Quantitative One-stage Assays for
Factors V and X
A R T H U R L. BARSON, P H . D . , AND M A R Y L. FLANAGAN,
B.S.
Warner-Lambert Research Institute, Morris Plains, New Jersey 0 7950
ABSTRACT
Babson, Arthur L., and Flanagan, Mary L.: Quantitative one-stage assays for
Factors V and X. Am J Clin Pathol 64: 817-819, 1975. The relative
sensitivities of the prothrombin time, Russell's viper venom time and
activated partial thromboplastin time were determined in one-stage quantitative assays for factor V and factor X. Sensitivity was defined as the slope of
the reference curve. The prothrombin time was the most sensitive test for
factor X and the least sensitive for factor V. The Russell's viper venom time
was the most sensitive test for factor V and the least sensitive for factor X.
The activated partial thromboplastin time approximated the sensitivity of
Russell's viper venom for factor V and had intermediate sensitivity for factor
X. T h e data indicate that factor V is best quantitated by the Russell's viper
venom time and factor X by the prothrombin time, exactly the opposite of
what is customarily done. (Key words: Factor V; Factor X; Russell's viper
venom; Prothrombin time; Activated partial thromboplastin time; Sensitivity; Quantitative assay.)
of factor V activity is destroyed. 3 Factor X is
individual coagulation factors by the one- usually measured by Russell's viper venom
stage method depends upon having sub- using as substrate charcoal-filtered plasma
strate plasmas lacking the factor being deficient in factors VII and X.2 Unlike
measured. Customarily, a tenfold dilution tissue thromboplastin, Russell's viper
of the test plasma is mixed with an equal venom is not sensitive to factor VII.
volume of factor-deficient plasma, and the
While simple in theory and practice,
clotting time of the mixture is compared one-stage factor assays often are very
with those obtained with various dilutions imprecise because of lack of sensitivity, as
of normal plasma in factor-deficient shown by flat reference curves. The senplasma, the 1:10 dilution representing sitivity of the assay method is reflected by
100% of the factor measured. Factor V is the slope of the reference curve (log
usually measured by a prothrombin time clotting time/log factor concentration),
using as substrate a plasma from a con- Both factor V and factor X participate in
genital factor V deficiency or a normal the intrinsic as well as the extrinsic clotplasma that has been held at 37 C. until ting systems, and both are measured by
Russell's viper venom. T o determine the
THE
QUANTITATIVE
MEASUREMENT
Received January 28, 1975; received revised manuscript April 24, 1975; accepted for publication
most
April 24 1975
Address reprint requests to Dr. Babson.
measuring each or these factors, we prepared reference curves for the prothrom817
sensitive one-Stage
•
•
r .•
r
procedure
for
^
_
818
BABSON AND FLANAGAN
200r
Q
O
A.J.C.P. —Vol. 64
200r
APTT
100-
u
5
RVV
©
100
SO
25
12.5
6.25
3.13
FACTOR V CONCENTRATION, % OF NORMAL
100
SO
25
12.5
6.25
3.13
FACTOR X CONCENTRATION, % OF NORMAL
FIG. 1. Standard reference curves for factor V
assay by Russell's viper venom time (RVV), prothrombin time (PT), and activated partial thromboplastin time (APTT).
FIG. 2. Standard reference curves for factor X
assay by Russell's viper venom time (RVV), prothrombin time (PT), and activated partial thomboplastin time (APTT).
bin time (PT), Russell's viper venom time for the APTT, and a 1:1 mixture of
(RVV), and the activated partial throm- Russell's Viper Venom Reagent and Plateboplastin time (APTT) using the same lin was used for the RVV. All reagents
substrates and normal plasma. Congenital were added automatically by the Coagfactor-deficient plasmas rather than arti- A-Mate Dual Channel.
ficial substrates were used since the decreased factor VIII in heated plasma
Results
would affect the A P T T and the lack of
Reference curves were calculated by
factor VII in charcoal-filtered plasma
regression
analysis. Figure 1 shows the
would affect the PT.
