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 Percent Activity (outdated?)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.



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.



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 .