DIAGNOSIS AND CONTROL OF T H E HEMOPHILIOID STATES W I T H
T H E PARTIAL THROMBOPLASTIN T I M E (PTT) TEST
N. F. RODMAN, JR., M.D., EMILY M. BARROW, M.A., AND JOHN B. GRAHAM, M.D.
Department of Pathology, University of North Carolina, Chapel Hill, North Carolina
In recent years it has become apparent
that there are several heritable bleeding disorders in humans other than hemophilia
(AHF deficiency). The term "hemophilioid
states" has been suggested as the generic
name for this group of disorders.7 The
existence of a variety of plasma deficiency
states makes urgent the development of
simple laboratory tests for specific diagnosis
and rational therapy. The purpose of this
communication is to describe a procedure
which meets this need and to document its
utility.
I t was shown many years ago by Mills20
that crude cephalin suspensions consistently
give longer clotting times with hemophilic
than with normal plasma. It has been known
since at least 1935, when Quick's prothrombin time test was published,23 that crude
whole brain extract clots hemophilic plasma
as rapidly as normal plasma. This dichotomy
of effects has given rise to the concept of
different types of thromboplastin, "complete" (like whole brain) and "partial" 13
(like cephalin extract). There is little fundamental understanding of the reasons for
these differences, but the differences have
provided a basis for simple tests of great
utility in the diagnosis and control of the
hemophilioid diseases.
The partial thromboplastin time (PTT,
or cephalin clotting time), a 1-stage clotting
test in which the crude cephalin extract of
brain is used as thromboplastin, was originally described by Langdell and his associates.13 Plasma deficient in antihemophilic
Received, January 27, 1958; revision received,
February 19; accepted for publication March 3.
Dr. Rodman is Resident Pathologist; Mrs.
Barrow is Research Assistant in Pathology; and
Dr. Graham is Associate Professor of Pathology.
These investigations were supported (in part)
by a research grant (H-3140) from the Public
Health Service, Department of Health,Education,
and Welfare.
factor (AHF), plasma thromboplastin component (PTC), factor V (proaccelerin,
labile factor), plasma thromboplastin antecedent (PTA), and Stuart factor have been
shown previously to be abnormal in this
test. 3, n It has also been reported that plasma
deficient in Hageman factor has a prolonged
partial thromboplastin time16 and that
plasma deficient in SPCA (serum prothrombin conversion accelerator, factor VII,
proconvertin) is normal in this test. 5 ' 16 The
present communication describes our experience with the PTT in the diagnosis of these
deficiencies, in control of therapy, and in
detection of circulating anticoagulants.
MATERIALS AND METHODS
Blood from patients and controls was
obtained from the antecubital vein. The
blood was mixed either with 1.34 per cent
sodium oxalate in a ratio of 9 parts blood to
1 part oxalate solution or with 3.2 per cent
sodium citrate in a ratio of 8 parts blood to
1 part citrate solution. The anticoagulants
were buffered by dissolving oxalate or citrate
powder in 5 per cent imidazole at pH 7.2519
rather than distilled water. Blood was
centrifuged for 15 min. at 3200 X g, and the
supernatant plasma withdrawn by pipet.
Plasmas were usually tested on the day
the blood was obtained. They were stored
occasionally, however, for several days at
- 2 0 C. with little change in the PTT. It
appears that the PTT of citrated plasma
varies less during storage than that of
oxalated plasma, probably because of
slower deterioration of AHF 21 and factor
V.4 After several hours at room temperature,
the PTT of oxalated plasma sometimes increases significantly while that of citrated
plasma usually changes only slightly.
The brain extract for use in the prothrombin time test and the cephalin extract for use
in the P T T test were prepared as follows:
bovine, canine, and human brain have been
525
526
RODMAN ET AL.
found to be satisfactory starting material.
The meninges were removed, and the brain
washed under tap water. About 100 Gm. of
wet brain were covered with acetone and
allowed to stand for 1 hr. The tissue was
gently mashed under acetone in a mortar
with a pestle. The supernatant was decanted,
.and the process repeated until the tissue
became flaky and the acetone remained clear.
The tissue was then ground into small
particles and spread on a flat pan to dry.
Usually 18 to 20 Gm. of dry powder are
obtained from 100 Gm. of wet brain tissue.
The dry brain extract when resuspended in
saline in a concentration of 25 mg. per ml.
was used as the thromboplastin in prothrombin time tests.
Cephalin was prepared by placing 18 to
20 Gm. of the dry brain extract in an Erlenmeyer flask, covering with ether, and stoppering. The dry material was suspended by
shaking and allowed to stand overnight at
room temperature. The following morning,
the supernatant was filtered until clear, and
the clear filtrate evaporated to dryness with
a stream of air. The dry residue was washed
twice with 40 ml. of acetone at room temperature and twice with 40 ml. boiling
acetone, then air-dried. The cephalin obtained from 100 Gm. of fresh brain (18 to 20
Gm. brain extract) usually amounted to 2
to 3 Gm. of pale yellow, waxy material. Of
this material 3 Gm. were suspended in 3 ml.
of saline, and the volume brought to 100 ml.
by the slow addition of saline with careful
mixing. In our experience satisfactory
material has been white and opaque in the
3 per cent solution. When a yellowish tint
has been present, the material has often been
inhibitory and unsatisfactory. The 3 per
cent stock suspension was stored in lots of
1 ml. at —20 C. Stock suspensions have been
stored at — 20 C. for 1 yr. or more without
significant loss of activity. Before use the 3
per cent stock suspension is diluted to 0.3
per cent with saline. The dilute suspension
also has been stored in desired quantities at
— 20 C. for long periods without serious
deterioration.
