Coagulation Time of whole blood

Coagulation Time ( Clotting Time) CT.

Clotting time was used as a screening test to measure all stages in the intrinsic coagulation system and to monitor heparin therapy . It is however, a time-consuming test, has poor reproducibility, is sensitive only extreme factor deficiencies, and is insensitive to high doses of heparin. It is therefore, of limited use in todays laboratory . Severe hemophilia, a fibrinogenemia, and sever fibrinolytic states cause a prolonged clotting time, as do circulating anticoagulants ( inhibitors), and heparin.

 The normal range for the test described below is 5 to 15 min. but each laboratory should determine its own normal values.

Reagent & equipment 1.Water bath, 37C. 2.Glass test tube. 3.Stopwatch. 4.Plastic syringe and 20-gauge needle.

 Specimen Fresh whole blood , 4 ml . Principle The coagulation time of whole blood is the length of time required for a measured amount of blood to clot under certain specified conditions.

Procedure
1. Label 3 glass test tube with patient name and number them, #1, #2, and #3. 2. Perform a clean, unromantic venipuncture using a 20-gauge needle and with drawn 4 mL of blood. 3. Remove the needle from the syringe, and carefully place 1 mL of blood in test tube #3, 1mL in tube #2, and 1mL in tube #1.

The last 1 mL of blood may be discarded. Start the stopwatch as soon as the blood is placed in tube #3.

Procedure
4. Place the three test tubes in a 37C water bath. 5. At exactly 5 min., title test tube #1 gently to a 45 angle. Repeat this procedure every 30 seconds, until the test tube can be completely inverted without spilling the contents (that is, until the blood is completely clotted). 6. Record the time it took the blood in test tube #1 to clot.

Procedure
7. 30 sec. after the blood in test tube #1 is clotted. Proceed with tube #2, and repeat the preceding procedure, tilting the test tube every 30 seconds, until a clot is formed. Record the results. Repeat this procedure for test tube #3. 8. Since agitation and handling speed up coagulation, the clotting time test tube #3 is the reported result.

Discussion
1. Poor venipuncture technique, causing hemolysis or tissue thromboplastin to mix with the blood, shortens the clotting time. 2. Bubbles entering the syringe when the blood sample is being obtained increase the rate of coagulation. Unnecessary agitation of the blood shortens the coagulation time. 3. Always tilt the tube in the same direction and at the same angle so that the blood is moving in the same pathway up the side of the tube each time. 4. At the completion of the clotting time, one tube should remain in the 37C water bath to be checked after 2 and 4 hours for clot retraction. Also, this same tube may be allowed to remain in the water bath overnight and checked the next day for clot lysis.

Clotting time - capillary method (Extrinsic)

Apparatus :
1. Sterile disposable pricking needle or lancet. 2. Stop watch 3.Dry glass capillary tube (narrow diameter 1 top 2 mm, minimum 10 cm long.) 4. Cotton Swab of absorbent cotton. 5.Spirit wetted, cotton swab. 6. 70 % v/v ethyl alcohol

PROCEDURE
1. Apply alcoholic 70 % v/v to the clean finger with cotton swab. Allow it to dry naturally. 2. Prick the finger with usual aseptic precautions. Immediately stop watch is started. 3. Dip one end of the capillary into blood drop gently without pressure. 4. Allow to fill the capillary with blood by lowering the end of fitted capillary. (Do not suck the blood) around th of its length undipped. 5. After every 30 seconds, using stopwatch, break a small piece of capillary. 6. Repeat breaking at regular time intervals, till fibrin thread appears at the broken end of capillary tube. Do not pull away the cut pieces ling apart and bristly. 7. Record time interval between pricking finger and first appearance of fibrin thread at the broken ends of capillary tube. That is clotting time of blood.

Clotting time of whole blood

Clotting Time - Slide Method (Intrinsic)

The surface of the glass tube initiates the clotting process. This test is sensitive to the factors involved in the intrinsic pathway The expected range for clotting time is 4-10 min.

Clot retraction
This test measures the amount of time it takes for a blood clot to pull away from the walls of a test tube. It is used to evaluate and manage blood platelet disorders, including Glanzmann's thrombasthenia

Glanzmann's thrombasthenia
Is an abnormality of the platelets It is an extremely rare coagulopathy in which the platelets lack glycoprotein IIb/IIIa . Hence, no fibrinogen bridging can occur, and bleeding time is significantly prolonged.

Clot retraction test

This is a measure of platelet function. The test is done in blood to which no anticoagulant has been added and is allowed to clot. Clot retraction is then looked for.
Clot retraction becomes abnormal in conditions like Fibrinogen deficiency (congenital or acquired) Thrombocytopenia < 1ooooo Thrombosthenia Polycythaemia

Procedrure
1. At the completion of the clotting time one tube should remain in the 37 water bath 2. Inspect the clot at 1,2,4 and 24 hr for retracted clot

Interpretation of results
The clot will retract from the walls of the tube until the red cell mass occupies 50% of the total volume of blood in the tube There is a variable degree of retraction or there is no retraction at all Results are reported as the length of time it took for the clot to retract
2-4 hrs is reported as normal After 4 hrs is reported as poor After 24 hrs is reported as no retraction

Clot lysis test

The whole blood clot lysis time is used to detect increase fibrinolysis This test is only able to detect high increase in fibrinolytic activity

Procedure
1. At the completion of the clotting time one tube should remain in the 37 water bath 2. A second tube is placed in the refrigerator as soon as clotted as a control 3. The tube is test for the disappearance of clot after 4, 8 and 24 hrs 4. If the sample becomes fluid in less than 48 hrs , the blood is poured out onto a piece of filter paper to be sure for clot disappearance

Interpretation
The disappearance of clot before 48 hrs means increase fibrinolysis After 48 hrs clot may be
37 C tube disappear Still intact Refrigerated tube Comment disappear Still intact Low fibrinogen Abnormal fibrinolysis (No clot lsis after 48 hrs) Nomal fibrinolysis

disappear

Still intact