Vol. 40, No. 4
Printed in U.S.A.
T U B AMKRICAN JOUHNAL OF CLINICAL PATHOLOGY
Copyright © 1960 by The Williams & Wilkins Co.
A WHOLE BLOOD DILUTION TEST FOR COAGULABILITY
D A V I D G R E E N , M . D . , AND LOUIS A. KAZAL, P H . D .
Cardeza Foundation,
Jefferson Medical
College Hospital,
A few years ago Tocantins14 observed that
the recalcification time of freshly prepared
citrated plasma decreased with storage.
The present studies were designed to measure the development of coagulant activity
in such plasma by means of the modified
thromboplastin generation test of Hicks
and Pitney. 6 In preliminary experiments
freshly prepared citrated plasma was incubated at 3S C. and serially tested during
a 90-min period (Fig. 1-4). There was a rapid
increase in the capacity of the plasma to
generate thromboplastin, reaching a maximum 30 min. after venipuncture. The loss of
activity after 30 min. was thought to be related to an increase in pH during the incubation at 38 C. If the dilution of plasma
in buffered saline solution prepared for the
initial Hicks-Pitney test was incubated and
serially tested, the pH remained constant
and no loss of activity occurred (Fig. IB). A
further modification, employing diluted
native whole blood instead of citrated
plasma, made possible a new test which was
simple, rapid, and reproducible. Information
was obtained about the earliest phases of
coagulation in normal, experimental, and
pathologic states. The interpretation of these
data in reference to contact activation and
thromboplastin generation is discussed in
this report.
Philadelphia,
Pennsylvania
Test plasma was prepared by immediately
ceiitrifugmg the citrated blood for 1 min. at
1S00 r.p.m. in a table model angle centrifuge. Substrate plasma was prepared by
centrifuging the citrated blood for 30 min. at
3000 r.p.m. in a refrigerated centrifuge at
4C.
Buffered saline solution (BPSS). A stock
solution containing 120 ml. of 0.4 N HC1 in
0.S5 per cent NaCl solution, and 125 ml.
of 0.8 M imidazole buffer (Eastman Kodak,
Rochester, New York) in 0.S5 per cent NaCl
solution, was diluted to 1 part, plus S.3
parts of 0.85 per cent NaCl solution. The pH
of the final solution was 7.25 to 7.35.
Barbital buffet'. Barbital buffer was prepared according to the method of Owren.12
Chloroform extract of brain. Chloroform
extract of brain was prepared according to
the method of Bell and Alton.1
The screening test for thromboplastin
generation. The screening test was performed
according to the procedure of Hicks and
Pitney, 6 with the exception that a 5 per cent
dilution of plasma in BPSS was used instead of the 10 per cent dilution of plasma
in barbital buffer originally described.
The Factor VIII assay. The Factor VIII
assay was performed according to the
method of Biggs and Macfarlane, 2 with
certain modifications.6
The whole blood dilution test. Immediately
MATERIALS AND METHODS
after venipuncture, 2.0 ml. of blood were
Collection of blood and preparation of
placed in a siliconized clotting tube and 0.2
plasma. Blood was collected through 19ml. was pipeted into a plastic tube (Styrene
gage needles into siliconized syringes contest tubes, 17 by 100 mm., Joseph E.
taining 19 per cent sodium citrate in the
Frankle & Company, Philadelphia, Pennratio of 10 ml. of blood to 0.2 ml. of citrate
sylvania) containing 1.8 ml. of BPSS and
solution, and thoroughly mixed by inversion.
thoroughly mixed. Both tubes were incubated at 3S C. The blood in the siliconized
Received, October 2, 1065.
D r . Croon's present address is Blood Coagulatube was observed until clotting occurred
tion Research Council, Churchill Hospital, Oxand this silicone clotting time was recorded.
ford, England.
The 10 per cent whole blood dilution in
This work was supported in part by United
BPSS was tested at 5, 20, 40, 60, and 90
States Public Health Service grants IIE03544 and
min. of incubation or longer, depending on
HE00374 from the National Heart, I n s t i t u t e , Nathe results of the testing procedure. Testing
tional I n s t i t u t e s of Health.
