Purpose
The Activated Partial Thromboplastin Time (APTT) assay is useful for three reasons: as a screening test for inherited or acquired deficiencies of the intrinsic pathway, to detect the lupus anticoagulant, and to monitor heparin therapy.
Principle
The test measures the clotting time of plasma after the activation of contact factors and the addition of phospholipid and CaCl2, but without added tissue thromboplastin, and so indicates the overall efficiency of the intrinsic pathway. Plasma is first preincubated for a set period with a contact activator such as kaolin. During this phase of the test, factor XIla is produced, which cleaves factor XI to factor Xla, but coagulation does not proceed beyond this in the absence of calcium. After recalcification, factor Xla activates factor IX and coagulation follows. PTT is affected by decreased levels of intrinsic pathway components (factors VIII, IX, XI, XII, prekallikrein, and high-molecular-weight kininogen), as well as decreased levels of common pathway components (fibrinogen, prothrombin, factor V and X). Factors VII and XIII are not measured by the PTT assay. It is also sensitive to the presence of circulating anticoagulants (inhibitors) and heparin.
Equipment & Reagents
1. PPP (platelet-poor plasma) prepared from the patient’s plasma was centrifuged at about 3000 rpm for ten minutes, and the platelet-poor plasma was separated into a plastic tube using a plastic Pasteur pipette.
2. PPP prepared from control plasma in the same way.
3. Kaolin 5 gram/L in barbitone buffered saline, pH 7.4.
4. CaCl2-0.025 mol/L stored at 4°C.
5. Phospholipid
5. Citrate vial
6. Centrifuge
7. Cuvette / glass tube
8. Waterbaths
9. Refrigerator
6. Micropipettes & pasteur pipette
5. Stopwatch
Sample
Citrated blood (9 volume blood: 1 volume 3.2% tri-sodium citrate)
Calibration Procedure
1. Mix equal volumes of the phospholipid reagent and kaolin suspension. Leave to warm up in a glass tube in the water bath at 37°C.
2. Place 0.1 ml of plasma in another glass tube. Add 0.2 ml of the kaolin-phospholipid reagent and start the stopwatch. Leave at 37°C for 10 minutes with occasional shaking.
3. At exactly 10 minutes, add 0.1 ml of pre-warmed calcium chloride and start a second stopwatch.
4. Record the time taken for the mixture to clot.
5. Perform the test on the patient’s plasma in duplicate and, also in duplicate, on the normal control plasma. Repeat the test if duplicate measurements differ by more than 5%.
Expression of Results
Express the results as the mean of the paired clotting times.
Normal Range
30-40 seconds
Interpretation of Results
The common causes of a prolonged APTT are as follows:
1. Disseminated intravascular coagulation (DIC)
2. Liver disease
3. Massive transfusion with plasma-depleted red blood cells.
4. Administration of or contamination with heparin or other anticoagulants.
5. A circulating anticoagulant (inhibitor).
6. Deficiency of a coagulation factor other than factor VII.
Interference & Cross-reaction
The actual times depend on the rengents used and the duration of the pre-incubation period, which varies according to the manufacturer’s recommendations for different reagents. These variables also greatly alter the sensitivity of the test to minor or moderate. deficiencies in the contact activation system. 31-
Sources of Error in Tests
- Error in blood collection: incorrect ratio of blood to citrate.
- Wrong anticoagulant for blood collection.
- Delay in separating plasma from cells.
- Delay in performing tests.
- Tests not performed with plasma and reagents kept at 37oC.
- Reagents detoriating: incorrectly storedor out of date.
- Technique is not constant. It is essential to use the same technique for incubation time, temperature, and pipetting.
- Inability to detect beginning of clot and/or slow response when using stopwatch.
Quality Control
1) Run control plasma daily. Take a mean of control values of 10 days, calculate standard deviation and CV%. The daily control value obtained subsequently should be within ±SD of control value.
2) When collecting samples in commercial citrate vials check the clotting times obtained from sample in vials with sample collected into lab prepared 3.2% sodium citrate for each batch of commercial vials.
Safety Precaution
Every test sample is regarded as potentially infectious material, and no universal safety precautions have to be taken.
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