIMARY HEMOSTASIS):

  • Platelet adhesion occurs when platelets contact exposed collagen, initiating release of calcium and shape change (from discoid to spiky forms).

  • Platelets aggregate via fibrinogen and ionized calcium, forming a platelet plug.

  • A sufficient platelet count and normal platelet function are required; thrombocytopenia (low platelets) or thrombocytosis (high platelets) affect hemostasis.

ROLE OF COAGULATION FACTORS; SECONDARY HEMOSTASIS

Coagulation factors (I–XIII) are plasma proteins, mostly produced in the liver, many vitamin K–dependent. An activation cascade occurs through intrinsic, extrinsic, and common pathways, culminating in the formation of a stable fibrin clot. Ionized calcium is required for the coagulation factors to work together.

Key points:

  • There are three pathways: Intrinsic, Extrinsic, and Common.

  • The intrinsic pathway involves factors XII, XI, IX, VIII (in order of activation).

  • The extrinsic pathway involves factor VII and the tissue factor complex.

  • The common pathway involves factors X, V, II (prothrombin), and I (fibrinogen), ultimately forming fibrin.

  • A platelet plug is initially formed but is unstable without a fibrin clot; secondary hemostasis stabilizes the clot with fibrin.

FIBRINOLYSIS

Fibrinolysis dissolves clots that are no longer needed after vessel repair. The main players are:

  • Plasminogen: the inactive form in circulation; binds to clots and is activated to plasmin by tissue plasminogen activator (TPA).

  • Plasmin: the active enzyme that degrades fibrin.

Laboratory test for fibrinolysis specifically includes the D-dimer test, which detects products of fibrin breakdown.

LABORATORY TESTS: HEMOSTASIS AND COAGULATION

The two most common routine tests are aPTT (activated partial thromboplastin time) and PT (prothrombin time). These tests measure clotting times in seconds and help assess intrinsic/common (aPTT) and extrinsic/common (PT) pathways. Prolonged results indicate coagulation abnormalities that warrant further investigation. In addition, specialized tests are used to identify specific factor or platelet deficiencies:

  • Fibrinogen assay

  • Factor assays (e.g., VIII or IX deficiency/Hemophilia)

  • D-Dimer

  • Thrombin time

  • Platelet function assays

Reference ranges and diagnostic/therapeutic purposes:

  • aPTT: typically 25{-}40\,s; measures intrinsic and common pathways; prolonged suggests factor deficiency or acquired coagulopathy; used to monitor heparin therapy.

  • PT: typically 10{-}13\,s; measures extrinsic and common pathways; prolonged suggests factor VII deficiency, liver disease, or vitamin K deficiency; used to monitor warfarin therapy via INR.

  • INR (international normalized ratio): INR = \left( \dfrac{PT{\text{patient}}}{PT{\text{normal}}} \right)^{ISI}; normal range 1.0{-}1.4; therapeutic INR for warfarin usually about 2.0{-}3.0 depending on indication.

The laboratory also reports the tissue factor–dependent PT reagent, which includes tissue factor and phospholipids; the extrinsic pathway activation leads to prothrombin activation to thrombin, converting fibrinogen to fibrin and forming a clot.

SPECIALIZED LABORATORY TESTS

  • Fibrinogen assay: measures the ability of fibrinogen to form fibrin after exposure to thrombin.

  • Factor assays: identify specific factor deficiencies (e.g., VIII or IX deficiency).

  • D-Dimer: detects fibrin degradation products; indicates fibrinolysis activity.

  • Thrombin time: measures the time for fibrinogen to convert to fibrin.

  • Platelet function assays: assess platelet aggregation capacity and function.

LABORATORY METHODS FOR COAGULATION TESTING: CLOT DETECTION

Clot detection methods include:

  • Visual (tubes, water bath, reagents) to observe clot formation.

  • Mechanical/Magnetic methods: use movement of a metal ball between magnets or movement of probes; fibrin formation restricts movement and marks the endpoint (clot time).