BaSCi adsorbed plasma was prepared as
described by Langdell and co-workers.13
The plasma eluate was prepared from the
Vol. 29
BaS0 4 "button" from the preceding procedure. The "button" was suspended in a
volume of 3.2 per cent sodium citrate equal
to that of the original plasma, at 5 C. for 30
min. with occasional stirring. This mixture
was centrifuged and the supernatant
dialyzed for 3 or more hours against normal
saline. Eluates of oxalated serum were pre
pared by a similar technic.
The partial thromboplastin time.(PIT or
cephalin clotting time) is defined as the
clotting time at 37 C., pH 7.4, of a mixture
consisting of 0.1 ml. citrated or oxalated
plasma, 0.1 ml. 0.02 M CaCl 2 , and 0.1 ml.
0.3 per cent cephalin extract. A convenient
way to perform the test is to place test tubes
containing, respectively, cephalin extract
and 0.02 M CaCl 2 solution in a water-bath
at 37 C. The plasma to be tested is kept at
room temperature. First, 0.1 ml. plasma is
placed in a clean, dry 10 by 75 mm. test tube.
Next, 0.1 ml. 0.3 per cent cephalin extract is
added to the tube; then 0.1 ml. CaCl2. A
stop watch is started when the CaCU is
added. The tube is agitated gently, and the
end point consists of the formation of a fibrin
clot. When plasma mixtures or dilutions are
to be tested, they are mixed or diluted
immediately before the addition of cephalin
and calcium.
Prothrombin times were performed by the
method of Quick,22 using thromboplastin
from human brain.
In the Stypven-cephalin clotting time a
mixture of equal quantities of 0.3 per cent
cephalin and Stypven* (Russell's viper
venom) was used as the thromboplastin.
The Stypven was diluted 1:10,000 prior to
mixing with cephalin, using the distilled
water supplied with the dried material.
Two-stage prothrombin determinations
were done by the method of Smith, Warner
and Brinkhous as described by Wagner and
associates.24
Prothrombin utilization was determined
as described by Graham and co-workers.8
The thromboplastin generation test of
Biggs and Douglas2 was performed with the
modifications described by Graham and
Barrow.6
* Burroughs Wellcome & Company.
June 1958
527
HEMOPHILIOID:STATES
Whole blood clotting times were determined by the method of. Lee and White, as
described by Jaques.12.
HBSULTS
Clinical Experience with the Partial
Thromboplastin Time
• .
-
.
T A B L E 1'
CLINICAL E X P E R I E N C E W I T H P A R T I A L T H R O M B O PLASTIN T I M E ( P T T ) T E S T
1
Type of Plasma Tested
No. of
Determinations
P I T (Sec.)
Mean S.D.
S.E.
Coefficient
of •
Variation
Per Cent
Our experience with the partial thrombo- Normal control
17
76 13 0.9
206
plastin time as used routinely in a clinical Normal control +
S4
11
9 1.0
85
coagulation laboratory over a period of
hemophilioid. paapproximately 1 yr. is summarized in
tient without'
Table I. During this period a single preparacirculating anticoagulant' (1:1
tion of human brain cephalin was used by 5
mixture)'' .
different workers. A total of 206 determina74
6 0.9 • S
45
tions were performed on samples of normal Suspected • •
"bleeder,'.'
never
plasma obtained for routine methodologic
control of the test, our policy being to use a '. positively: _•_ '
diagnosed. . : •
fresh control plasma in conjunction with
each test plasma. Some of the control plasmas were obtained by ourselves in the time of the' mixture was actually less than
laboratory, others by medical students and that of the control alone.
house officers on the wards. It will be seen
Data on 45 patients suspected on clinical
in the top line of Table 1 that despite the grounds of having a hemorrhagic diathesis,
studiedly uncontrolled venipunctures the but never definitely established as having 1
coefficient of variation was not excessive.
of the plasmatic clotting defects, are shown
Experience with 85 patients known to in the bottom line of Table 1. It will be
have one or another of the hemophilioid noted that the average PTT of these patients
diseases, but lacking independent evidence was actually somewhat less than that of the
of circulating anticoagulants, is summarized control group with a coefficient of variation
similarly in the middle line. It will be noted of 8 per cent. However, the difference bethat the average PTT of the mixture of tween the means of the control group and
patient's plasma with control plasma was 8 the group of suspected "bleeders" was not
sec. longer than the average for the controls statistically significant (t = 0.9S99, 249
alone (top line). The smaller coefficient of d.f., v > 0.05).
