Activated Partial Thromboplastin Time (APTT / aPTT / PTT) Test Procedure

Principle

     Platelet-poor plasma is added to an equal volume of partial thromboplastin reagent and warmed to 37°C for an exact incubation time. Pre-warmed (37°C) calcium chloride reagent (0.025M) is added to this mixture to activate the coagulation cascade. The time required for clot formation is recorded. Clot formation may be detected by optical or electromechanical methods using manual, semi-automated, or automated devices.

Reagents and Equipment

1. Partial thromboplastin reagent: consists of phospholipids and a contact activator
   
2. Calcium chloride reagent, 0.025M
   
3. Fibrometer system
   a. Fibrometer
   b. Thermal prep block, 37 + 1°C
   c. Automatic pipet
4. Coagulation cups, non-wettable surface
   
5. Fibro-tips

Quality Control

Quality control materials (normal and abnormal) with established control limits should be run. NCCLS recommends controls be tested at the beginning of each testing day, followed by testing during each subsequent shift or with each batch of assays.

Specimen

Whole blood anticoagulated with 3.2% sodium citrate is the specimen of choice. The specimen should be processed to obtain platelet-poor plasma.

Procedure (Fibrometer Testing)

1. Pre-warm a sufficient quantity of calcium chloride reagent to 37°C for the number of tests to be performed.
   
2. Pipet 0.1 mL patient sample or control sample into labeled coagulation cup using the automatic pipet.
   
3. To each sample cup, add 0.1 mL of partial thromboplastin reagent using the automatic pipet. Carefully mix the contents of each cup.
   
4. Allow sample-partial thromboplastin reagent mixture to pre-warm to 37°C for 1-2 minutes. No longer than five minutes.
   
5. Pipet 0.1 mL pre-warmed calcium chloride reagent into coagulation cup containing sample-partial thromboplastin reagent mixture using the automatic pipet (switch in the "on" position). When the calcium chloride reagent is dispensed the timer will automatically start. Alternatively, the timer may be started by touching the timer plate. Reagent should be forcibly added to ensure mixing of reagent and sample.
   
6. The timer will stop when clot formation occurs.
   
7. Record time taken for clot formation.
   
8. Each sample (patient or control) should be run in duplicate.

Results

The average of the duplicate partial thromboplastin times are recorded to the nearest second.

Reference Interval

Each laboratory should establish its own reference interval following a recommended procedure. The reference interval should be re-established with changes in instrumentation, reagent lot number, or at least once a year.

Comments 

1- Partial thromboplastin reagent consists of phospholipids and a contact activator. Kaolin, celite, silica, and ellagic acid are examples of activators available. Care should be taken in choosing the partial thromboplastin reagent, since reagents vary in their sensitivity or insensitivity to lupus anticoagulant.

2- Each laboratory should determine the optimal incubation time for partial thromboplastin reagent and sample based on its assay procedure. The longer the incubation time, the shorter the partial thromboplastin times due to increased contact activation. Excessive heating (>5 minutes) will result in loss of Factor V and Factor VIII.

3- Precision between duplicate measurements is said to be acceptable if the difference between duplicates is 10% or less of the mean of the duplicates.

4- The APTT has been used to monitor heparin therapy. However, newer methodologies such as Factor Xa inhibition assay are replacing this use of the APTT.

5- The APTT is prolonged in:

• Inherited single factor deficiencies of factors XI, X, IX, VIII, V, II, and I.
• Disseminated intravascular coagulation (DIC).
• Presence of circulating inhibitors like lupus-like anticoagulant.

6- If the patient's hematocrit exceeds 55%, NCCLS recommends adjusting the amount of anticoagulant used in the collection tube to prevent over-anticoagulation of the specimen. This correction formula is:

C = (1.85 x 10^-3) X (100 - Hct) X V

Where: C = volume of sodium citrate, V = volume of whole blood drawn, Hct = patient's hematocrit.

  

Note
 
Potential sources of error

• Associated with specimen inappropriate ratio of anticoagulant to blood 
  failure to correct citrate volume if hematocrit >55%
  clotted, hemolyzed, icteric, or lipemic specimen
  delay in processing or testing
  inappropriate storage
  
• Associated with reagents incorrect preparation of reagents
  failure to properly store reagents
  use of reagents beyond reconstituted stability time
  use of reagents beyond expiration date
  contaminated reagents
• Associated with procedure
  incorrect temperature
  incorrect incubation times
  incorrect volumes of sample, reagents, or both
  improperly functioning instrument