SOME CLINICAL OBSERVATIONS ON THE PROTHROMBIN
TEST*
BEN FISHER, B.Sc.
From the Department of Medicine, University of Illinois College of Medicine,
Chicago, Illinois
The prothrombin test has become clinically useful with the advent of anticoagulant therapy. There is, however, still a great deal of confusion concerning
the indications for this test and the interpretation of its results. This is especially true of the reports by various authors on studies using Dicumarol (3,
3'-methylene-bis-[4-hydroxycoumarin]) as an anticoagulant in vivo. The necessity for less ambiguity in the interpretation of the prothrombin test has been
stressed in a previous communication.4
Indications for the use of the prothrombin test fall into three goups: in differentiating bleeding states due to hypoprothrombinemia from those caused by
other conditions; as a diagnostic aid in the differentiation of obstructive jaundice
(stone or stricture of the ductus choledochus); and in following the course of
therapy with Dicumarol. The prothrombin test should not be used as a routine
liver function test.
In the majority of reports in the literature concerning prothrombin levels,
the results of the test are given in terms of "seconds" or of "per cent", without
any mention of the method used. In some instances, the prothrombin time of a
"normal patient" is given. The use of normals, in this sense, is fallacious, as will
be shown in this report. In reviewing the literature and testing the proposed
methods, a great variation was seen in the type of procedure and also in the manner of expressing the results. Thus, there is no real standardization of technics
or of prothrombin values, which may lead to confusion in connection with anticoagulant therapy. While some of these methods can be used as a "relative
index" of the prothrombin concentration, most of them are not accurate enough
to follow that course of Dicumarol therapy. This is true of the so-called "bedside" and "rapid" prothrombin methods, especially those using blood from a
ringer puncture.
The many suggested prothrombin methods and modifications can be divided
into two large groups: the whole blood and the plasma methods. The latter
can be further divided into those employing whole plasma and those employing
diluted plasma. Each method will be discussed in turn.
According to Howell,7 prothrombin determinations are based on the following
reactions: (1) Prothrombin + thromboplastin + Ca++—> thrombin, (2) Thrombin + fibrinogen—^fibrin.
From these reactions, it is seen that if the thromboplastin, Ca + + , and fibrinogen
are constant or controlled, the production of a fibrin clot can be used as the
stoichiometric point in a quantitative titration, with the prothrombin as a
* Received for publication, March 26, 1947.
471
472
FISHER
variable. In all of the methods this is based on a time factor: the time required
for formation of the clot, after the proper constituents entering into the reaction
have been mixed. This time must, of course, be converted into suitable equivalents to express the amount of prothrombin in the blood. To report the seconds required for the clot to form does not allow for the variability of the different
methods or reagents.
W H O L E BLOOD METHODS
The whole blood methods are used because they are easy, and a minimum
of equipment is needed. The simplest of these is the "slide test"; 12 a drop of
blood from a finger or an ear puncture is added to a drop of thromboplastin
emulsion on a slide and the mixture is stirred, or a pin is drawn through it, until
a fibrin clot forms. There are many variations of this procedure,1 •10 and all are
subject to error in that the drop cannot be kept constant and the admixture of
various amounts of tissue fluid cannot be avoided. This is important when
working with so small an amount of blood and so complex a mechanism.
Smith's "bedside" one-stage test,16 which was first reported by Quick,12 is
performed in the same manner as the Lee-White test for coagulation time, with
the addition of thromboplastin. It represents, then, a method for determining
accelerated coagulation time rather than the prothrombin time. Smith's
test is widely used in small hospitals and laboratories, but has disadvantages
which will be discussed together with the errors of whole blood methods in
general.
Another whole blood technic which has been used is Kato's microprothrombin
method. 7 It was developed for use primarily in pediatric practice since it uses
blood from a finger or a heel puncture. The method, however, entails the use of
special microhemopipets, which require some previous training in their manipulation before accurate results can be obtained. Kato stated that his method is but
a procedure for determining an accelerated clotting time. 9
It can be seen that there are many disadvantages and inaccuracies connected
with the whole blood procedures. Since almost all of these technics use blood
which is not treated with an anticoagulant, it is necessary to perform repeated
venipunctures or finger tip punctures in order to carry out more than one determination. Accuracy of any degree cannot be obtained with a single determination on a whole blood specimen, since, as will be shown later, there is some
degree of statistical deviation of a given sample even with more accurate methods.
