A Precise, Continuous Recording Clot Timer
Based on a Thermometric Detection System
WILLIAM D. BOSTICK, B.S., AND PETER W. CARR, P H . D .
Department of Chemistry, University of Georgia, Athens, Georgia 30602
ABSTRACT
Bostick, William D., and Carr, Peter W.: A precise, continuous recording
clot time based on a thermometric detection system. Am. J. Clin. Pathol.
60: 330-336, 1973. A new plasma and blood coagulation time detector is
described. The instrument utilizes a conventional glass probe thermistor
mounted in a vibratory mixer as the detection element. Both the initiation
and the end point of the coagulation process are registered on a strip-chart
recorder. A precision of 2 to 4% in end-point time has been obtained in
both the normal and therapeutic prothrombin time ranges. The end point
as determined by this method has a correlation coefficient of 0.98 at the
99.5% confidence interval with the Fibrometer method.
for the instrumental detection and determination of clotting time
have been introduced over the past decade.
That these devices have not been entirely
satisfactory is witnessed by the increasing
variety of physicochemical principles employed in the design of the most recently
available instruments. 1 - 13 Generally, the
presently available automatic or semiautomatic systems fall into two categories: those
which detect the onset of clotting, i.e., are
sensitive to the end-point condition but
produce no other type of information about
the sample, and those which produce a
continuous record of some characteristic of
the sample. The well-known electromechanical system *•7-8 is an example of the first
type, and the photoelectric clot timer3> e<12
is representative of the second.
NUMEROUS DEVICES
The purpose of this paper is to describe
a prototype, mechanically simple clot timer
Received November 20, 1972, received revised
manuscript January 4, 1973; accepted for publication January 12, 1973.
This work was carried out in the Department of
Chemistry at the University of Georgia, Athens,
Georgia 30601, and was supported by a grant from
the National Institute of Health (GM 17913).
which has the following characteristics: (a)
inherently monitors the temperature of the
test sample; (b) unambiguously indicates
the instant of reagent addition without use
of any ancillary switches in the pipet; (c)
records a physical property of the plasma
prior to and after coagulation; (d) provides
a sharp, precise indication of a coagulation
time for both clear and turbid or opaque
samples. T h e present device is similar to
the photoelectric system in that it produces
a permanent continuous recording on the
sample; however, in contrast to the photoelectric method, the procedure correlates
closely with the Fibrometer system in the
abnormal prothrombin time range, 6 ' 12 and
is not subject to interference from hyperbilirubinemic, lipemic, or hemolyzed samples.6
Detection of the coagulation end point is
based upon the drastic change in rate of
ohmic heat dissipation from a thermistor
upon the conversion of plasma sol to gel.
Power ( ^ 5 to 10 milliwatts) is dissipated
from the thermistor in the sol by vibrating
the thermistor at 60 Hz at extremely small
amplitudes. Clot formation insulates the
330
September
1973
THERMOMETRIC COAGULATION TIMER
331
Fie. 1. Thermometric coagulation
detector.
thermistor or severely decreases the heat
dissipation rate. This results in a rapid internal self-heating of the thermistor, which
can be measured by use of an ordinary
VVheatstone bridge circuit.
Unlike some technics, such as the Fibrometer, endpoint time resolution for a
sample is not limited by an instrumental
period of sampling, but rather by the kinetics of clot formation and the time constant and resolution of the recorder. We
have found that the vibratory motion of
the thermometric detector at the frequency
and amplitude used this study does not
have any deleterious effect upon clot formation.
Materials and Methods
Apparatus
The experimental apparatus (Fig. 1) consists of (1) the thermometric clot detector
and (2) associated circuitry, (3) a vibratory
mixer, (4) a constant-temperature water
bath, and (5) a strip-chart recorder.
The Thermometric Clot Detector. A detail of the detector is shown in Figure 2.
The sensing element is a 2,000-ohm glass
probe thermistor (Veco 32A223 ®). A Teflon
disk impeller (0.325 in diameter) is forcefitted over the thermistor probe, and the
assembled unit mounted in the chuck of
the vibratory mixer.
1
Victory Engineering Co., Springfield, New Jersey.
