Bleeding Time, Clotting Time, Prothrombin Time/PTT

Platelets (Thrombocytes)
• Function – interact with injured vascular wall structures, plasma proteins, other circulating blood cells
a. Adhesion: adhere to basement membrane of injured blood vessel b. Release action: release of alpha & dense granules, & lysosomes c. Aggregation: formation of secondary, irreversible platelet aggregation

• Coagulation:
– Starts with activation of Thromboplastin, from injured tissue

• Fibrinolytic System:
– Endothelial cells
• Heparin

– Antithrombin

Drugs & Medications (?) 6. Family History 5.Evaluation of Coagulation Factor Disorders • Medical History: 1. Other Systemic diseases • Physical Exam: . Symptoms 2. Location of Bleeding (?) 4. Onset of Bleeding 3.

Platelet count *If abnormal add: 1.Laboratory Screening Tests 1. Bleeding Time 2. Partial Thromboplastin Time (PTT) 3. Clotting Time . Prothrombin Time (Protime/PT) 2.

VII. V. X • Test of choice for monitoring anticoagulant therapy by Vit K antagonists (II. Prothrombin time /Protime/PT • Screen for deficiencies in: Extrinsic Pathway – FI. FII.I. IX. X) • Principle: Thromboplastin + Platelet-poor plasma & Ca++ react w/ FVII to activate FX --convert Prothrombin to Thrombin . VII.

Prothrombin ratio (PT divided by mean of Ref range multiplied by 100) 4. Patient time in sec w/ Ref range 2. Percent Activity (outdated?) .• Specimen source: – Citrated platelet-poor plasma (PPP) – Reference Range: Values differ • 10 – 12 seconds or 12 – 14 seconds • Reporting: 1. Patient time in sec w/ control time 3.

Comparison of Patient value with the normal value = result is normalized to an international reference preparation (IRP) of thromboplastin .Widely used nowadays .5.Values usually provided by manufacturers of reagents . International Normalized Ratio (INR): .

• Interpretation of results: – Sensitivity: detect deficiencies of <40% .50% of normal – Prolonged PT indicates abnormality in: • • • • Common pathway Extrinsic pathway Abnormality may be acquired or hereditary Factor inhibitors .

Celite. or ellagic acid . micronized silica. Activator . Partial Thromboplastin Time (PTT) • Test of choice for factor deficiencies of the Intrinsic & Common Pathway • Test of choice for monitoring Heparin therapy • PTT Reagent: 2 ocmponents 1.Kaolin. Platelet substitute (phospholipid) – prepared from brain & plant phospholipids 2.II.

1mL warm CaCl2. .• Specimen source: – Citrated platelet-poor plasma (PPP) • Reagents: – Phospholipid w/ Activators (PTT reagent) – 0.1mL) + 0.1mL PTT reagent. + 0.025 M CaCl2 • Procedure: PPP (0. incubate at 37°C for 3-5min.

along with reference range. instrument used – Lower limit of 20 sec to an upper limit of 45 sec . method.• Reporting: – Reported in seconds. • Reference range: vary according to reagent. to the nearest tenth.

• Interpretation: – Prolonged PTT in the absence of Heparin use indicate: • Factor deficiency • Acquired circulating anticoagulant (e.g. anti-FVIII) – Prolonged PTT if factor measured is <40% to 50% of normal .g. Lupus inhibitor) • Antibody to a specific Factor (e.

c. Sample collection & preparation a. d. e. Improper collection & specimen processing Incorrect anticoagulant-to-plasma ratio Hemolysis Platelets in plasma sample Unexpected heparin contamination .• Sources of Error: 1. b.

c. Failing light source Fluctuations in temperature Loss of calibration of tubing Contamination .2. Incorrect dilution 3. Improper storage b. Reagent preparation a. Water impurities c. Instrumentation a. b. d.

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Platelet Count • Formed in the BM from Megakaryocytes • Difficult to count: a. c. d. Small & difficult to discern Attached to surfaces/particles in diluting fluid Disintegrate easily Form clumps with other platelets .III. b.

• Specimen: – Capillary blood from finger puncture • Values are lower because of clumping at puncture site – Venous blood – EDTA (Lavender tube) as anticoagulant of choice .

Automated Method a. Unopette system b. Hemacytometer 2.• Method of Determination: 1. Optical methods b. Impedance methods . Manual Count a.

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1 mm X 106 = Platelets X 109/L Platelet Reference Range: 150 – 450 x 109/L .Calculation: Manual Platelet Count Ave # of Plt in 4 squares X 1mm2 x 100 0.

• Clinical Significance: – Thrombocytopenia: • Platelet values lower than normal – Thrombocytosis: • Platelet values higher than normal .

Platelet Aggregation Studies 3.TESTS FOR PLATELET FUNCTION 1. Platelet Closure time 2. Automated Platelet Function Analysis .

– Instrument and test cartridge system which simulates the process of platelet adhesion and aggregation following a vascular injury.Platelet closure time: – Assess platelet-related primary hemostasis. greater accuracy & reliability than Bleeding Time (BT). – Rapid evaluation of platelet function on samples of anticoagulated whole blood. – Sensitive to platelet adherence and aggregation abnormalities . – The time required to obtain full occlusion of the aperture is reported as the closure time (CT) in seconds. allows discrimination of aspirin-like defects and intrinsic platelet disorder. .

Platelet Aggregation Studies: – Measure response of platelet during hemostatic process • Change in shape • Increase in surface adhesiveness • Platelet plug formation – Use of Aggregating Agent – Measurement: Turbidimetry Method .

• Most common method: Capillary Tube Method. • Normal value: 5 to 8 minutes.CLOTTING TIME • Time required for a blood sample to coagulate/clot in vitro under standard conditions. – Affected by calcium ion levels and many diseases. • Time taken for blood to clot reflects time required for the generation of thrombin .

Plasma VIII:vWF deficiencies 3. Platelet numbers & function 2. Vessel wall composition • Standardization of Wound size is a problem • Employs BP cuff inflated at 40 mmHg .BLEEDING TIME • Principle: BT is the time it takes for a standard wound to stop bleeding • Comprehensive test of platelet action in vivo & is sensitive to the following abnormalities: 1.

• Reference range: – 2 to 9 minutes • Considerations for the Test: – – – – Volar skin must be dry Adequate incision/proper depth Site should not touch filter paper Aspirin& aspirin-containing meds must be discontinued 1 week prior to testing – NSAIDS must be discontinued 24 hrs prior to testing .