455
456
G R E E N AND
SERIAL TESTING OF NORMAL
UNDILUTED CITRATED PLASMA
INCUBATED AT 38°C
100
Vol. 46
KAZAL
SERIAL TESTING OF A 5% DILUTION
OF CITRATED PLASMA IN BPSS
INCUBATED AT 38°C
100 r—
80
60
6 NORMALS
40
7 MIN.
REACTION TIME
20
10
100
INCUBATION TIME (Minutes)
FIG. 1. A, the modified screening test (Hicks-Pitney5) was performed
serially on normal citrated plasma during a 90-min. time interval; B, the results
obtained by serially testing a 5 per cent dilution of citrated plasma are presented. Six normal plasmas were studied.
WHOLE BLOOD DILUTION TEST
100 r
80 [
1o 60
10% DILUTION NATIVE BLOOD IN BPSS, 38*C
"
Ihh
w
.
GEOMETRIC MEAN
( 2 0 Normals)
1 RANGE-2 STD. DEV
5
S
CLOTTING TIME
was performed by simply adding 0.3 ml. of
0.02 M CaCl2 to a 0.3-ml. aliquot of the
whole blood dilution and starting a stopwatch. After a 5-min. reaction time, 0.1 ml.
of this mixture and 0.1 ml. of normal substrate plasma were added to 0.1 ml. of 0.02
M CaCl2, a second stopwatch was started,
and the clotting time was recorded. A clot
subsequently formed in the reaction mixture. The rate of generation of thromboplastic potential is defined by the slope of
the curve described by the serial clotting
times. The amount of thromboplastin
actually generated is reflected by the clotting time itself.
-
40
5 MIN. REACTION TIME
20
llimi»' Ijjii p|j||l[]TlnrffiM
•
RESULTS
Whole blood dilution test. The BPSS dilutions of native whole blood from 20 normal
donors were prepared and tested as described in the Methods section. The data
are summarized in Figure 2. The geometric
mean of values at each time interval has
been computed and the range of 2 standard
deviations is shown. During the first 40 min.
of incubation, there was a rapid increase in
the generation of thromboplastic activity.
At the end of 90 min., it was possible to
20
40
60
80
INCUBATION TIME (Minutes)
100
FIG. 2. The buffered saline solution (BPSS)
dilutions of native whole blood from 20 normal
donors were incubated at 38 C. and tested serially
during a 90-min. period. The geometric mean and
the range of 2 standard deviations are illustrated.
shorten the substrate clotting time to 23
sec. or less in all but 1 of the samples.
A concentration of iO per cent native
whole blood in BPSS was used because fibrin
formation was not observed with this degree
Oct. 1966
COMPARISON OF A 5% DILUTION
WITH THE STANDARD 10% DILUTION
100
«
c
o
80
- - • - 5% DILUTION,
7 MIN. REACTION TIME
60
— o - 10% DILUTION,
S MIN. REACTION TIME
<J
Q>
S 40
UJ
\ - - - .
Z
P
K
O
-I
20
10
457
A DILUTION T E S T O F C L O T T I N G
20
40
60
80
100
INCUBATION TIME (Minutes)
F I G . 3. The whole blood dilution test was modified by using a 5 per cent dilution. T h e curve obtained is compared with t h a t of the s t a n d a r d 10
per cent dilution.
of dilution. At concentrations greater than
20 per cent the blood would clot during the
90-min. test period; concentrations less than
10 per cent required longer reaction times
after recalcification in order to obtain clotting times within the usual range. The activity curves of 5 per cent and 10 per cent
concentrations, however, were identical
(Fig. 3). I t was observed that blood at all
concentrations clotted rapidly when calcium
was added during the testing procedure.
The blood dilutions were usually incubated at 3S C. Incubation at 4 C. was performed in 6 experiments, and the mean of
values obtained was compared with the
mean of 20 experiments at 38 C. (Fig. 4/1).
