Activated Partial Thromboplastin Time (aPTT) Mr. Mohammed A. Jaber

Objective The PTT measures functional activity of the intrinsic and common pathways. The PTT can evaluate for inherited or acquired deficiencies in these pathways. The PTT is also used to monitor heparin therapy. Heparin is given to people to prevent blood clotting. Heparin acts to accelerate Antithrombin which inhibits the actions of thrombin.

Objective It is also the single most useful procedure available for routine screening of coagulation disorders. It measures deficiencies mainly in factors VIII, IX, XI, and XII, but can detect deficiencies of all factors except III and VII. The PTT can also detect inhibitors of blood coagulation, such as the lupus-like anticoagulant. The reference values for the activated PTT test, using manual methods, are 28.0-35.0 seconds.

principle The principle of this test relies on the assumption that during anticoagulation the Calcium present in the blood is bound to the anticoagulant. After centrifugation, the Plasma contains all the intrinsic coagulation factors except calcium (removed during anticoagulation) and the platelets (removed during centrifugation).

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.

principle The activator provides a negatively charged surface for activation of factor XII. After a specific incubation time of citrated plasma with the APTT reagent which allows for optimum activation of contact factors, calcium chloride is added. The time required for the plasma to clot is the activated partial thromboplastin time.

Clinical Significance: A long partial thromboplastin time indicates a significant defect in at least one of the plasma procoagulants. The PTT time is abnormal with deficiencies XII, XI, IX, VIII, PK, HK (intrinsic pathway) and X, V, II, I or XIII (common pathway)

Clinical Significance: When the partial thromboplastin time is used in combination with the prothrombin time, most procoagulant disorders can be classified. Plasma with a long prothrombin time and a normal partial thromboplastin time is deficient in factor VII. If both the prothrombin time and the partial thromboplastin time are long, the plasma is deficient in X, V, II, I or XIII.  If the prothrombin time is normal but the partial thromboplastin time is long, the Plasma is deficient in factor XII, XI, IX, or VIII.

Clinical Significance: In addition to screening for coagulation defects, the APTT test is used to monitor heparin therapy. In general the PTT of a patient on heparin therapy should be 1½ to 2½ times normal.

Procedure: Pre- warm a sufficient quantity of calcium chloride reagent to 37oC for the number of tests to be performed. Pipet 100 µL or 0.1 mL of normal control into a labeled test tube. Into each test tube, add 100 µL or 0.1 mL of partial thromboplastin reagent. Incubate the control/ partial thromboplastin mixture at 37oC for a minimum of three (3) minutes.

Procedure 100 µL APTT reagent 100 µL plasma Incubation at 37° C Add 100 µL CaCl2 100 µL APTT reagent 100 µL plasma Incubation at 37° C 3-5 min. Time to clot formation

Procedure: Forcefully add in100 µL or 0.1mL of calcium chloride into the control/ partial thromboplastin mixture and start the stop watch immediately. Mix the tube once, immediately after adding the calcium reagent. Allow the tube to remain in the water bath, approximately 20 seconds, mixing occasionally. After 20 seconds, remove the tube from the water bath/heat block. Wipe off the outside of the tube. Gently tilt the tube back and forth until a visible clot forms.

Procedure: Stop the stop watch immediately and record the time in seconds. Carry out 1 significant figure passed the decimal point. For example, if your result is 30.31, report as 30.3 seconds. Repeat the procedure for a second run of control. Record the time and average the two results if they match appropriately. If not, repeat a third time and average the two that match within acceptable limits. Be sure and cross out any values you aren’t using for the final calculation. Include measurement unit of seconds on report sheet.

Repeat steps 2-8 for the patient sample. Procedure: Repeat steps 2-8 for the patient sample. For PTT, results should match within 2 seconds for results less than 40 seconds. Results over 40 seconds should match within 3 seconds.

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 Delay in testing or processing Inappropriate storage

Sources of Error Associated with storage Incorrect preparation of reagents Failure to properly store reagents Use of reagents beyond reconstituted stability time or expiration date Contaminated reagents

Sources of Error Associated with procedure Incorrect temperature Incorrect incubation times Incorrect volumes of sample, reagents or both

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