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General Approach in

Investigation of Haemostasis

Lecture 10:

One Stage Factor V Assay


One Stage Assay
 When Prothrombin time (PT) and activated
partial Thromboplastins time (APTT) are
prolonged, a one-stage assay is used to detect
a deficiency of factor II, factor V, or factor X.
 If PT is abnormal but APTT is normal, factor VII
may be deficient.
 The percentage of factor activity is determined
by the amount of correction of the PT when
specific dilutions of patient plasma are added
to the factor-deficient plasma.
 These results are obtained from an activity
curve made using clotting times of dilutions of
normal reference plasma and specific factor
deficient plasma.
:Principle
 The assay of a clotting factor relies upon
measuring the degree of correction of the
Prothrombin time (PT) when plasma is added
to a plasma sample specifically deficient in
the factor to be measured.
Reagents and Equipment
 PT / PTT Reagent
 Specific factor-deficient plasma (II, V, VII, and
X)
 Imidazole buffered saline, pH 7.3 ± 0.1
 Normal reference plasma (commercial
reference plasma with known factor levels)

 Note: It is recommended that the factor-


deficient plasma utilized be verified as having
less than I% activity for the specific factor
being measured and close to 100% activity of
all other factors.
Procedure

 Preparation of the activity curve.


 Procedure for testing patient plasma.
Preparation of the activity
curve
1. Prepare 1:10, 1:20, 1 :40, 1:80, 1:160, 1:320,
1:640, and 1:1280 serial dilutions of the
normal reference plasma with Imidazole-
buffered saline.
 The 1: 10 dilution is considered 100% factor
activity.
 It is recommended that at least five
dilutions be used to prepare the factor
activity curve, although it is common to use
seven or eight dilutions (Table).
Buffered
Tube Amount of plasma
Saline Dilution % of Factor
No. (mL)
(mL)
1 0.1 0.9 1:10 100
0.5 of tube no.
2 0.5 1:20 50
1
0.5 of tube no.
3 0.5 1:40 25
2
0.5 of tube no.
4 0.5 1:80 12.5
3
0.5 of tube no.
5 0.5 1:160 6.25
4
0.5 of tube no.
6 0.5 1:320 3.13
5
0.5 of tube no.
7 0.5 1:640 1.56
6
0.5 of tube no.
8 0.5 1:1280 0.78
2. Warm Thromboplastins to 37°C.
3. Perform the following test procedure on each
dilution.
1) Add 0.05 mL of specific factor-deficient
plasma to 0.05 mL of the diluted normal
reference plasma and warm to 37°C for the
2 min.
Preparation of the activity

2) Add 0.2 mL of commercial Thromboplastins


to the sample and determine the clotting
time.
curve
4. Plot results on 2 x 3 cycle log graph paper,
with percent factor activity on the x axis and
seconds on the y axis. Draw a best-fit line.
 The curve will demonstrate a plateau at the
least concentrated dilutions and should be
plotted as such, demonstrating the end of
sensitivity for the assay.
Preparation of the activity

 If using an automated analyzer, the curve is


generally constructed internally and stored
for a specified length of time.
curve
A graph shows the results of a 1-stage PT-based factor X assay with varying factor X
levels - note the parallel lines.
The reference plasma is shown in red. The axes are both logarithmic and the dilutions
are plotted on the X-axis and the clotting time in seconds on the Y axis.
Procedure for testing patient
plasma
1. Warm Thromboplastins to 37°C.
2. Prepare I: 10 and I :20 dilutions of citrated
patient plasma with Imidazole-buffered
saline.
If a third dilution is desired,
Prepare 1:5 Dilution for test plasma expected
to have reduced level and 1:40 dilution for
test plasma expected to be normal
3. Add 0.05 mL of specific factor-deficient
plasma to 0.05 mL of diluted patient plasma.
4. Add 0.2 mL of Thromboplastins to the sample
and determine the clotting time.
5) Repeat steps 3, 4, and 5 on the 1 :20 and .
1:40 dilution of patient plasma, multiplying
the measured result by 2 or 4 respectively to
correct for the dilution ratio when compared
with the I:10 dilution. The results of the 1:
10, 1 :20 and 1:40 dilutions should agree
within 15%. Report the average of the
results.
 Read the percent activity directly from the
Procedure for testing patient

activity curve.

A “Blank” should also be tested as follows:


- 0.1 ml buffer , 0.1 ml Factor V deficient
plasma
- The clotting time for the blank reflects the
plasma

quality of the deficient plasma and should be


equivalent to less than 1%.
Note:
 Inhibitors will often have a "dilutional" effect,
demonstrating nonparallel curves with
increasing dilutions.
 This should be considered if the results of the
1: 10, 1 :20 and 1:40 dilutions do not agree
within 15%. In this case, results should not be
averaged, but further dilutions such as 1 :80,
Procedure for testing patient

and I: 160 performed until results of two


consecutive dilutions match within 15% and
measure within linearity of the calibration
curve.
 Specific volumes required for adding factor-
deficient plasma, diluted patient plasma, and
Thromboplastins reagent may vary depending
on the automated analyzer used.
plasma
Reference Ranges
 The reference ranges for the factors are as
follows:
factor II: 80% to 120% of normal
factor V: 50% to 150% of normal
factor VII: 65% to 140% of normal
factor X: 45% to 155% of normal.

 Laboratories should establish their own reference


ranges although it is probable that many do not
and choose to use either published ranges or
those provided with a commercial standard.
Discussion
 If you find a low FV result (and similarly if you find a low
FVIII result) you should request a FVIII (or FV) assay to
exclude these rare autosomally inherited disorders.
 If you find a low FVII result make sure that the test was
performed using human TF in the PT. Some FVII gene
mutations e.g. FVII Padua can give rise to varying factor
levels depending upon the source of TF used in the PT.
Wherever possible, human recombinant TF should be
employed as this gives a result that more closely relates to
the situation found in vivo.
 If you find a low level of a vitamin K dependent clotting
factor - consider the possibility that that the patient is on
warfarin, has true vitamin K deficiency or may have a
mutation within the genes involved in encoding the
proteins involved in the vitamin K cycle.
 Remember - PT-based factor assays will be reduced in
patients with vitamin K deficiency and in patients on oral
vitamin K antagonists such as warfarin.
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