The generation of coagulant activity was
similar at both temperatures, but the
amount developed was greater with incubation at 3S C. If the whole blood was diluted
in barbital buffer or in saline solution incubated at 4 C , the curve of activation was
similar to that obtained with BPSS (Fig.
EFFECT OF INCUBATION
AT 4°C
EFFECT OF VARIOUS
DILUENTS
100
60
100 " 0
20
60
100
INCUBATION TIME (Minutes)
F I G . 4. Modifications of the whole blood dilution t e s t are
compared with the standard procedure. A, the dilution was
incubated a t 4 C. T h e curve obtained represents the geometric
mean of values from 6 normal donors; B, saline solution a t 4 C. and
38 C. and barbital buffer a t 38 C. were used as diluents. E a c h
curve represents the mean of values from 3 experiments.
458
G K E E N AND
4B). An acceleratory effect of saline solution
incubated at 38 C. was consistently demonstrated, however. Each curve represents the
mean of 3 separate experiments.
eu
'
i
•
i
i
i
Correlation of the whole blood dilution test
with the silicone clotting time. A positive
correlation was found between the dilution
clotting times in the eai'ly periods of incuba-
r—
-*1
| 3 MIN. INCUBATION )
60
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40
UJ
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o
z
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60
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P<0.05
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60
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80
| 60 MIN. INCUBATION*)
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P<0.2
40
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INCUBATION |
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Vol. 46
KAZAL
P-029
P<0.30
80
20
40
60
80
DILUTION CLOTTING TIME (Seconds)
F I G . 5. Correlation of dilution clotting time with silicone
clotting time.
COMPARISON OF HEMOPHILIA B AND HEMOPHILIA A
200
200
200
| HEMOPHILIA Bf
— o — PRE-TRANSFUSION C.T. 250' I
— a — POST-TRANSFUSION C.T. 90' I
— • — NORMAL CONTROLS C.T 35'
I
I
40
20
— o — B . F . C.T. 4 3 0 '
— • — F.A. C.T. 39tf
— D — D.C. C.T, 150'
--0--D.C.
A.
U
10
-•— R.R
C.T. 3 6 0 ' \
PR C.T Btf
R.B C.T 36C
1
— O — J.B. CT. 220
— c — J.R CT 180'
—A —
— O —
10 MIN. REACTION TIME
W
Q
10
100
200
300
"0
100
200 " 0
100
200
300
INCUBATION TIME (Minutes)
F I G 0. A, three patients with hemophilia B were studied. T h e clotting times a t a 5-min
reaction time are recorded. B, another patient with hemophilia B was studied before and
after transfusion. T h e curve obtained from normal controls is shown for comparison. C, five
p a t i e n t s with hemophilia A of varying degrees of severity were studied. T h e clotting times a t
a 10-min. reaction time were recorded.
Od.
1966
A DILUTION TEST OF CLOTTING
tion and the silicone clotting time performed
simultaneously on 20 donors (Fig. 5). Those
individuals who had long silicone clotting
times also had prolonged dilution clotting
times after 5 and 20 min. of incubation (p
values of 0.01 and 0.05, respectively). This
EFFECT OF TRANSFUSION (Tx) ON AHG LEVELS
AND ON DILUTION CLOTTING TIMES
( 4 0 ' INCUBATION TIME) IN A PATIENT
WITH MILD HEMOPHILIA (GRADEII).
100
30
60
90
I20'24hrs.
0
POST-TRANSFUSION (Minures)-H
F J G . 7. Another patient with mild hemophilia A
was studied before and after transfusion. F a c t o r
V I I I levels are compared with the dilution clotting times (5-min. reaction time).
450
degree of coiTelation was not found with the
longer periods of incubation.
The whole blood dilution test in patients
with hemophilia B and hemophilia A. Curves
of the dilution clotting times in 2 patients
(B. F. and F. A.) with severe hemophilia B,
and in 1 patient (D. C.) with mild hemophilia B, are shown in Figure 6-4. Figure GB
illustrates the correcting effect of transfusion in another patient with moderately
severe hemophilia B. A normal dilution
clotting curve is included for comparison.
