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Laboratory investigations in bleeding disorders FINAL.pptx

Most important presentation for dmlt and mbbs on lab investigation of bleeding disorders

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1 Laboratory Investigations in Hemorrgic Disorders
INTRODUCTION • Bleeding disorders constitute an important group of disorders in haematology. • Bleeding disorders result when the blood's ability to form a clot at the site of blood vessel injury is impaired. • They are classified as disorders of primary hemostasis (platelet or vascular disorders) or disorders of secondary hemostasis (coagulation protein disorders). 2
3 Bleeding disorders: • Excessive bleeding can result from : 1)Increased fragility of vessels 2)Platelet deficiency or dysfunction 3)Derangement of coagulation • The evaluation of a patient suspected of having a bleeding disorder can be divided into two parts: - Clinical evaluation–history, physical examination, family history - Laboratory evaluation–screening tests, specific tests.
4 CLASSIFICATION OF BLEEDING DISORDERS  Bleeding disorders are classified as: - Disorders of primary hemostasis - Disorders of secondary hemostasis
5 A) DISORDERS OF PRIMARY HEMOSTASIS: 1) Vessels wall abnormalities - Congenital e.g. Ehlers-Danlos syndrome - Acquired e.g. Henoch-Schönlein purpura 2) Platelet abnormalities: - Quantitative: Thrombocytopenia (e.g. ITP, drug-induced, congenital) - Qualitative: Platelet function disorders i) Inherited, e.g. Glanzmann thrombasthenia, Wiskott-Aldrich syndrome, Bernard Soulier syndrome ii) Acquired, e.g. Uremia drugs
6 B) DISORDERS OF SECONDARY HEMOSTASIS: (COAGULATION SYSTEM) - Congenital: Hemophilia A,B; von Willebrand disease; other coagulation factor deficiencies[XI, VII, II, V, X] - Acquired: Vitamin K deficiency, Liver disease, Disseminated intravascular coagulation
7 AQUIRED BLEEDING DISORDERS DISEASE • Liver disease and cirrhosis • Gastrointestinal malabsorption • Shock/Sepsis/Malignancy • Renal disease • Lymphoproliferative disorders • Amyloidosis PATHOPHYSIOLOGY - Decreased synthesis of coagulation factors, thrombocytopenia - Vitamin K deficiency - DIC, increased consumption of coagulation factors and platelets - Acquired platelet dysfunction - Acquired autoantibodies to specific coagulation factors(inhibitors) - Acquired factor X deficiency, blood vessel infiltration
8 DIAGNOSTIC APPROACH TO THE BLEEDING DISODERS A) Clinical evaluation: i) History ii) Physical examination iii) Family history B) Laboratory evaluation: i) Screening tests ii) Confirmatory tests.  The evaluation of a patient suspected of having a bleeding disorder can be divided into two parts:
9 LABORATORY EVALUATION OF COAGULATION SYSTEM • Screening tests: 1. Prothrombin Time 2. Activated Partial Thromboplastin time 3. Thrombin Time • Confirmatory tests: 1. Reptilase time 2. Mixing tests 3. Fibrinogen assay(Modified Claus Assay)
10 Screening tests for hemostasis
11 TESTS TO EVALUATE CIRCULATING INHIBITORS • Platelet neutralizing procedure • Lupus anticoagulant & antiphospholipid antibodies
12 LAB INVESTIGATIONS OF FIBRINOLYTIC SYSTEM • Detection of FDPs using latex agglutination method • D-dimer test • Euglobin clot lysis
13 SPECIMEN COLLECTION 1.Type of Vacutainer: - Blood is collected in a light blue-top vacutainer, which contains 3.2% sodium citrate as the anticoagulant. 2.Blood-to-Anticoagulant Ratio: - A strict 9:1 ratio of blood to sodium citrate is required to ensure valid coagulation test results.
14 3.Venipuncture Technique: - Blood should be drawn using a clean, atraumatic venipuncture to avoid hemolysis and activation of coagulation pathways. - For accurate coagulation studies, it's also important to avoid traumatic venipuncture, which may release tissue thromboplastin and interfere with test results.
15  Special Note – Torniquet use: - In sample collection for bleeding disorders, tourniquet application is generally avoided or minimized, and if used, it should be applied briefly and gently. - Prolonged or tight tourniquet application can cause hemoconcentration, activate platelets, and alter coagulation test results (especially PT, aPTT, and D-dimer). - If necessary, a tourniquet may be applied loosely for no more than 1 minute and released immediately after venipuncture begins. - For platelet function tests or bleeding time, tourniquets are strictly avoided, as they can activate platelets artificially.