Multiple determinations (two or three) should be carried out, and their average
taken, in order to procure values of greater accuracy.
Comment can be made on the two whole blood methods which are used more
frequently (Smith's and Kato's), and on their variations. Equal volumes of the
patient's and "normal" bloods are used, and a ratio of the relative amounts of
prothrombin is assumed. This is sometimes expressed as "clotting power" or
"prothrombin index". It must be remembered, however, that the prothrombin
fraction of the blood is in the plasma, and that the red blood cells replace a certain
volume of this plasma. This would not be significant were it not for the differ-
OBSERVATIONS ON P R O T H R O M B I N
TEST
473
ence in hematocrit values of the patient and of the "normal". Thus, equal
total volumes of whole blood do not always provide equal amounts of plasma and
of red blood cells.
Although Abramson, Weinstein and White 1 stated that they consider blood to
be 1:1 suspension of cells and plasma, it is erroneous to follow this formula. I
have observed patients with portal cirrhosis who have given prothrombin values
of over 100 per cent (compared with tests run on blood of "normal" individuals
at the same time) by the Smith's bedside test, yet have given consistently low
values (30 to 40 per cent) by Quick's method (Table 2). Patients with cirrhosis
frequently show a secondary anemia and a low hematocrit, especially before any
therapy has been instituted. These tests have been interpreted to mean that
although equal volumes of whole blood were used, cirrhotic patients had more
plasma per unit of whole blood than normal. Therefore, although they may have
less prothrombin per unit of plasma, these patients have sufficient plasma to
compensate when aliquot amounts of plasma are not used. Furthermore, it is
difficult to judge the end point of the reaction, using whole blood, since the opaque
color masks the clot formation. The sensitivity of these tests approximates that
of clotting time methods, which are recorded in terms of half-minutes. The
whole blood methods, are usually expressed in terms of the "normal" blood run
at the same time; this makes the determination variable, depending on the hematocrit and prothrombin values of the "normal" used. The values are then represented by a linear curve, whereas serially diluted plasmas give a hyperbolic
curve. Hauser6 and Quick14 have reported the occurrence of familial hypoprothrombinemia which was refractory to vitamin K. This increases the hazard
of using different "normals" as a standard each time a determination is run. It
is believed, therefore, that whole blood methods and this method of expression of
prothrombin levels often have sources of errors.
PLASMA METHODS
Plasma prothrombin methods are believed to be more accurate, since aliquot
portions of plasma are used and the prothrombin is contained in the plasma.
These methods can be divided into "whole" and dilute plasma procedures, and
considerable controversy has been waged over the sensitivity and accuracy of
one method as opposed to another. It should be remembered that a single
prothrombin level has no more significance than a single determination of the
basal metabolic rate; it is best to follow the patient over a short period of time
and notice the trend of the prothrombin values procured.
DILUTED
3
2
PLASMA
15
Brambel, Allen et al., Shapiro et al., and several other investigators have
proposed modifications of plasma prothrombin methods in which the plasma is
diluted to 50 or 12.5 per cent before carrying out the procedure. They claim
that these dilutions increase the sensitivity of the test by increasing the time of
formation of the clot. Therefore, they claim that differences in time of clot
formation are more significant. These "sensitized" procedures have been the
474
FISHER
basis for claims that xanthine and salicylate drugs produce hypoprothrombinemia
and for the relationship of hyperprothrombinemia to thrombosis.
Plasma dilutions of the order of 10 to 12.5 per cent, made with 0.9 per cent
saline solution, show great variability in clotting, since the fibrinogen and electrolyte concentrations are similarly reduced. The quality of the clot is also
poor. It is not feasible for small laboratories to prepare, by adsorption methods,
prothrombin-free plasma (which is an ideal diluent), since this would involve
more time and work than is desirable for the preparation of reagents for a simple
laboratory test. As is shown in Table 1, the standard deviation at 10 per cent
is much greater than that at 50 to 100 per cent. Prothrombin levels of 60 to
100 per cent are usually not significant in whole-plasma methods since there is
very little change in clotting time; levels below this (30 to 50 per cent which is
the range concerned in Dicumarol therapy) are sufficiently prolonged to observe
delays in clot formation. Therefore, there is no advantage in using diluted
plasma in routine prothrombin determinations, since the dilution adds another
source of error.