Note the close correlation between the
silicone clotting times (as shown in the
legends) and the dilution clotting curves.
The longer silicone clotting times are associated with the most abnormal dilution
clotting curves.
Five patients with hemophilia A were
studied. Patient P. P. was classified as
having moderately severe hemophilia, and
the others as having very severe hemophilia. The curves were all very similar
(Fig. QC), despite the fact that some of the
patients were tested while they were bleeding or immediately after transfusion. A dual
defect was found. There was an initial lag
phase of approximately 90 min. before
thromboplastic potential increased, and
the amounts of activity finally generated
were very small. Suitable clotting times were
recorded only after incubation of the reac-
TABLE 1
R E L A T I O N O P D I L U T I O N C L O T T I N G T I M E TO S I L I C O N E C L O T T I N G T I M E AND PUOTIIUOMUIN
T I M E IN 5 P A T I E N T S WITH HYPOPKOTHROMBINEMIA
Dilution Clotting Time at
Subject
5 min.
18 min.
33
15-70
100
>00
>C0
97
102
>G0
X>0
20
13-52
SO
>G0
XiO
40
174
245
115
40 min.
60 min.
90 min.
20
12-3S
37
17
12-25
25
15
10-23
22
32
153
43
181
20
130J
min.
sec.
Normals (20)*
Cirrhosis
Cirrhosis
Cirrhosis
Coumadin
Coumadin
Coumadin
Coumadin
Silicone
Clotting
Times
* Geometric mean and range of 2 s t a n d a r d deviations,
t Clot lysed a t 90 min.
| 30 sec. a t 300 min.
35
17-53
30
IS
2Sf
40
7(i
25
Prothrom
sec.
11.013.5
19.9
20.0
42.0
27.0
22.0
per cent
22
13
<10
22
3
14
20
.460
GREEN AND KAZAL
Vol.
46
TABLE 2
BLOOD D I L U T I O N T E S T IN V A R I O U S CLINICAL C O N D I T I O N S
Patient
Normals (20)
Idiopathic thrombopenic purpura
Drug-induced thrombopenia
"Capillary bleeding"
Acute leukemia
Acute leukemia (repeat)
Hemophilia in a femalef
Dilution Clotting
Time*
Plastic Clotting Timet
Platelets
sec:
min.
per cu. mm.
13-52
28
24
36
35
32
104
49
168
15-30
15
37 (silicone)
23
18
19
15
18
22
200,000-400,000
20,000
25,000
Normal
Normal
Normal
6,000
* 20-min. readings,
f Polystyrene plastic tubes.
| P a t i e n t with a history of bleeding and transfusion; father is a hemophiliac, m o t h e r ' s father is a hemophiliac.
tion mixture for 10 min. instead of the usual
5 min.
A sixth patient with mild hemophilia A
was studied before and after transfusion
(Fig. 7). In order to prevent the diluting
effect of direct transfusion, 1000 ml. of the
patient's blood were removed by phlebotomy, and the red cells were separated, suspended in 1000 ml. of fresh compatible
normal plasma, and infused. Serial Factor
VIII assays and dilution clotting times (5min. reaction time, 40-min. incubation
time) were performed before and at 30 min.,
120 min., and 24 hr. after the transfusion.
I t is evident from the figure that the dilution
clotting time reflected the various levels of
Factor VIII as determined before, 120 min.
after, and 24 hr. after the plasma transfusion.
Relation of dilution clotting time to silicone
or plastic clotting time, prothrombin time, and
platelet count in patients with various clinical
disorders. Five patients with long 1-stage
prothrombin times secondary to either cirrhosis or Coumadin therapy had abnormally long dilution clotting times (Table
1). In 3 of these patients the silicone clotting times were normal. Two patients with
thrombocytopenia had normal dilution clotting times (Table 2). One man with acute
leukemia and marked thrombocytopenia had
a prolonged dilution clotting time on initial
examination but 24 hr. later the test was
normal. Three patients referred because of
"capillary bleeding" (epistaxis, easy bruising) had normal coagulation profiles (including tourniquet test, bleeding time, and
partial thromboplastin time), and the dilution clotting time was also normal. One
woman with mild classic liemophilia (see
footnote, Table 2) had a normal plastic
clotting time but the dilution clotting time
was definitely prolonged.
