MICRO-PROTHROMBIN TEST WITH CAPILLARY
WHOLE BLOOD
A MODIFICATION OF QUICK'S QUANTITATIVE METHOD*
KATSUJI KATO
From the Department of Pediatrics, University of Chicago
The discovery by Dam and Schonheyder (1934) of an antihemorrhagic factor, called vitamin K, and its possible etiologic
relation to hemorrhagic diatheses so prominently associated with
certain diseases, has recently given rise, both among clinicians and
laboratory workers, to great interest in the study of the blood
coagulation system. An increasing number of publications
clearly demonstrate that deficiency in this fat-soluble vitamin is
responsible for the bleeding tendency in conditions such as
obstructive jaundice (Quick, Stanley-Brown and Bancroft,
1935; Warner, Brinkhous and Smith, 1938; Butt, Snell and
Osterberg, 1938), liver injury (Smith, Brinkhous and Warner,
1937), hemorrhagic disease of the newborn (Brinkhous, Smith
and Warner, 1937; Waddell and Guerry, 1939), and sweet clover
disease of chicks and cattle (Roderick, 1931; Almquist and Stokstad, 1935; Quick, 1937). It is conceivable that additional
observations on this subject will continue to be published, since
it is extremely important to ascertain whether or not many other
obscure types of bleeding may have for their etiology the deficiency of this vitamin.
The immediate and most constant effect of either partial or
total lack of vitamin K appears to be a depression on the prothrombin level of the blood, resulting in prolongation of clotting
time, as clinically manifested by a tendency to bleed. The lack
of a proper method of quantitative measurement of prothrombin
in the blood has been responsible for the relatively retarded
* Received for publication July 1, 1939.
147
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KATSttfl KATO
development of our knowledge concerning the mechanism of
normal and abnormal blood coagulation. As pointed out by
Quick and others, the so-called Howell's prothrombin test (1914)
does not actually measure the amount of prothrombin, in as much
as it merely determines the coagulation time of recalcified plasma.
The elaborate titration methods developed by Eagle (1935) and
followed by Warner, Brinkhous and Smith (1936) are rather
complicated and thus do not lend themselves readily to clinical
procedure. The simple one-stage method, first described by
Quick and his associates (1935) and more recently recommended
by Ziffen, Owens, Hoffman and Smith (1939), may be performed
at the patient's bedside, yet obvious mechanical difficulties in
carrying out such a delicate test at the bedside render the procedure hazardous if not impracticable (Quick, 1939). The most
suitable method is that of Quick, Stanley-Brown and Bancroft
(1935), which is being used by an increasing number of observers
in the quantitative study of prothrombin content of the blood.
One difficulty in the performance of the Quick's prothrombin
test is that it calls for 4.5 cc. of venous blood. In older children
and adults venipuncture requires no second thought; but in small
subjects, particularly prematurely or full-term newly born infants,
and in small animals, the necessity of obtaining so large an
amount of blood offers a real obstacle, especially if the test is to
be repeated at frequent intervals on the same individual. The
micro-prothrombin test here proposed is an attempt to modify
the excellent method developed by Quick and his coworkers, by
using small quantities of capillary whole blood, instead of plasma
from venous blood as described by the originators of the test.
MICRO-PROTHROMBIN TEST
The heel in premature and newborn infants, the ventral surface of the big
toe in older infants, and the finger tips or ear lobe in children are suitable
locations for obtaining capillary blood for the test.
A deep puncture is made. The blood, allowed to flow freely without squeezing, is received into the hollow of a hanging-drop slide of a type recommended
for obtaining blood samples for determining the sedimentation rate, packed
cell volume and icteric index (Kato, 1938). The hollow of the slide is previously
MICKO-PKOTHROMBIN
TEST
149
coated with 20 c. mm. of a 2 per cent double oxalate solution, having the following composition:
Potassium oxalate..
Ammonium oxalate
Distilled w a t e r . . . .
0.75 gram
1.25 grams
100.00 cc.
The oxalate mixture is first allowed to dry in the slide at room temperature
before receiving the blood. Approximately 0.20 cc. of blood is collected and
at once thoroughly mixed with the dry oxalate by agitating the mixture with a
fine glass rod, or by rotating the slide several times. The slide is then placed
FIG. 1. UTENSILS REQUIRED FOR PERFORMANCE OF MICBO-PROTHROMBIN T E S T
A, oxalated capillary blood in the well of a hanging drop slide, placed in a
moist chamber; B, thromboplastin suspension; C, calcium chloride solution;
D, porcelain spot plate; E, glass rod; F, three combination micro-hemopipettes
in a moist chamber, such as a Petri dish with a moist filter paper, until the
observer is ready to perform the test.
For the performance of the micro-prothrombin test, exactly 10 c. mm. each
of freshly made thromboplastin suspension and of iV M calcium chloride solution are measured out into combination microhemopipettes (Kato, 1938) and
then mixed together in the well of a clean hanging drop slide. The most convenient utensil for mixing the reagents is a white porcelain spot plate (fig. 1),
since it is provided with twelve circular depressions which enable the worker to
perform several tests in rapid succession. If only one combination .microhemopipette is available, the inner bore of the pipette must be thoroughly rinsed
with normal saline after each use. Finally, 10 c. mm. of well-homogenized
oxalated whole capillary blood are quickly added to the above mixture, the
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KATSXJJI KATO
observer simultaneously clickmg the stop watch. The temperature of the
reagents, just prior to use, must be brought to that of the room in which the
tests are performed. The thromboplastin suspension and calcium chloride
solution are prepared according to the procedures described below.
