Materials Needed:

Plasma sample (fresh or frozen plasma from the patient)

Reagent (activated partial thromboplastin time [aPTT] reagent)

Factor VIII-deficient plasma (to use as a standard or control for comparison)

CaCl2 (calcium chloride solution) – to help initiate the clotting process

Coagulometer or clotting analyzer to measure clot formation time

Calibrators or standard factor VIII-deficient plasma with known factor VIII activity levels

Sample Preparation :

Collect plasma from the patient using appropriate anticoagulants (usually citrate or EDTA).

Ensure the plasma is processed and stored correctly (freeze at -80°C if not testing immediately).

Pre-incubation of Plasma :

Thaw the plasma sample (if frozen), and keep it on ice until ready for testing.

If the plasma is not immediately tested, make sure to use fresh plasma, as prolonged storage can affect the clotting properties.

Reagent Preparation :

Prepare the aPTT reagent and ensure it is at the correct temperature, typically room temperature or 4°C as per the manufacturer’s guidelines.

Reconstitute or prepare calcium chloride (CaCl₂) according to protocol specifications.

Performing the Assay :

Add the patient’s plasma sample to a test tube (typically 50 µL).

Add aPTT reagent (as specified by the manufacturer) to the plasma sample (usually around 50 µL).

Mix gently and incubate the mixture for a specified time (typically 3-5 minutes at 37°C).

After the incubation, add calcium chloride (CaCl₂) to the mixture (about 50 µL) to initiate clotting. This step is critical to activate the intrinsic pathway of coagulation.

Clot Detection :

The clotting time is measured using a coagulometer or clotting analyzer.

The instrument will detect the time it takes for the sample to form a clot. This will be compared against standard or control samples with known Factor VIII levels (usually Factor VIII-deficient plasma, which provides a baseline).

Factor VIII Activity Calculation :

The clotting time for the patient sample is compared to the clotting time for the normal or control plasma, as well as to a standard curve generated from Factor VIII-deficient plasma with known Factor VIII activity levels.

Factor VIII activity is calculated as a percentage of the normal plasma value.

Factor VIII activity - (Patient clotting time​/Normal clotting time) x 100%

Interpretation of Results :

Normal Factor VIII activity is typically 50%-150%.

For Hemophilia A patients, activity is usually significantly lower (often

Hi Dr. Nicolas Poirier,

Thank you so much for your response. I will try it once.

Best,

Aabid