EFFECT OF HEMORRHAGE
IRRESPECTIVE OF PLATELET COUNT
IOO
80
60
ui
--•--
J.W.. PLATELET COUNT 25,000/mm3,
CLOTTING TIME 3 7 '
. . . . . . . M.P, PLATELET COUNT 9 0 0 , 0 0 0 / m m ' ,
CLOTTING TIME 18'
— o — NORMAL CONTROLS
40
s
\ .
z
20
10
20
40
60
80
IOO
INCUBATION TIME (Minutes)
F I G . 8. T h e whole blood dilution test was
performed in 2 patients with clinical bleeding.
T h e curve obtained from normal controls is
shown for comparison.
Oct. 1966
461
A D I L U T I O N T E S T O F CLOTTING
Effect of hemorrhage irrespective of the
platelet count. Figure S illustrates the effect
of active bleeding. Patient J. W. had hematuria and melena secondary to a drug-induced thrombocytopenia. Patient M. P. was
bleeding from a large infected wound slough
and had thrombocytosis. The initial examination in both cases revealed maximal
coagulant potential despite the marked
difference in platelet counts.
DISCUSSION
The experimental data presented in the
preceding sections demonstrate an increasing
capacity to generate thromboplastin in
diluted whole blood or citrated plasma
during a 90-min. observation period. This
potentiation of coagulant activity was independent of calcium concentration, but the
actual generation of measurable thromboplastin required calcium. These early
changes in the whole blood dilution may
represent the in vivo counterpart of in vitro
"contact activation." The relatively similar
activation curves obtained at 4. C. and 3S C.
are consistent with the observation that the
reactions related to the early stages of
thromboplastin generation are not affected
by low temperature.11
Contact activation occurs when blood or
plasma is exposed to a foreign surface. I t is
mediated by Factors XI 13 and XII. 3 ' "• 15
An intermediate is produced, which has been
designated as "activation product." 15 This
substance is a protein and becomes firmly
adherent to the foreign surface. Activation
product formation is not dependent on the
presence of calcium. The activating surfaces
in the whole blood dilution described in the
present experiments may be provided by the
formed blood elements. The function of the
activation product would then be to bind
the plasma factors to these surfaces. Borchgrevink and Owren4 found that Factors V
and VIII are indeed tightly bound to the
surface of the platelet, and Husom 7,8 indicated that early thromboplastin formation
may occur here, although more recently
latridis and Ferguson9 demonstrated the
presence of only Factors V, XI, XII, and
fibrinogen in the washed platelet plasmatic
atmosphere. Stasis of the formed elements
of whole blood in the test tube, however,
may make their surf aces more < accessible
to the contact factors.
The whole blood dilution test reflects the
earliest reactions of blood coagulation/The
silicone clotting time is also sensitive to the
events in the initial stages of clotting.10 The
results obtained with the whole blood dilution study in 20 normal donors during the
early periods of incubation correlated well
with simultaneously performed silicone
clotting times.
In most bloods examined, both normal
and pathologic, thromboplastinogenic capacity increased with incubation; however,
2 patients who were actively bleeding
showed maximal activation prior to incubation. This suggests that contact activation
and factor binding had occurred in vivo. On
the other hand, the patients with severe
hemophilia A and B demonstrated delayed
generation of thromboplastic potential, indicating impaired contact activation and
factor binding or interaction.
The amounts of coagulant eventually
generated (as measured by the final clotting times) were directly dependent on the
concentration of the classic clotting factors,
and were abnormal in hemophilia, hypoprothrombinemic states, and with increased
dilution of the whole blood (<10 per cent).
SUMMARY A N D
CONCLUSIONS
A method which permits study of the
earliest phases of coagulation has been
described. Blood is diluted with buffered
saline solution immediately after venipuncture. The dilution does not generate thromboplastin or thrombin unless calcium is
supplied, but its potential to generate
coagulant activity increases with incubation.