16 4. Sample Handling: - Tubes must be gently inverted several times to mix and transported promptly to the lab; clotting or delay can lead to inaccurate results. 5. Plasma Preparation: - Samples are centrifuged at 1500–2000g for 10–15 minutes to obtain platelet- poor plasma, essential for tests like PT, aPTT, and factor assays.
17 6. Storage Guidelines: - If not tested within 2 hours, plasma should be frozen at -20°C or lower to preserve coagulation factor stability.
18 SCREENING TESTS
19 1. PROTHROMBIN TIME • Principle: - Tissue thromboplastin and calcium are added to plasma & clotting time is determined. - Assesses the extrinsic and common coagulation pathways, mainly factors I, II, V, VII, X. • Collection: - Light blue-top vacutainer (3.2% sodium citrate). • Equipment: - Water bath at 37°C - Test tubes - Stopwatch
20 • Reagents: - Thromboplastin reagent: contains tissue factor and phospholipids - Calcium Chloride 0.025 moL/liter • Method: - Deliver 0.1ml of plasma in a glass test tube kept in water bath at 37°C. - Add 0.1ml of thromboplastin reagent and mix. - After 1 minute, add 0.1ml of calcium chloride solution. Immediately start the stopwatch record the time required for clot formation. • Normal Range: - 11–16 seconds; INR: 0.8–1.2.
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22 • Prolonged In: - Vitamin K deficiency - Warfarin therapy - Liver disease - DIC - Inherited deficiency of factors in extrinsic & common pathways. • Advantages: - Widely available, standardized with INR. • Disadvantages: - Affected by sample handling; limited for intrinsic defects.
23 INR – International Normalized Ratio • Standardized form of Prothrombin Time (PT) used to monitor oral anticoagulant therapy (e.g., warfarin). • Calculated using the formula: INR = (Patient PT / Mean Normal PT) ^ ISI, where ISI is the International Sensitivity Index of the thromboplastin reagent. • High INR indicates increased bleeding risk, while low INR suggests inadequate anticoagulation. • Essential for dose adjustment of warfarin and for preventing thromboembolic events.
24 ISI – International Sensitivity Index • ISI reflects the sensitivity of thromboplastin reagent compared to an international reference standard. • It is a numerical value assigned by the manufacturer and is critical for standardizing PT results across different laboratories. • The lower the ISI, the more sensitive the reagent. • ISI is used in the INR formula: INR = (Patient PT / Normal PT)^ISI — to adjust for reagent variability and allow reliable warfarin monitoring.
25 2.ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) • Principle: - Plasma is incubated with an activator (which initiates intrinsic pathway of coagulation by contact activation). - Phospholipid and calcium are then added & clotting time is measured. - Evaluates intrinsic and common pathways (factors VIII, IX, XI, XII). • Collection: - Light blue-top tube (citrated plasma).
26 • Equipment: - Water bath at 37°C - Test tubes - Stopwatch • Reagents: - Kaolin 5gm/liter(contact activator) - Phospholipid - Calcium Chloride 0.025 mol/liter
27 • Method: - Mix equal volumes of phospholipid reagent and calcium chloride solution in a glass test tube and keep in a water bath at 37°C. - Deliver 0.1ml of plasma in another test tube and add 0.1ml of kaolin solution. - Incubate at 37°C in the waterbath for 10 mins. - After exactly 10 mins, add 0.2ml of phospholipid-calcium chloride mixture, start the stopwatch and note the clotting time.
28 • Normal Range: - 30–40 seconds. • Prolonged In: -Hemophilia A/B -Heparin therapy -Lupus anticoagulant -DIC
29 • Advantages: - Sensitive to most factor deficiencies. • Disadvantages: - Affected by pre-analytical variables (e.g., poor sample quality).
30 3)Thrombin Time (TT) • Principle: - Assesses the final step of coagulation i.e. measures conversion of fibrinogen to fibrin by thrombin. - Thrombin is added to patient’s plasma & time required for clot formation is noted. • Collection: - Citrated plasma. • Equipment: - Water bath at 37°C - Test tubes - Stopwatch
31 • Reagent: - Thrombin solution • Method: - Take 0.1ml of buffered saline in a test tube and add 0.1ml of plasma. - Note clotting time after addition of 0.1ml of thrombin solution. • Normal range: - +/- 3 seconds of control
32 • Prolonged In: • Hypofibrinogenemia, dysfibrinogenemia, afibrinogenemia • Heparin therapy • DIC • Advantages: Simple, rapid. • Disadvantages: Non-specific; abnormal in presence of heparin or FDPs.
