In this exercise, two tests will be performed to screen for defective clotting factors. The formation of thrombin in the plasma samples will be inhibited by an anticoagulant. The anticoagulant used, either citric acid or oxalic acid, removes calcium ion (Ca2+) from the plasma. The removal of Ca2+ has an anticoagulant effect because calcium is a necessary cofactor in the activation of a number of the clotting factors. This inhibition can be easily reversed by adding calcium ion during the clotting tests.

Clotting time or Coagulation time The time required for blood to clot in a glass tube. The normal range for the test described below is 5 to 15 min. but each laboratory should determine its own normal values. Reagent & equipment water bath, 37C. Glass test tube. Stopwatch. Plastic syringe and 20-gauge needle. Specimen: fresh whole blood , 4 ml .

Procedure Label 3 glass test tube with patient name and number them, #1, #2, and #3. Add 1 ml of blood in each tube. The last 1 mL of blood may be discarded. Start the stopwatch as soon as the blood is placed in tube #3. Place the three test tubes in a 37°C water bath. 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).

Record the time it took the blood in test tube #1 to clot. 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.

Clotting time - capillary method Material Sterile disposable pricking needle or lancet. Stop watch Dry glass capillary tube (narrow diameter 1 top 2 mm, minimum 10 cm long.) Cotton Swab of absorbent cotton. Spirit wetted, cotton swab. 70 % v/v ethyl alcohol

Clotting time of whole blood

Clotting Time - Slide Method 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.

Terms Why is PPP essential? Platelet –Poor plasma (PPP) <10 x 10 9 /L. Why is PPP essential? Contains platelet factor 4(heparin neutralizer). Contains phospholipid (affects lupus anticoagulant and factor assay testing). Contains proteases (affect testing for vWF). Platelet-Rich plasma (PRP) 200-300 x 10 9 /L.

PT Reagent Calcium ions and Thromboplastin from brain tissue (Rabbit). Thromboplastin (Tissue Factor) protein-lipid complex found in tissues outside blood vessels.

Principle The procedure uses a tissue thromboplastin reagent with CaCL (Calcium Chloride) to provide a one step procedure for evaluating plasma clotting. The normal values for the prothrombin time range from 10.0 to 13.0 seconds. An elevated prothrombin time may indicate the presence of Vitamin K deficiency, DIC, liver disease, Presence of FSP’s

Performing a PT test Pre-warm PT reagent and sample to 37 oC Add 100 L sample to tube Add 200 L of PT reagent to tube Start timer Record time to clot in seconds If the results from run 1 and run 2 are within + 1 second from each other, average the two results and report with appropriate units. If results are not within required limits, a third run should be performed and average the two that match within acceptable limits. Calculate INR

Expected PT Values Mean Normal Plasma = 10 to 14 seconds. A high sensitivity (Low ISI) PT will give a high normal PT value (13 to 15 seconds). Oral anticoagulant monitoring = Target INR of 2.0 to 3.0. INR of greater than 5 or 5.5 = unacceptable high risk of bleeding.

PTT Reagents Phospholipids and a surface activator Calcium Chloride reagent added to start the reaction. APTT reagent mimics the surface of a platelet.

Principle Under carefully controlled conditions and with properly prepared reagents, calcium, partial thromboplastin and an activator (such as kaolin, celite, micronized silica or ellagic acid) are added to the plasma to be tested, which eliminates the variability of activation by glass contect. The partial thromboplastin reagent stimulates activated platelet surfaces by providing a phospholipid platelet surface on which enzymatic reactions can occur. which eliminates the test’s sensitivity to platelet number and function.

Sample Citrate tube; 3.2% citrate tubes Ratio of anticoagulant to blood should be 1:9 After centrifugation, the Plasma contains all the intrinsic coagulation factors except calcium (removed during anticoagulation) and the platelets (removed during centrifugation).

Procedure Prewarm sufficient volumes of CaCl2 (0.025 M) to 37oC Label 2 test tube & 2 control tubes Add 0.1 ml (100µL) of sample or control to the appropriate tube Add 0.1 ml of APTT reagent to each tube Incubate at 37oC for 5 minutes Add 0.1 ml (100µL) of prewarmed CaCl2 & start stop watch immediately Record the time at which clot is detected

Expected APTT Values Normal Range: 25 to 43 seconds Slightly Elevated: 45 to 65 seconds Extremely Elevated > 70 seconds

What does the test result mean? APTT prolongations are caused by either factor deficiencies (especially of factors VIII, IX, XI, and/or XII), Inhibitors (most commonly, lupus anticoagulants{ immunoglobulin that binds to phospholipids and proteins associated with the cell membrane}) Therapeutic anticoagulants such as heparin. DIC Liver disease

Sources of error Associated with specimen Inappropropriate ratio of anticoagulant to blood Failure to correct citrate volume if hematocrit > 55% Clotted, hemolyzed or lipemic samples Lack of PPP

3. Associated with procedure 2. Associated with reagent Incorrect preparation of reagents Failure to properly store reagents Use of reagents beyond reconstituted stability time or expiration date Contaminated reagents 3. Associated with procedure Incorrect temperature Incorrect incubation times Incorrect volumes of sample, reagents or both

Defective Factor PT APTT V Abnormal VII Normal VIII IX X XI XII