Home › Biochemistry › Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Biochemistry JoVE (Open Access) Citable · DOI

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

DOI: 10.3791/3822-v
What you'll learn
  • ✓ Perform one-stage FV activity assay
  • ✓ Perform two-stage FV activity assay
  • ✓ Measure FV activity in normal and DIC plasma
  • ✓ Interpret clot formation absorbance curves
Protocol

Biopharma Insights This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

Difficulty
intermediate
Total time
~2-3 hrs/sample set
Biosafety
BSL-2

Steps

1
Prepare reagents and plasma samples

Prepare FV-deficient plasma, thromboplastin, calcium chloride, and test plasma samples for the microplate assay.

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2
Run one-stage microplate FV assay

Mix diluted plasma with FV-deficient plasma and reagents, then monitor absorbance at 405nm during clot formation in a kinetic microplate reader.

▶ 03:07
3
Run two-stage microplate FV assay

Perform a two-stage assay with an initial activation step followed by clot formation, measuring absorbance continuously.

▶ 09:27
4
Measure FV in normal and DIC plasma

Apply the microplate assay to determine FV activity in normal human plasma and in samples from patients with disseminated intravascular coagulation.

▶ 11:31
5
Analyze and interpret results

Analyze the absorbance curves to calculate FV activity and compare results between normal and pathological samples.

▶ 12:15
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