33 4)Bleeding Time (BT) •Principle: - Measures primary hemostasis—the time taken for bleeding to stop after a standardized skin incision; reflects platelet function and vascular integrity. •Collection: - Requires capillary blood, usually using a template method (Ivy's method preferred). •Procedure: - Standardized incision made on the forearm; blotting paper used every 30 seconds until bleeding stops.
34 • Normal Range: - 2–7 minutes (Ivy method). • Prolonged In: - Thrombocytopenia - von Willebrand disease - Uremia - Aspirin use • Advantages: - Simple, inexpensive bedside test. • Disadvantages: - Poor reproducibility, operator dependent, largely replaced by platelet function analyzers.
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36 5)Clotting Time (CT) •Principle: - Measures intrinsic pathway of coagulation—time taken for whole blood to clot in a test tube. •Collection: - Venous or capillary blood in a glass tube, no anticoagulant. •Procedure: - Capillary method (Duke's) or tube method (Lee-White); observe clot formation at intervals.
37 •Normal Range: - 6–10 minutes (Lee-White method). •Prolonged In: - Hemophilia A/B - Severe liver disease - DIC •Advantages: - Inexpensive. •Disadvantages: - Non-specific, poor sensitivity and standardization; obsolete in modern practice.
38 6) PLATELET COUNT  Various methods are used to perform platelet count: 1. Manual Platelet Count (Hemocytometer Method) • Uses a Neubauer chamber and diluted blood (usually 1% ammonium oxalate). • Platelets are counted under a phase contrast microscope. • Time-consuming and less accurate; used in resource-limited settings or when automated counts are doubtful.
39 2. Peripheral Blood Smear Estimation: • Semi-quantitative method: Estimate platelet count by counting platelets per oil- immersion field. • 10–20 platelets/HPF = ~150,000–400,000/µL. • Useful for confirming automated count accuracy, detecting giant platelets, or platelet clumps.
40 3. Impedance (Coulter) Method • Most common method in automated hematology analyzers. • Based on electrical resistance: as cells pass through an aperture, each platelet causes a pulse. • Differentiates platelets by size (2–20 fL).
41 4. Optical/Light Scatter Method: • Uses laser light scatter and flow cytometry principles. • Differentiates platelets from other cells based on size, granularity, and light scatter patterns. • More accurate in distinguishing platelets from RBC fragments or microcytes.
42 5. Fluorescence Flow Cytometry • Uses fluorescent dyes that bind specifically to platelets. • Highly sensitive and accurate; useful in cases with platelet clumping, giant platelets, or thrombocytopenia. 6. Immunological Methods (e.g., CD41/CD61 tagging) • Flow cytometry-based method using monoclonal antibodies against platelet-specific antigens (e.g., GPIIb/IIIa). • Very accurate, especially in diagnosing immune thrombocytopenia.
43 Incorrect platelet counts Spurious low platelet count Spurious high platelet count • Large or giant platelets, • Platelet satellitism • EDTA-induced platelet clumping • Previous contact of platelets with foreign surfaces such as dialysis membranes • Abnormal amounts of plasma proteins in various paraproteinemias • Platelet agglutinins • Microspherocytes • Fragmented red blood cells • Cytoplasmic fragments from leukemic blasts • Pappenheimer bodies • Electronic noise due to poor earthing of instrument • Circulating micro-organisms like Candida spp.
44 D-dimer •Principle: - Measures fibrin degradation products—indicates recent fibrin formation and breakdown. •Collection: - Citrated plasma. •Procedure: - ELISA, latex agglutination, immunoturbidimetry.
45 •Interpretation: - Elevated in active clot breakdown. •Normal Range: - <500 ng/mL (varies by method). •Increased In: - DVT, PE - DIC - Post-surgery, trauma
46 Clot solubility test • Patient plasma is recalcified to induce a clot; • Clot is then suspended in 5 mol/L urea (or 1% monochloracetic acid) for 24 hours. • Principle of the factor XIII screening test is that clots cross-linked by factor XIII resist denaturation by high concentrations of urea or acid. • Deficiency of factor XIII results in premature clot lysis .
47 QUALITY CONTROL • Quality Control – General Measures: - All tests and controls should be performed in duplicates. - Ensure operator skill through proper training. - Required instruments must be properly calibrated. • Equipment-Specific QC: i) Centrifuge: - Serviced and calibrated every 6 months or after breakdown - Placed on sturdy platform to reduce vibrations - Balanced sample load during use
48 ii) Water Baths: - Maintain temperature at 37°C (check in all 4 corners) - Use transparent walls for better visualization - Fill with distilled water to indicated level iii) Pipettes: - Must be calibrated - Use disposable tips to avoid contamination