  • Optical (photo-optical) methods: detect changes in optical density or turbidity as clot forms; endpoint recorded as clotting time.

SODIUM CITRATE AS AN ANTICOAGULANT; COAGULATION SAMPLES

  • Sodium citrate (3.2% buffered) chelates calcium, preventing clotting during sample collection.

  • After collection, clotting is initiated in the assay by adding calcium back to the sample along with tissue factor in PT testing.

  • The typical blood-to-anticoagulant ratio for accurate coagulation testing is 9:1 (9 parts blood to 1 part citrate). The specimen must be mixed gently (usually 3–4 inversions) to prevent activation of coagulation.

  • Incorrect handling includes under-filling, excessive inversions, clots, or microclots, which can falsely prolong clotting times or cause invalid results.

COAGULATION SAMPLES: HANDLING AND STORAGE

  • PT samples should be tested within 24 hours if stored at room temperature; aPTT samples should be tested within 4 hours; for heparin monitoring, aPTT samples should be tested within 60 minutes of collection.

  • Plasma must be prepared as platelet-poor plasma (PPP) by removing buffy coat. PPP can be stored frozen at \(-20^{\circ})C for up to 2 weeks or at \(-70^{\circ})C for longer durations.

  • PPP preparation requires careful handling to avoid disturbing the buffy coat and ensuring a proper 9:1 ratio with citrate during collection.

PLATELET POOR PLASMA PREPARATION (PPP)

Steps:

  1. Centrifuge the citrate tube.

  2. Use a transfer pipette to remove plasma, avoiding the buffy coat; transfer the top 2/3 to an aliquot tube.

  3. Centrifuge the aliquot at 1500 g for 10 minutes.

  4. Transfer the top 3/4 of the plasma to another aliquot tube (do not disturb the bottom).

  5. Label with patient ID and indicate PPP.

  6. Freeze according to established protocols.

INTERFERING FACTORS IN CLOT-BASED TESTS; SOLUTIONS

Common problems and solutions:

  • Tube not filled to minimum volume can falsely prolong PT; recollect with correct volume.

  • Hematocrit > 55% reduces plasma volume and affects the 9:1 ratio; adjust anticoagulant volume accordingly.

  • Clots or visible microclots: recollect.

  • Lipemia or icterus can affect optical tests; use mechanical methods or correct for interference if possible.

  • Hemolysis can affect results; recollect.

  • Incorrect reagent or QC dilutions: correct dilutions and repeat test.

QC materials and reagents:

  • Reagents and QC materials may be lyophilized and require reconstitution with specified diluents; some reagents require refrigeration while others are room temperature.

  • QC materials are best used within 48 hours after reconstitution; allow reagents to sit for 15–30 minutes before use; some tests require refrigerated storage; automated instruments may have onboard refrigerated reagent sections.

AUTOMATED COAGULATION ANALYZERS; QC

  • Modern coagulation testing uses automated and semi-automated analyzers capable of running several assays on platelet-poor citrated plasma.

  • Different reagents are required for different tests; each reagent lot should be quality controlled before patient testing.

PROTHROMBIN TIME (PT) AND INR; INTERPRETATION

PT evaluates extrinsic and common pathways. Common clinical uses include:

  • Pre-surgical screening

  • Monitoring warfarin therapy (often with INR)

Factors influencing PT include deficiencies in the extrinsic and common pathways, warfarin therapy, liver disease, and vitamin K deficiency.

Principle: In citrated plasma, adding thromboplastin and CaCl2 activates the extrinsic pathway, leading to prothrombin activation to thrombin and conversion of fibrinogen to fibrin clots. The PT is reported in seconds; a separate INR calculation standardizes results across laboratories:
INR = \left( \dfrac{PT{\text{patient}}}{PT{\text{normal}}} \right)^{ISI}
Normal INR range is typically 1.0{-}1.4; therapeutic INR for many indications is about 2.0{-}3.0.

ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT)

The aPTT assesses the intrinsic and common pathways and is used to monitor unfractionated heparin therapy.

  • It uses partial thromboplastin reagents with a phospholipid component and an activator (e.g., silica).