This enhanced thromboplastic capacity is
thought to be dependent on the binding of
plasma factors to the surfaces of the formed
elements of the blood. This binding is
mediated by the contact factors.
The whole blood dilution test has demonstrated that the rate of generation of thromboplastic potential is increased in bleeding
patients and impaired in Factor VIII and
IX deficiency (hemophilia A and B). The
amount of coagulant activity ultimately
462
GREEN AND KAZAL
generated was decreased in Factor VIII
and I X deficiency and in patients with
prolonged 1-stage prothrombin times. I t is
hoped that the experimental model described in this paper will be a useful adjunct
in the study of contact activation and
thromboplastin generation.
Acknowledgments. D r . Allan J . Erslev, Director
of the Cardeza F o u n d a t i o n , made valuable comments and gave generous support to this investigation. D r . R u t h R . Holburn and Miss M a r g a r e t
DeSipin performed the F a c t o r V I I I assays, and
provided continuing encouragement and thoughtful criticism. Instruction for the statistical analysis was provided by D r . H y m a n Menduke.
REFERENCES
1. Bell, W. N . , and Alton, H . G.: A brain extract
as a s u b s t i t u t e for platelet suspensions in
the thromboplastin generation test. N a ture, 174: 880-881, 1954.
2. Biggs, R., and Macfarlane, R. G.: H u m a n Blood
Coagulation and its Disorders. E d . 3. Oxford, Blackwell Scientific Publications, 1962,
p. 401.
3. Biggs, R., Sharp, A. A., Margolis, J., H a r disty, R. M., Stewart, J. and Davidson, W.
M . : Defects in the early stages of blood coagulation. A report of 4 cases. Brit. J.
H a e m a t . , 44: 177-191,1958.
4. Borchgrevink, C. F . and Owren, P . A.: T h e
hemostatic effect of normal platelets in
hemophilia and factor V deficiency. Acta
med. scandinav., 170: 375-383, 1961.
Vol. 46
5. Hicks, N . D . and Pitney, W. R.: A rapid
screening test for disorders of thromboplastin generation. Brit. J. H a e m a t . , 8:
227-237, 1957.
6. Holburn, R. R.: Personal communication.
7. Husom, 0 . : T h e particulate n a t u r e of intrinsic
prothrombinase. Scandinav. J . Clin. & L a b .
Invest., 13: 212-215, 1961.
8. Husom, 0 . : Intrinsic prothrombinase: investigations on the possibility of its formation
by successive coating of intermediates on
activated surfaces. Scandinav. J. Clin. &
L a b . Invest., 13: 210-224, 1961.
9. Iatridis, P . G. and Ferguson, J. I I . : T h e plasmatic atmosphere of blood platelets. Evidence t h a t only fibrinogen, AcG. and activated Hageman factor are present on the
surface of platelets. T h r o m b . & D i a t h .
Haemorr., IS: 114-125, 1965.
10. Jaques, L. B . , Fidlar, E., Feldsted, E. T., and
MacDonald, A. G.: Silicones and blood
coagulation. Canad. M. A. J., 55: 26-31,
1946.
11. Nour-Eldin, F . : H y p o t h e r m i a and blood coagulation. A c t a ' h a e m a t . , 29: 218-225, 1963.
12. Owren, P . A.: A q u a n t i t a t i v e one-stage
method for the assay of prothrombin.
Scandinav. J. Clin. & L a b . Invest., / : 81-83,
1949.
13. Ratnofif, O. ID. and Colopy, J. E . : Familial
hemorrhagic t r a i t associated with deficiency
of a clot promoting fraction of plasma. J.
Clin. Invest., 34: 602-613, 1955.
14. Tocantins, L. M . : Personal communication,
1958.
15. Waaler, B . A.: Contact activation in the intrinsic blood clotting system. Scandinav. J.
Clin. & Lab. Invest., 11 (supp.. 37): 1-133,
1959.