The statistical data can be used to devise a
variation, 11 per cent, may be related to the
fact that most of the venipunctures were test for the presence of a circulating antiperformed by ourselves under relatively coagulant in a patient known to have a
controlled conditions. That the 8-sec. hemophilioid disease. First, an unknown
difference between the means of the 2 sets of but suspected plasma, a control plasma and
determinations is real can be shown in either a mixture of equal parts of the 2 are tested
of 2 ways. It can be shown to be statistically with the PTT test. If the PTT of the mixture
significant at the 1 per cent point by a " t " is more than 26 sec. longer than the control
test (t = 5.089, 289 d.f., p < 0.01), or, more (difference between control and mixture
crudely, it can be seen from the standard equals 8,. plus 2 S. D. about the mixture
errors of the 2 means that the tails of the 2 equals 18), it may be assumed with confidistributions overlap only slightly. In dence that the patient has a circulating
individual instances the PTT of the patient- anticoagulant. Actually, a plasma may concontrol mixture was less than 8 sec. longer tain a low-titered anticoagulant when the
than the control, as might have been ex- prolongation of the PTT of the mixture over
pected from the variation noted in the last that of the control is in the 15 to 25 sec.
column of Table I. Occasionally, the clotting range.
528
Vol. 29
RODMAN ET AL.
Diagnostic Use of the Partial Thromboplastin
Time Test
The specific diagnosis of a clotting factor
deficiency depends at present upon the failure of the unknown plasma or serum to
correct the clotting defect of a sample with a
known deficiency. We have found that this
principle has great utility when the P T T is
applied in conjunction with a battery of
known, congenitally deficient plasmas. If a
test plasma fails to correct the prolonged
PTT of a known deficient plasma, the test
plasma may be assumed to have the specific
deficiency, provided the prolonged P T T of
the test plasma is corrected by normal control plasma and, therefore, does not contain
an anticoagulant. Conversely, if the test
plasma corrects the deficient plasma, it may
be assumed to contain the factor known to
be deficient. This positive test is a less
specific test than the negative one, since
contaminants such as traces of thrombin13
may produce apparent correction of a
deficient plasma. The danger of misinterpreting an apparent correction can be minimized by using a normal control plasma for
comparison with the unknown test plasma.
Similar but less specific tests may be devised
by use of mixtures of test plasma with
BaS0 4 -adsorbed plasma (rich in AHF and
factor V), serum or serum eluate (rich in
PTC, Stuart factor, and SPCA), and plasma
eluate (rich in prothrombin, PTC, Stuart
factor, and SPCA).
Patient 1. PTC deficiency. J. J. T. is a
40-year-old white man with a severe bleeding
tendency whose maternal uncle and 3
brothers died of hemorrhage. He has had
numerous joint hemorrhages, hematuria,
epistaxis, bleeding from gums, and prolonged
bleeding after tooth extraction.
He has a prolonged whole blood clotting
time, impaired prothrombin consumption,
normal prothrombin time and normal
2-stage prothrombin concentration. His
serum is abnormal and his plasma normal in
the thromboplastin generation test.
Table 2 shows diagnostic data on this
patient obtained with the PTT. The
patient's clotting time was markedly prolonged, yet corrected to within 1 sec. of the
control value by mixing with an equal
TABLE 2
DIAGNOSIS
OP PLASMA
THROMBOPLASTIN COM-
PONENT DEFICIENCY WITH T H E PARTIAL THROMBOPLASTIN T I M E T E S T
PTT*
Type of Plasma (ml.)
Normal
BaSOi- Normal
Normal Patient
plasma
adsorbed eluate
AH Ft
deficient
PTCf
deficient
72
263
73
0.1
0.05
0.1
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Sec.
81
269
0.05
0.05
59
61
0.05
0.05
0.05
0.05
81
83
0.05
0.05
84
450
* P a r t i a l thromboplastin time t e s t .
t Antihemophilic factor.
| Plasma thromboplastin component.
volume of control plasma. It was not corrected by mixing with BaS0 4 -adsorbed
plasma, but was corrected by mixing with
the eluate from the same plasma. Further,
the patient's plasma corrected AHF-deficient
plasma completely, but failed by 366 sec. to
correct PTC-deficient plasma. This patient,
deficient in PTC, did not have a circulating
anticoagulant, as the P T T of the mixture of
his plasma and control plasma was only 1
sec. greater than that of the control plasma.
Patient 2. AHF deficiency. G. M. T. is a
30-year-old white man who has had bleeding
episodes since early childhood. These have
included hemorrhages into muscles and
major joints, hematuria, and retropharyngeal hematoma. He has a normal prothrombin time, 92 per cent prothrombin by the
2-stage method, impaired prothrombin consumption, and a prolonged whole blood
clotting time.
Table 3 exemplifies the diagnosis of a case
of AHF deficiency with the PTT. The
patient's partial thromboplastin time was
very long but corrected to within 20 sec. of
June 1958
529
HEMOPHIMOID STATES
TABLE 3
DIAGNOSIS
CIENCY
OF
WITH
TABLE 4
ANTIHEMOPHILIC
THE
PARTIAL
TIME
FACTOR
DEFI-
THROMBOPLASTIN
DIAGNOSIS
OF
SPCA*
PROTHROMBIN
TIME
THROMBOPLASTIN
TEST
PLUS
Normal
PTCt
Deficient
AHFJ
Deficient
0.1
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
TIME
TEST,
WITH
THE
THE
PARTIAL
AND
STYPVEN
CEPHALIN
54
128
0.05
0.05
0.05
0.05
0.05
0.05
I-Stage Clotting T e s t s
(Sec.)
