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

Evaluation of Coagulation Factor Disorders

Medical History:
1. Symptoms 2. Onset of Bleeding 3. Location of Bleeding (?) 4. Family History 5. Drugs & Medications (?) 6. Other Systemic diseases

Physical Exam:

Laboratory Screening Tests

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

I. Prothrombin time /Protime/PT

Screen for deficiencies in: Extrinsic Pathway
FI, FII, V, VII, X

Test of choice for monitoring anticoagulant therapy by Vit K antagonists (II, VII, IX, X) Principle: Thromboplastin + Platelet-poor plasma & Ca++ react w/ FVII to activate FX --convert Prothrombin to Thrombin

 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/ Ref range 2. Patient time in sec w/ control time 3. Prothrombin ratio (PT divided by mean of Ref range multiplied by 100) 4. Percent Activity (outdated?)

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

- Values usually provided by manufacturers of reagents

 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

II. 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. Platelet substitute (phospholipid) prepared from brain & plant phospholipids 2. Activator - Kaolin, Celite, micronized silica, or ellagic acid

 Specimen source:
Citrated platelet-poor plasma (PPP)

Reagents:
Phospholipid w/ Activators (PTT reagent) 0.025 M CaCl2

Procedure: PPP (0.1mL) + 0.1mL PTT reagent,

incubate at 37C for 3-5min, + 0.1mL warm CaCl2.

 Reporting:
Reported in seconds, to the nearest tenth, along with reference range.

Reference range: vary according to reagent, method, instrument used

Lower limit of 20 sec to an upper limit of 45 sec

 Interpretation:
Prolonged PTT in the absence of Heparin use indicate:
Factor deficiency Acquired circulating anticoagulant (e.g. Lupus inhibitor) Antibody to a specific Factor (e.g. anti-FVIII)

Prolonged PTT if factor measured is <40% to 50% of normal

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

2. Reagent preparation
a. Improper storage b. Water impurities c. Incorrect dilution

3. Instrumentation
a. b. c. d. Failing light source Fluctuations in temperature Loss of calibration of tubing Contamination

III. Platelet Count

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

 Specimen:
Capillary blood from finger puncture
Values are lower because of clumping at puncture site

Venous blood EDTA (Lavender tube) as anticoagulant of choice

 Method of Determination:
1. Manual Count
a. Unopette system b. Hemacytometer

2. Automated Method a. Optical methods b. Impedance methods

Calculation: Manual Platelet Count

Ave # of Plt in 4 squares X 1mm2 x 100 0.1 mm

X 106 = Platelets X 109/L

Platelet Reference Range: 150 450 x 109/L

 Clinical Significance:
Thrombocytopenia:
Platelet values lower than normal

Thrombocytosis:
Platelet values higher than normal

TESTS FOR PLATELET FUNCTION

1. Platelet Closure time 2. Platelet Aggregation Studies 3. Automated Platelet Function Analysis

Platelet closure time:

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

CLOTTING TIME Time required for a blood sample to coagulate/clot in vitro under standard conditions. Most common method: Capillary Tube Method. Affected by calcium ion levels and many diseases.

Normal value: 5 to 8 minutes.

Time taken for blood to clot reflects time required for the generation of thrombin

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. Platelet numbers & function 2. Plasma VIII:vWF deficiencies 3. Vessel wall composition

Standardization of Wound size is a problem Employs BP cuff inflated at 40 mmHg

 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