WHOLE PLASMA
The two whole plasma technics which are used are the one-stage method of
Quick13 and the two-stage method of Warner, Brinkhous, and Smith.17 The
two-stage method can be discussed briefly by stating that, although it is quite
accurate as a medium in the study of the blood clotting mechanism and other
complex research projects, it is beyond the scope of the average small hospital
laboratory.
Quick's one-stage method has been shown to be an accurate and relatively
simple procedure. It is accurate enough to follow Dicumarol therapy, and can
be learned in a short time. By this method, serial dilutions of plasma (using
0.9 per cent saline or prothrombin-free plasma), are plotted to produce a hyperbolic curve; the concentrations of prothrombin being plotted on the abscissa,
and the clotting times, in seconds, on the ordinate. Quick has calculated the
following formula to fit this curve:
% of prothrombin = p „
302
_
_
in which 302 and 8.7 are constants and P.T. is the patient's prothrombin time
in seconds. One hundred per cent is the value of prothrombin represented by
plasma which will clot in eleven to thirteen seconds, using an active thromboplastin preparation.
Much work has been done on the preparation of a suitable thromboplastic
substance for use in the prothrombin test. Rabbit and beef lungs have been
used as sources of the material, as well as rabbit brain (acetone-dehydrated according to the method of Quick). Fullerton 6 and Page and Russell11 have used
Russell's viper venom, which shows thromboplastic activity, and a good degree
of stability in the crystalline state. We have obtained good results with the
product from rabbit brain prepared by Difco.
O B S E R V A T I O N S ON P R O T H R O M B I N
LABORATORY
475
TEST
STUDIES
Three laboratory studies are included in this report. For all of the studies,
blood was drawn by venipuncture and oxalated in the proportion of 0.1 cc. of
0.1 M sodium oxalate to 0.9 cc. blood. The specimens were centrifuged for
fifteen minutes at moderate speed, and the supernatant plasma drawn off for use
in the tests. Thromboplastin emulsion was prepared by incubating one ampule
TABLE 1
STANDARD D E V I A T I O N OF P R O T H R O M B I N R E A D I N G S BY Q U I C K ' S M E T H O D I N R E L A T I O N
TO P E R C E N T A G E O F P R O T H R O M B I N
PER CENT OF PROTHROMBIN I N
DILUTED PLASMA
PROTHROMBIN TIME I N SECONDS
10
20
30
40
50
100
STANDARD DEVIATION
±5.3
±3.8
±2.9
±2.0
±1.6
±0.9
74.2
37.5
27.1
22.1
19.0
14.4
TABLE 2
COMPARISON OF S M I T H ' S AND Q U I C K ' S M E T H O D S O F P R O T H R O M B I N T E S T S I N 5 P A T I E N T S
WITH P O R T A L C I R R H O S I S AND I N 3 " N O R M A L " I N D I V I D U A L S
SMITH'S METHOD
*N. B.
O. L.
*R. B.
F. W.
*E. L.
M. K.
A. G.
J. L.
QUICK'S METHOD
Reading in
Seconds
Per Cent of
Normal
Reading in
Seconds
Per Cent of
Prothrombin
23.8
21.4
20.5
17.0
25.8
24.0
28.9
24.6
100.0
111.2
100.0
120.6
100.0
107.5
89.3
104.9
14.2
21.1
14.0
26.4
16.9
19.8
16.1
19.3
94.3
29.9
100.0
19.6
51.2
34.3
59.2
36.4
HEMATOCRIT,
RED CELLS
54.0
44.0
48.0
48.0
41.0
34.0
* Represents "normal" subject.
(0.15 gm.) of Difco acetone-dehydrated rabbit brain with 2.5 cc. of oxalate-saline
mixture, (90 cc. of 0.9 per cent saline solution and 9 cc. of 0.1 M sodium oxalate)
in a water bath from twelve to fifteen minutes at from 45 to 48 C. The emulsion
was centrifuged slowly for three minutes to settle the large particles, and then
decanted through a small piece of cotton. The concentration of calcium chloride
used to re-calcify the plasma was 0.025 M.