T y p e of thromboplastin
65
88
0.05
0.05
T y p e of Plasma (ml.)
Sec.
55
208
75
0.1
0.05
TEST,
PTT*
T y p e of Plasma (ml.)
Normal Normal
BaSOj- plasma
Patient
adeluate
sorbed
DEFICIENCY
0.05
11.5
36.9
11.4
S2
66
70
5.S
6.0
5.6
0.05 0.05 0.05
0.05
13.2
13.9
S7
S4
6.2
6.2
11.1
11.S
5S
52
6.0
6.4
0.05 12.1
0.05 36.S
63
55
6.2
6.0
71
199
0.05
Patient S. SPCA (factor VII, •proconvertin)
deficiency. Through the courtesy of Dr.
Harold A. Wurzel of the University of
0.1
0.1
0.05
the control plasma by mixing with the latter.
The patient's prolonged P T T was corrected
almost as well by the BaSOu-adsorbed
plasma as by whole normal plasma, but was
not corrected by the eluate from adsorption
of this plasma. (The partial correction of the
patient's PTT by the eluate may have resulted from a trace of thrombin in this
eluate, as on other occasions there has been
no correction by similar eluates.) His plasma
corrected PTC-deficient plasma but not
AITF-deficient plasma, thus indicating
specific AHF deficiency. The fact that his
PTT was not completely corrected by any
of the above reagents suggests a mild circulating anticoagulant, a distinct possibility in
view of the hundreds of transfusions he has
received. However, it will be noted that the
P T T of the patient-control mixture was
only 20 sec. longer than the control plasma,
thus not falling outside our limit for specific
diagnosis of anticoagulants.
Nor- Normal
Stuart
mal
Brain
de- plasma BaSOi- extract
adficient
eluate sorbed
Stypven
plus
cephalin
67
87
* P a r t i a l thromboplastin time test,
t Plasma thromboplastin component.
% Antihemophilic factor.
Patient
Cephalin
Normal
0.05
0.05
0.05
0.05
0.05
* Serum
prothrombin conversion accelerator.
Pennsylvania we obtained plasma from a
patient, previously unreported. She is a
23-year-old white woman who has been a
"bleeder" since infancy. She has been considered deficient in SPCA, since her prothrombin time has been consistently prolonged, not corrected by BaSCi-adsorbed
plasma but corrected by the eluate, and her
prothrombin by a 2-stage assay has been
normal. Both her plasma and serum are
normal in the thromboplastin generation
test.
Table 4 shows data obtained in the prothrombin time, the partial thromboplastin
time, and the Stypven-cephalin clotting
time. The prothrombin time of the patient's
plasma is prolonged; it is corrected by mixing
with Stuart factor-deficient plasma and by
plasma eluate, but not by the supernatant of
BaSO^adsorbed plasma. The partial thromboplastin time and Stypven-cephalin times
of every mixture containing this patient's
plasma are normal.
Patient 4. Stuart defect. A. G., a patient
previously reported by Dr. Charles Barnett
of Fredericksburg, Virginia,1 as having factor
530
RODMAN ET AL.
VII deficiency, is a 32-year-old Negro man
who has had transfusions for excessive
bleeding following tooth extraction and
laceration of the right forearm. Minor cuts
have always been difficult to stanch.
Table 5 illustrates diagnostic data using
the prothrombin time, PTT, and the Stypvencephalin time. I t will be noted that his
clotting time was prolonged in each of these
tests. The patient's plasma failed in the
prothrombin time to correct known Stuartdeficient plasma but did correct SPCAdeficient plasma. The patient's prolonged
clotting time in all 3 of these tests was not
corrected by the BaSOvadsorbed plasma.
Although the specific data are not shown,
the patient's clotting tests were corrected by
plasma eluate.
Patient 5. Hageman deficiency. L. C , a
17-year-old white boy, has had no bleeding
episodes despite tonsillectomy at age 5,
appendectomy at age 15, and a tooth extraction. Minor cuts have not been troublesome,
and he does not bruise easily. He was found
by routine study prior to operation to have
TABLE 5
D I A G N O S I S OF STUART FACTOR D E F I C I E N C Y WITH
THE
PROTHROMBIN T I M E T E S T ,
THROMBOPLASTIN
TIME
TEST,
THE P A R T I A L
AND STYPVEN
PLUS CEPHALIN
l-Stage Clotting Tests
Type of Plasma (ml.)
Type c f thromboplastin
NorSPCA* mal Brain CephaPa- Stuart
de- BaSO(- extract
deNormal tient
lin
ficient ficient adsorbed
Stypven
plus
cephalin
55
13.6
154.4 >300
61
14.0
6.2
34.3
6.4
72.6
13.8
66.0
247
63
468
21.S
6.2
38.1
36.9
13.4
19.4
66
50
51
6.0
6.0
S.4
0.05 14.4
65
0.05 158.6 >300
6.4
39.0
0.1
0.1
0.05 0.05
0.05
0.1
0.05
0.05 0.05
0.05
0.05
0.05
0.05
0.1
0.05
0.05
* Scrum prothrombin conversion accelerator.