experimental points and the calculated
curves for factor V and Figure 2 shows
Materials and Methods
the curves for factor X. Table 1 gives the
slopes of calculated curves along with
All determinations were performed on
their standard errors. The slopes are a
a Coag-A-Mate Dual Channel using reameasure of the sensitivity to varying activgents and congenital factor-deficient plasity levels.
mas supplied by General Diagnostics,
The slope of the RVV reference curve
Morris Plains, New Jersey 07950. Blood
for
factor V was more than twice the slope
was collected from several normal people
in one-tenth volume of 3.8% sodium
citrate and the plasma was pooled. Dilutions of normal pooled plasma were made
in 0.85% sodium chloride. Each reaction
mixture consisted of 0.1 ml. of factor V or
factor X deficient plasma, 0.1 ml. of
diluted normal plasma, and 0.2 ml. of
reagent. Simplastin Automated was used
for the PT, Automated A P T T was used
Table 1. Calculated Slopes and Standard
Errors of Reference Curves in
Figures 1 and 2
Assay Procedure
PT
APTT
RVV
Factor V
.152 ± .012
.310 ± .022
.340 ± .017
Factor X
- . 3 8 3 ± .021
- . 2 6 8 ± .020
- . 2 0 7 ± .014
December 1975
819
FACTOR V AND X ASSAYS
of the PT curve. The slope of the A P T T
curve for factor V was not significantly
different from the RVV curve, but both
were significantly (p < 0.05) higher than
the PT curve. T h e clotting times for the
A P T T were inconveniently long, and the
instrument did not detect clot formation
below 0.6% factor V (6% on the reference
curve). For factor X curves, all three
slopes differed significantly (p < 0.05).
The sensitivity of the PT was almost twice
that of the RVV.
Discussion
It would appear that the customary use
of the PT to quantitate factor V and the
RVV to quantitate factor X is just opposite
what should be used to maximize the
sensitivity of the one-stage quantitative
assays. The A P T T offers no particular
advantage for either factor. Russell's viper
venom's great sensitivity to factor V would
make it the reagent of choice for factor V
assays. Since the RVV is not sensitive to
factor VIII, aged plasma would provide a
factor V-deficient substrate which is
equally useful and less costly for this assay
than a congenital factor V-deficient
plasma. We have found reference curves
with aged plasma and congenital factor
V-deficient plasma to be comparable.
Borchgrevink and associates1 described
a two-step assay for factor V using Rus-
sell's viper venom in which the diluted test
plasma is mixed with a substrate prepared
from normal plasma previously incubated
with Russell's viper venom and cephalin
until factor V activity is destroyed. After
exactly 2 minutes' incubation, the clotting
reaction is initiated with calcium chloride.
During the 2-minute incubation, factor V
is presumably converted to factor Va. We
have compared this more complex twostep assay with the one-stage assay described herein and have found essentially
identical reference curves. The 2-minute
activation time does not appear to have any
effect on the sensitivity of the procedure.
There is less argument for switching
from the RVV to the PT to quantitate
factor X. Although the PT would provide
greater sensitivity, it requires congenital
factor X-deficient plasma, while the RVV
can also use charcoal-treated bovine
plasma as the substrate. We have found
that both substrates give similar factor X
reference curves when tested with Russell's viper venom.
References
1. Borchgrevink CF, Pool JG, Stormorken H: A
new assay for factor V ( p r o a c c e l e r i n accelerin) using Russell's viper venom. J Lab
Clin Med 55:625-632, 1960
2. Denson KWE: T h e specific assay of ProwerStuart factor and factor VII. Acta Haematol
25:105-120, 1961
3. Denson KWE: H u m a n blood coagulation,
Haemostasis and Thrombosis. Edited by R.
Biggs. London, Blackwell, 1972, pp 6 3 4 - 6 3 6