1. Ten "normal" patients, free from liver disease or blood dyscrasias, were
studied. Five of them were men and five were women; two were negro and eight
were white. The plasmas were diluted with 0.9 per cent sodium chloride solution to provide prothrombin concentrations of 100, 50, 40, 30, 20 and 10 per
476
FISHER
cent. Three determinations, by Quick's method, were performed at each dilution, on each plasma sample. When plotted, these values gave the characteristic
hyperbolic curve. Using the formula, the standard deviation at each dilution
'D =
N":
100
90-
80-
70-
i- bO•£
to 5 0 -
o
40-
o
a.
30-
20-
Quick's method*
Smiths method*
10-
MARCH. 1946
4
8
12
FIG. 1. Comparison of curves for Prothrombin Time as determined by Quick's and
Smith's methods in a patient with postoperative pulmonary embolism who received
Dicumarol therapy.
* Quick's Method: 13 seconds = 100 per cent of prothrombin
* Smith's Method: 17 seconds = 100 per cent of normal
Date
3-3-46
3-4-46
3-5-46
3-7-46
3-9-46
3-10-46
Amount of Drug Administered
200 mg. Dicumarol
300 mg. Dicumarol
50 mg. Dicumarol
5 mg. Vitamin K
50 mg. Dicumarol
50 mg. Dicumarol
was calculated for the series (Table 1). This study shows that the standard
deviation increases in inverse proportion to the concentration of the plasma, and
indicates the factor of greater variability at low concentrations. When acknowledging the occurrence of 3 <rD in 99 per cent of determinations, according to
OBSERVATIONS ON PROTHROMBIN TEST
477
the bell-shaped curve of distribution, it is seen that the deviation may be significant at the lower concentrations of plasma.
2. A second study concerned Quick's method using whole plasma and Smith's
"bedside test" using whole, unoxalated blood. The values for Smith's test were
computed as a percentage of "normal" blood which was tested at the same time:
Prothrombin time of patient . , , . _
~ .
,
=
-.
f
X 100 = % of normal
lime of normal
This is the usual calculation employed with the bedside and simple tests. For
Quick's method, the percentages were computed using his formula. Since our
100 per cent values averaged to thirteen seconds, a correction for this was made in
the formula:
% of prothrombin = (p T
302
_ 2) _ 9
Five patients with a clinical diagnosis of portal cirrhosis were studied (Table
2). This study shows that considerably higher values are obtained using
Smith's method. This finding is in keeping with the remarks made concerning
whole-blood methods in the beginning of this paper.
3. The third study was made in a case involving Dicumarol therapy. A
patient with suspected postoperative pulmonary embolism was seen on the
obstetric service. She was given Dicumarol, and prothrombin tests were made
by both Quick's and Smith's methods. The usual calculation for each method
was made and compared (Fig. 1). At a few points in the plotted curve, the
values for Smith's test followed those for Quick's method, although they were of
a higher order; the trends were somewhat similar.
The Dicumarol was administered with relation to the values obtained by
Quick's method, and was safely discontinued in ten days. At no time did the
patient exhibit any hemorrhagic manifestations.
SUMMARY
A discussion of prothrombin tests and their values is presented. It is suggested
that whole plasma be used in the perf oftnance of the prothrombin test rather than
whole blood or diluted plasma since sources of error caused by dilution of the
fibrinogen and electrolytes and by masking of the end point by whole blood, are
obviated. Aliquot portions of whole plasma give a better means of comparison
without the introduction of extraneous factors. It is also suggested that prothrombin levels be expressed in terms of per cent of prothrombin, as in Quick's
method, rather than in seconds or in per cent of prothrombin time as determined
in a "normal" subject.
Some laboratory studies are presented with a comparison of findings with
Smith's "bedside test" and Quick's one-stage method.
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Am.
478
FISHER
2. A L L E N , J . G., J U L I A N , O. C , AND DRAGSTEDT, L. R . : Use of serial dilutions in determi-
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•
•
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