Vol. 29
a prolonged whole blood clotting time (29
rain.). He has a normal bleeding time,
negative tourniquet test, impaired prothrombin consumption, abnormal thromboplastin generation test, and normal prothrombin by 2-stage assay.
Table 6 shows diagnostic data using the
partial thromboplastin time. In this test the
patient's PTT was greatly prolonged, but
corrected by control plasma as well as by
both the BaSCVadsorbed plasma and the
eluate. The patient's plasma corrected
plasmas specifically deficient in AHF, PTC,
Stuart factor, and SPCA. His plasma failed
by 78 sec. to correct plasma known to be
specifically deficient in the Hageman factor.
Thus, this patient was established specifically as having the Hageman trait solely by
the P T T test.
The Partial Thromboplastin Time Test for
Evaluation of Therapy
The PTT test has been found very helpful
in day-to-day evaluation of the status of
patients receiving transfusion therapy. The
following examples demonstrate its usefulness.
PTC deficiency. The clinical history of
J. J. T., patient 1, has been described above,
and the diagnostic data were shown in
Table 2. Because of severely carious teeth
and gum disease it was thought desirable to
extract all his teeth. For the first stage, he
was admitted to the hospital for preparation
by transfusion and extraction of 6 teeth.
Figure 1 illustrates daily partial thromboplastin time data, with the patient, control,
and the patient plus control (equal parts
mixture) values, during a period of about 1
month while 6 teeth were being removed.
Extractions were performed on 3 separate
occasions, as shown. A single tooth was
extracted on March 14 and 20, and on
March 26, 4 teeth were removed. He was
given plasma before extraction on each
occasion, with a significant shortening of
the PTT. The extractions were not undertaken until after it had been determined by
the P T T that a good response had been
obtained from transfusion. During and after
each procedure there was minimal bleeding.
Only after the 3rd extraction, involving 4
r
June 1958
531
HEMOPHILIOID STATES
TABLE 6
DIAGNOSIS O F H A G E M A N D E F I C I E N C Y WITH P A R T I A L THROMBOPLASTIN T I M E T E S T
Type of Plasma (ml.)
Normal
Patient
Normal
BaSO.,adsorbed
Normal
plasma
eluate
AHK*
deficient
PTCt
deficient
Sec.
Stuart
deficient
SPCAt
deficient
Hageman Partial thromdeficient§ boplastin time
72
30S
74
0.1
0.05
0.1
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Prothrombin
time
12.5
17.7
13.9
66
76
0.05
0.05
52
57
0.05
0.05
70
So
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
77
SI
0.05
0.05
S4
03
0.05
0.05
63
6S
0.05
0.05
72
150
* Antihemophilic factor.
t Plasma thromboplastin component.
t Scrum prothrombin conversion accelerator.
§ Kindly supplied by Dr. Oscar Ratnoff.
teeth, were additional postoperative plasma
transfusions required to control oozing. It
will be noted also that this was the only
period during which there was immediate
and significant postoperative prolongation
of the PTT. I t should be noted, finally, that
the clotting times of the mixtures of patient's
with control plasma were never more than
26 sec. longer than the control clotting time
alone, indicating no significant inhibitory
activity generated by the patient in response
to transfusion.
AHF deficiency. W. W. R. is a 26-year-old
white man with classic hemophilia whose
first severe bleeding was into the jaw at 6
months of age. There have been numerous
subsequent episodes of bleeding including
hemarthroses, melena, a retropharyngeal
hematoma, and bleeding after tooth extrac-
tion. He was admitted to the North Carolina
Memorial Hospital on July 18, 1957, with
hemorrhage into the left hip. By assay on a
previous admission he had had 2 per cent
AHF.17
Figure 2 shows daily P T T data on the
patient's plasma, control plasma and a mixture of equal parts of the 2. Following admission on July 18, he received 6 units of
fresh plasma, and the P T T dropped from
256 sec. to 94. On less intensive plasma
therapy his P T T rose gradually to 230 sec.
on the 8th hospital day, July 25, a phenomenon which has been demonstrated in dogs
to be the result of loss of injected AHF. 14
Physical therapy was begun July 24 but
stopped on July 26 when pain recurred in
the left hip and extended into the lumbosacral region. An abscess developed in the
532
Vol. 29
RODMAN ET Ah.
PTC-DEFICIENCY
J.J.T.
220-
A Patient
x Control
e Mixture 1:1
200
PTT 180
(Sec.)
160
140
120
100x---x»
80-
A
* \
x
x-
o-i
°-&-<
x-x
^ - '
...©....©••
«4
X
60Units
«
Plasma
2
250 ml/unit i
I3'l4 15" 16" 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
1957
MARCH
Fio. 1. Daily partial thromboplastin times of patient's plasma, control plasma, and the mixture
of equal parts of patient's and control plasma. In each instance the date of each tooth extraction is
designated by an appropriate symbol..The first 2 extractions involved single teeth, and the third, on
March 26, involved 4 teeth.
deltoid region July 29 following an injection
of Demerol, and therapy by transfusion was
resumed. The abscess was treated conservatively and allowed to drain spontaneously.