  • The test is performed in two steps with an incubation period in between, then CaCl2 is added to initiate clotting.

  • Normal range is typically 25{-}40\,s; prolonged aPTT suggests factor deficiencies or anticoagulant effects.

POINT-OF-CARE TESTING (POCT) FOR COAGULATION

Small hand-held devices provide rapid PT/INR and aPTT testing at the patient bedside or clinic. Reference values can differ somewhat depending on the instrument and sample type (plasma, capillary blood, or whole blood).

  • Common POCT devices include HemoChron, ProTime, and CoaguChek systems.

CHECK-IN QUESTIONS (SUMMARY PROMPTS)

  • List the four interrelated systems that have a role in hemostasis.

  • Describe the blood vessels’ role in hemostasis.

  • Outline the role of platelets in clot formation.

  • What is the general reference range for PT in citrated plasma, and what is the preferred method of reporting PT results? Explain why.

  • What type and concentration of anticoagulant is used to collect blood for PT and aPTT testing?

  • Which hemostasis pathways are measured by the PT test? Which are measured by the aPTT test? What is the purpose of adding CaCl2 to the aPTT test?

PRACTICE QUIZ PREPARATION (ANSWERS)

  • Final result of primary hemostasis: b. Platelet plug

  • In the aPTT, CaCl2 is mixed with c. Phospholipid component of thrombin (phospholipids) and kaolinite-based activators are used in some reagents (commonly kaolin or silica as activator; the calcium chloride is added later in the assay).

  • The cells that contribute most to blood clotting: b. Platelets

  • The mineral required for coagulation: e. Calcium

  • For optimal PT/aPTT results, testing should be done within d. 4 hours of collection

  • PT assay measures: f. Extrinsic pathway and h. Thromboplastin

  • The test commonly used to monitor heparin therapy: l. PT/INR (with aPTT also used depending on protocol)

SUMMARY FOR EXAM PREP

  • Hemostasis is a coordinated process involving vascular constriction, platelet plug formation, coagulation factor activation, and fibrinolysis.

  • Primary hemostasis covers vascular response and platelet plug formation; secondary hemostasis covers coagulation cascades creating a fibrin clot.

  • PT/INR reflect the extrinsic and common pathways; aPTT reflects the intrinsic and common pathways.

  • D-dimer indicates fibrinolysis activity; fibrinogen and factor assays help pinpoint specific deficiencies.

  • Anticoagulants have distinct mechanisms (warfarin: vitamin K–dependent factors; heparin: thrombin and Xa; aspirin: platelet function).

  • Sample handling (citrate anticoagulation at 9:1 ratio, avoiding tissue fluid contamination, and proper PPP preparation) is critical for accurate results.

  • Quality control on reagents and automated analyzers ensures reliability of clot-based testing.

{PT}{ ext{normal}} \,=\ 10{-}13\,s, \quad {aPTT}{ ext{normal}} \,=\ 25{-}40\,s, \quad INR{normal} \,=\ 1.0{-}1.4 {INR} = \left( \dfrac{PT{ ext{patient}}}{PT_{ ext{normal}}} \right)^{ISI}
{ ext{Therapeutic INR}} \,\approx\ 2.0{-}3.0

Key procedural values to remember:

  • Citrate volume ratio: \dfrac{V{blood}}{V{citrate}} = 9:1

  • PPP preparation involves centrifugation at 1500\,g for 10\text{ min} and careful removal of plasma without disturbing the buffy coat.

  • PT testing window: within 24\,\text{h}; aPTT testing window: within 4\,\text{h}; heparin monitoring: within 60\,\text{min} of collection.

  • Storage for PPP: -20^{\circ}\text{C} up to 2 weeks; -70^{\circ}\text{C} for longer durations.

  • Clot detection: visual, mechanical/magnetic, and optical methods.

These notes summarize the content from the MLHP 106 Hematology and Transfusion Science module covering hemostasis, coagulation testing, sample handling, testing methods, and interpretation of PT, aPTT, INR, and related assays.