Suspension of thromboplastin. Remove the brain of a freshly killed rabbit
and strip off the pia and blood vessels. Macerate the organ in a mortar until
a fairly smooth paste is obtained. Add about 10 cc. of acetone and continue
to grind thoroughly. Decant the supernatant liquid and add another portion
of fresh acetone, triturating the tissue thoroughly after each addition of fresh
solvent. After repeating the process four times, the final product is obtained
in the form of a coarsely granular powder from which the last portion of acetone may be filtered off. The powder is then spread out in a thin layer on the
filter paper and thoroughly dried in the incubator over night. Approximately
0.2 gram of the dried powder is mixed with 5 cc. of normal saline and placed in
an oven with temperature at 45-50°C. for 10 minutes. Shake the mixture
vigorously for a few minutes and then let it stand at room temperature until
the powder has completely settled to the bottom of the container. Centrifugation of the suspension is inadvisable since the suspended particles of thromboplastin may be thrown down, producing a relatively inactive preparation. The
supernatant turbid liquid is the suspension of thromboplastin, and will remain
active for several days if kept in the refrigerator in a tightly stoppered bottle.
Calcium chloride solution. Dissolve 1.11 grams of anhydrous calcium chloride in 400 cc. of distilled water. This solution is permanent, but concentration
due to evaporation must be carefully avoided.
After the three ingredients have been mixed and the stopwatch clicked,
the mixture is agitated for 5-6 seconds with a fine glass rod to insure a thorough
mixing of the components. The formation of a gelatinous clot, evidenced by
development of fibrin with resulting fixation of the mass, is the end point of the
reaction and can be noted accurately by the stop-watch.
The average normal prothrombin time, as measured by this method, is
20 seconds, with a deviation of ± 2 .
The performance of a prothrombin test by the above method is
so simple that it may be included in routine hematological work,
since the same blood sample obtained for the determination of the
sedimentation rate, packed cell volume and icteric index may be
also used for the microprothrombin test. It is important, however, to remember that prothrombin undergoes relatively rapid
deterioration after the blood has been removed from the host;
hence this sensitive and delicate test should be carried out as
soon as possible after withdrawal of the blood. A delay of even
one hour may cause a noticeable error in the final results. The
MICRO-PROTHROMBIN
TEST
151
amount of blood sample as recommended above will be sufficient
to carry out three or four various dilution tests (25, 50 and 75
per cent) so that a check could be made on the dilution curve as
described below.
,
DILUTION CURVE FOR PROTHROMBIN
The clotting time of oxalated blood after the addition of thromboplastin and calcium chloride solutions is inversely proportional
1
1
1.
1
1
1
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0 Venous Plasma
1
1
"- 1
-
en
Seconds
60
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£
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| 2
0
^ ^ ^ t r r r s —
0
10
L
10
I
20
!
30
t
40
1
50
1
60
1
70
1
80
I
90
Prothrombin Concentration in Percentage
FIG. 2. PROTHROMBIN DILUTION CURVES CONSTRUCTED FROM DATA OBTAINED
BY PLOTTING THE COAGULATION TIME OF EACH DILUTION AGAINST THE
PERCENTAGE CONCENTRATION OF PROTHROMBIN IN THE ORIGINAL BLOOD
AND PLASMA.
For detailed explanation see the text
to the concentration of the prothrombin. Hence by plotting the
coagulation time of the blood against the percentage of dilution, a
curve of normal correlation may be obtained, as demonstrated
by Quick (1938). A curve so constructed may be used as a guide
to determine the concentration of prothrombin in any pathological blood. Since the suspensions of thromboplastin may vary
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KATSTJJI KATO
in their potency, even normal blood may give slightly varying
results for the prothrombin time. As a rule, the fresh thromboplastin preparation will give the shortest clotting time of the blood,
since the thromboplastic activity of the suspension decreases
with ageing. However, the accompanying chart (fig. 2) is shown
as illustrating average curves of prothrombin concentration 'as
determined by the micro-prothrombin test on both normal whole
capillary blood collected by the method here described and on
normal venous plasma prepared by the method of Quick. All
samples of blood and plasma were diluted with normal saline so
as to represent varying strengths of prothrombin concentration.
The chart demonstrates the remarkably close agreement
between the capillary whole blood curve (solid line) and the
venous plasma curve (broken line). Both samples of blood were
taken simultaneously from the same subject and the dilution tests
performed at the same time using the same reagents. It is to be
noted that the coagulation time of venous plasma gives consistently a lower value as compared with that of capillary whole
blood, a phenomenon easily explained on the basis of a relatively
higher prothrombin content of cell-free plasma per unit volume.
The close approximation of these two curves, however, is a sufficient proof that capillary whole blood may be safely used for the
performance of micro-prothrombin test here proposed.
SUMMARY
A new micro-prothrombin test is described, using small quantities of oxalated whole capillary blood instead of larger amounts
of venous plasma. The method is a simplified modification of the
original quantitative prothrombin test, devised by Quick, Stanley-Brown and Bancroft, which requires a veni-puncture. The
new test measures the concentration of prothrombin in the
oxalated blood in terms of its coagulation time upon recalcification
in the presence of excess thromboplastin; hence, though simplified,
it serves the same objective as the original method.
This new method requires only 10 c. mm. of whole blood for a
single test and hence is so simple to perform that repetition of the
test even in a premature infant is made easily practicable. A
MICBO-PROTHKOMBIN TEST
153
micro-prothrombin test may thus be included in routine hematological work, the blood sample obtained in the manner here
described being both sufficient in quantity and suitable in quality
for the determination of the sedimentation rate, packed cell
volume and icteric index, as well as of the prothrombin content,
all procedures being facilitated by the use of the combination
microhemopipette (Kato).
REFERENCES
Owing to their number references are omitted but will be included in author's
reprints.