When the abscess had healed, transfusions
were stopped and the patient's P T T lengthened into its usual range. This man never
showed evidence of a circulating anticoagulant, since the P T T of the mixture of his
plasma with control plasma was always
within a few seconds of the control value.
Demonstration of an Inhibitor with the
Partial Thromboplastin Time Test
P. W. B. is a 13-year-old white boy with a
severe deficiency of PTC. He has a lifelong
history of hemorrhages, including epistaxis,
hemarthroses, hematuria, and prolonged
bleeding with tooth extraction. He has a
normal prothrombin time and prolonged
P I T . His plasma corrects AHF-defieient
plasma but not PTC-deficient plasma in the
partial thromboplastin time test. He has
been described previously as a PTC-deficient
patient with a circulating anticoagulant
specific against PTC. 18
He was admitted to the North Carolina
Memorial Hospital in September 1956 with
increasingly severe headache and drowsiness
following a mild blow to the head. Despite
the fact that he had been shown a year
previously to have a circulating anticoagulant, he appeared on admission to have at
most a very weak inhibitor. It was decided
to treat him vigorously with fresh frozen
plasma. On this therapy the patient's PTT,
the daily determinations of which are shown
in Figure 3, became much shorter for several
days. During this period there was improvement in his level of consciousness. On the
7th day after institution of plasma therapy,
the patient's PTT became markedly pro-
oWne 1958
533
HEMOPHILIOID STATES
A H F - DEFICIENCY
W.W.R.
260
A Patient
x Control
o Mixture 1:1
PTT 220
(Sec.)
180140100-
x.^Q
.-x
"X^
""•&»••-;
•"!(•'
x-
-x'
"^
x
60H
6
Units
Plasma
250 ml/unit
54
3"
2
H
8 19 20 21 22 23 24 25 26 27 28 29 30 31 ' I
1957
JULY
2 3 4 5 6 7
AUGU9T
8 9 10
FIG. 2. Daily partial thromboplastin times of patient's plasma, control plasma, and a patientcontrol plasma mixture.
longed, as did the PTT of the patient-control
mixture. This prolongation of the mixture
was interpreted as indicating development
of a strong inhibitor. During this period and
subsequently the patient improved gradually, and several months later the only
residual defect was a mild speech disorder.
On July 31,1957, he was readmitted in mild
shock from a retroperitoneal hemorrhage
which had reduced his packed cell volume to
13 per cent. Figure 4 shows daily P T T data
during this admission. The first PTT on his
plasma was markedly prolonged, but there
was correction to within 15 sec. of the control value by mixing with control plasma.
This indicated that the previously demonstrated inhibitor had largely disappeared.
The patient's PTT was much shorter after
vigorous transfusion therapy until the reappearance of the inhibitor on August 7. With
the development of this potent inhibitor it
was felt that neither plasma nor whole
blood transfusions would be of further benefit. Fortunately the bleeding had ceased, and
he was discharged on the 10th hospital day.
The thromboplastin generation test has
been considered a reliable method for
demonstrating a circulating anticoagulant.10
Table 7 contains the thromboplastin generation test data on this patient, and at the
time (September 24, 1956) the P T T clearly
showed inhibitory activity. Both his plasma
and serum were defective in the thromboplastin generation test, the serum being
more defective than the plasma. In addition,
the patient's serum in an equal quantity
inhibited the action of control serum.
Plasma from this patient the same day
showed inhibitory activity detectable in
high dilution by the P T T test (Table 8). If
an increase greater than 26 sec. over the
control diluted with saline is taken as the
end point for clear demonstration of inhibitory activity, it would appear that the titer
at this time lay between 1:100 and 1:1000.
Relative Sensitivities of the PTT and the
Thromboplastin Generation Test in
Mild Deficiency States
We have found that the P T T is very
sensitive in demonstrating mild plasma clotting defects. The following examples demon-
534
Vol. 29
RODMAN ET AL.
640-1
62
PTC-DEFICIENCY WITH INHIBITOR
P.W.B.
°J
340320300280260240PTT
220 (Sec)"u
200180o.
16014012010080-
o-••<? /
\
a
/
,*—x-
X'
605Units 4Plosmo *"
250ml/unH|-
* /
» /
X'
v
^ °
-x'
..x--x---x^
,-x-
«
-x'
x
12
1956
15 16 17'18 19 20 2l'22'23'24'25'26'27'28 29'30l 1 2
3 4
SEPTEMBER
OCTOBER
FIG. 3. Daily partial thromboplastin times of patient's plasma, control plaasma, and the mixture
of patient's and control plasma in equal parts. The great prolongation of the mixture indicates the
appearance of the inhibitor.
strate this in patients deficient in
antihemophilic factor (AHF) and plasma
thromboplastin component (PTC).
Mild AHF deficiency. Recently a mild
hemophiliac with a negative history 1 of a
family described several years ago,"9 was
observed in connection with a plastic operation on the kidney. For several weeks after
this operation he bled persistently from the
operative site. Prior to operation the patient's plasma showed only a 1 sec. prolongation over control BaSCi-adsorbed plasma in
the thromboplastin generation test. This difference might have been attributed to
laboratory error had we not known independently that he was mildly deficient in
AHF. (An assay several years earlier had
revealed 35 per cent AHF.) By comparison
with control plasma in the PTT test the
patient's plasma repeatedly failed to correct
plasma severely deficient in AHF, the
differences ranging up to 93 sec. At the same
time in the P T T the patient's plasma corrected PTC-deficient plasma almost as well
as did the control plasma and never showed
evidence of a circulating anticoagulant.
Mild PTC deficiency. Similarly we have
had the opportunity of studying a patient
with a mild deficiency in PTC by both
methods. In the thromboplastin generation test the patient's serum was only 1.8
sec. longer than control serum. The patient's
PTT, nevertheless, was 18 sec. longer than
the control, and correction of plasma known
to be very low in PTC was 34 sec. better by
control than by the patient's plasma. At the
same time the patient's plasma corrected
AHF-deficient plasma as well as did the
control and showed no evidence of an anticoagulant. He was prepared by plasma
June 1958
535
HEMOPHIMOII) STATES
transfusions preoperatively a n d underwent
hemorrhoidectomy without complications.
If t h e thromboplastin generation t e s t alone
had been used to determine the pres-
TABLE S
DEMONSTRATION OF INHIBITOR IN HIGH TITER IN
PTC* DEFICIENT PATIENT WITH THE PARTIAL
THROMBOPLASTIN TIME
PTC-DEFICIENCY WITH INHIBITOR
ISOO- p
W
Patient's
Plasma
Diluted with
Saline
Clotting Mixtures
B
PTTt
(Cephalin)
I250sec.
80.0
103.7
>300
177.2
119.0
113.6
104.3
Control
Control and saline (1:1)
Control
plus
patient's
plasma (1:11
A Patient
x Control
o Mixture 1:1
1:10
1:100
1:1,000
1:10,000
1:100,000
* Plasma thromboplastin component.
t Preincubation for 15 min. at 37 C.
ence or absence of a deficiency in the last 2
patients, we might well h a v e concluded t h a t
both were normal. Using t h e P T T and
severely deficient plasmas as test substrates,
there was never d o u b t of the presence or t h e
nature of t h e defects.
DISCUSSION
T h e partial thromboplastin time is a very
useful clinical test because it is simple,
versatile, and sensitive. While determinations vary somewhat from d a y t o day, there
FIG. 4. Daily partial thromboplastin times of
patient's plasma, control plasma, and an equal
parts mixture.
TABLE 7
THROMBOPLASTIN GENERATION TEST DEMONSTRATING INHIBITORY ACTIVITY IN PTC* DEFICIENCY
AFTER TRANSFUSION
Incubation time (min.)
l
sec.\
Control plasma plus control serum
P a t i e n t ' s plasma plus pat i e n t ' s scrum
Control plasma plus pat i e n t ' s serum
P a t i e n t ' s plasma plus
control serum
Control plasma plus control serum and patient's serum (equal
parts)
30.0
>45
36.2
>45
32.6
2
3
4
5
sec.
6
7
8
9
10
11
sec.
sec.
11.4
11.6
sec.
sec.
sec.
sec.
10.3
9.7
10.0
10.2
40.4
35.3
27.8
30.0
31.2
29.S
27.6
28.2
2S.6
IS.4
14.9
15.2
15.2
15.S
IS.7
20.9
21.5
16.9
12.5
sec.
sec.
sec.
23.5
20.2
13.0
43.5
42.2
39.0
31.5
29.6
29.0
24.1
7.7J
* Plasma thromboplastin component.
f Clotting time.
I Italicized numbers represent minimum clotting times.
10.8
536
RODMAN ET AL.
is little variation with repeated determinations on a single plasma or on a series of
plasmas during a single day.
Preparations are simple, as little time is
required for thawing the cephalin, eluate,
serum, and known deficient plasmas. The
most time-consuming step is the preparation
of fresh BaS0 4 -adsorbed plasma, when it is
to be used. When performing daily tests on
patients previously diagnosed, it is a simple
matter of preparing the plasma from whole
blood and thawing the cephalin. The actual
determinations in such an instance, which
always include the P T T of the mixture of
patient's with control plasma for detection
of inhibitory activity, usually require only
10 min.
The versatility of the partial thromboplastin time in diagnosis has been exemplified in detail. Plasma deficient in all of the
blood coagulation factors mentioned above
with the single exception of SPCA have been
shown to have prolonged partial thromboplastin times. By the use of mixtures with
prototype plasmas a conclusive diagnosis
can be made easily. For this purpose we
maintain a battery of lyophilized, known
deficient plasmas. If such plasmas are not
available, a presumptive diagnosis can be
made by use of the P T T and prothrombin
time with BaS0 4 -adsorbed plasma and
eluate. For example, in a congenital male
bleeder whose prothrombin time is normal,
it should be suspected that the patient is
probably deficient in either AHF or PTC.
If his partial thromboplastin time is prolonged, and is corrected by mixing with
BaS0 4 -adsorbed plasma, AHF deficiency is
almost certainly present. Conversely if this
patient's prolonged partial thromboplastin
time is not corrected by BaSCvadsorbed
plasma but is corrected by mixing with the
eluate of either plasma or serum, PTC
deficiency probably exists.
I t might be argued that the PTT is of
greatest diagnostic value when used in conjunction with a battery of prototype deficient plasmas. This is true. It is our policy to
use such plasmas routinely rather than the
BaS0 4 -adsorbed plasma and eluate since
specific diagnosis ultimately requires plasma
mixtures anyhow. I t might seem that this
Vol. 29
policy introduces a difficulty which the
ordinary laboratory can not overcome.
There is no problem, however, once a prototype patient has been diagnosed. If he has
been properly handled, he will be willing to
donate his own plasma whenever it is needed.
We call in our prototype patients at intervals for donations, and lyophilize the plasma
obtained. Thus there is always on hand a
sufficient supply of the complete battery.
Most severe bleeders who will be encountered are men with either AHF or PTC
deficiency, and there is little drain on the
stocks of Stuart, SPCA and Hageman
plasmas once obtained. Furthermore, a
diagnostic laboratory has no need for the
more recondite types until 1 of these rare
types is encountered. When this occurs,
laboratories such as ours are glad to furnish
small samples to others. In the past year
many workers in other parts of the world
have been assisted in diagnosing the rarer
defects by receiving small samples of
lyophilized plasma. As a result, a number of
SPCA-deficient and Stuart factor-deficient
patients have been specifically identified and
have themselves become local prototypes.
As a guide to therapy the P T T helps
determine the status of each of 2 distinct
aspects of the patient's coagulation mechanism. First, the disappearance of the clotting
factor supplied by transfusion can be readily
followed. This disappearance is manifested
by the gradual prolongation of the patient's
P T T after transfusion is decreased or
stopped. This phenomenon is illustrated in
Figures 1 and 2. Secondly, by testing mixtures of a patient's plasma with control
plasma, the presence or absence of a circulating anticoagulant can be determined readily.
This is seen most strikingly (see Figs. 3 and 4)
when such daily mixtures suddenly show
significant prolongation over the control
PTT.
The sensitivity of the partial thromboplastin time in detecting mild deficiencies of
AHF and PTC is well illustrated by the last
2 patients. In each instance symptoms were
either mild or inapparent. Had the thromboplastin generation test alone been relied upon, we probably would have decided that no significant deficiency existed.
June 1958
537
H E M O P H I L I O I D STATES
By testing the ability of each patient's
plasma to correct plasmas with known,
severe deficiencies, however, the defects
were clearly demonstrated. It has not yet
been established that the partial thromboplastin time is as sensitive in detecting mild
deficiencies of PTA, Hageman, and Stuart
factors as detecting deficiencies of AHF and
PTC, but our data suggest that such is the
case.
SUMMARY
1. The partial thromboplastin time (PTT)
test is described in detail, including preparation of the reagents.
2. The partial thromboplastin times of
plasmas deficient in antihemophilic factor
(AHF), plasma thromboplastin component
(PTC), Stuart factor, and Hageman factor
are shown to be prolonged. It is also known
that such is also the case in factor V (proaccelerin, labile factor) deficiency. The conclusive diagnosis in each instance can be
demonstrated by experiments in which test
plasmas fail to correct the clotting time of
known deficient plasmas.
3. Plasma deficient in serum prothrombin
conversion accelerator (SPCA) is shown to
have a normal partial thromboplastin time.
4. The PTT test is shown to have considerable value in observing daily changes
in a patient's coagulation status.
5. Circulating anticoagulants can be diagnosed by a simple modification of the test, in
which unknown plasma is mixed with a
normal control plasma. The titer of the anticoagulant can also be determined if desired,
by mixing the plasmas in various ratios and
incubating prior to testing.
6. The sensitivity of the P T T test in
detecting mild deficiencies of AHF and PTC
is compared with another widely used
method. It would appear that the P T T test
is not only simpler to perform but also more
sensitive.
SUMMARIO I N
INTEIILINGUA
1. Le test del tempore thromboplastinic
partial (TTP) es describite in detalio, incluse le preparation del reagentes.
2. Le tempores thromboplastinic partial de
plasmas deficiente in factor antihemophilic
(FAH), componente de thromboplastina
de plasma (CTP), factor Stuart, e factor
Hageman esseva determinate e trovate prolongate. II es etiam cognoscite que le mesmo
vale in caso de deficientia de factor V (proaccelerina, factor labile). Le diagnose conclusive pote esser establite in le caso individual per experimentos in que le plasma sub
investigation non corrige le tempore de coagulation de un plasma a deficientia cognoscite.
3. Esseva trovate que plasma deficiente in
accelerator del conversion de prothrombina
serai (ACPS) ha un normal tempore thromboplastinic partial.
4. Es monstrate que le test de T T P es de
alte valor in le observation de alterationes
diurne in le stato coagulatori del patiente individual.
5. Anticoagulantes circulante pote esser
diagnosticate per un simple modification del
test, in que le non-cognoscite plasma es
miscite con un normal plasma de controlo.
Etiam le titro del anticoagulante pote esser
determinate, si desirate, per miscer le plasmas in varie proportiones e per incubar los
ante le test.
6. Le sensibilitate del test de T T P in le
detection de leve grados de deficientia de
FAH e de CTP es comparate con un altere
methodo que es usate extensemente. II pare
que le test de T T P es non solmente plus
simple in su execution sed etiam plus sensibile in su resultatos.
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