332
BOSTICK AND CARR
Fio. 2. Detail of clot detector.
Associated Circuitry. The transducer signal is measured by use of a Wheatstone
bridge circuit (Fig. 3).2 Due to the nature
of the end-point phenomenon, it is an advantage to power the bridge with a potential (EB) of at least 5 volts (d.c). In this
AJ.C.P.—Vol.
60
work, bridge potentials of approximately
10 volts (d.c.) were used.
Vibratory Mixer. To prevent excessive
self-heating of the thermistor in the plasma
sol prior to clot formation, it is necessary
to agitate the solution gently near the
thermistor surface. This can be optimally
achieved by use of a vibratory mixer
(Chemapec VIBRO-Mixer E-l t). The mixer
is operated at 85 VAC; operation of the
mixer at voltages drastically smaller or
greater than this value can result in loss
of end-point clarity or disruption of the
clot, respectively.
Constant-temperature Water Bath. The
temperature of the water in the baili (f>0
by 45 by 30 cm.) is regulated to ± 0.05 C.
by a temperature controlled (YSI Thermistemp Model 711) at a mean of 37.0 C. The
water is stirred by a mixer (Lightnin Mixer
Model L§). The bath is covered with % in.
diameter polypropylene balls (Techne'J) to
insulate the bath thermally and to reduce
evaporation of the water.
Strip-chart Recorder. In this work, an
inexpensive multispeed servo chart recorder
was used (Heath Model 1R-18M ||). This
recorder has a fixed sensitivity of 10 millivolts full scale and a maximum chart speed
of 5 sec. per in. The recorder sensitivity
may be conveniently varied by interposing
an operational amplifier (Burr Brown 3112/
12c**) between the Wheatstone bridge and
the recorder (Fig. 3). This has the additional desirable effect of presenting a low
input impedance to the recorder. The prothrombin times for this study were recorded at an effective sensitivity of 200
millivolts full scale and a chart speed of
5 sec. per in.
f Chemapec, Inc., Hoboken, N. J.
j Yellow Springs Instrument Co., Yellow Springs,
Ohio.
§ Mixing Equipment Co., Rochester, N. Y.
*i Techne, Inc., Princeton, New Jersey.
|| Heath Co., Benton Harbor, Mich.
** Burr-Brown Research Corp., Tucson, Ariz.
September 1973
333
T H E R M O M E T R I C COAGULATION T I M E R
To
2 K f l Thermistor
Fin. 3. Wheatstonc bridge and
operational amplifier circuits.
To Recorder
Methods
The One-stage Prothrombin
Time Test
Simplastin ft is the thromboplastin reagent used. For each run, 0.6 ml. of the
thromboplastin reagent is incubated for 3
min. in a glass test tube (13 X 40 mm.)
placed in the water bath. With the bridge
power and vibrator on, a recorder baseline
is established. Inherent in the nature of the
detection system, the location of the baseline is a direct measurement of the temperature of the test solution, and should
not vary substantially if the solution is
properly thermally equilibrated with a wellregulated water bath. With the chart on,
the test is initiated by the addition of 0.3
ml. of plasma, which likewise has been incubated in the bath.
Figure 4 is a recorder trace for a prothrombin time test, illustrating the interpretation of the end-point time as the linear distance (time) between points A and B
as indicated by measurement C. The recorder response at point A is due to elementary thermochemical effects and, primarily, intentional temperature mismatch
of the reagents. Point B, precisely located
by extrapolation of the linear portions of
the end-point break, represents the onset of
rapid self-heating of the thermistor due to
clot formation. Any fibrin strands adhering
ft Warner-Chilcott, Morris Plains, N. J.
to the detector are removed prior lo (lie
next analysis.
Results
Prothrombin
Time Test
Table 1 indicates the performance of the
thermometric clot detector with several coagulation control specimens in the normal
and therapeutic ranges, and illustrates the
obtainable precision and accuracy. The precisions of the thermometric end points
stated in Table 1 were computed at the
90% confidence intervals, as given below:
ts
Vn
(1)
/ n £ X » -- C(XX) 2
"V
n((nn--1l)
(2)
X±
where
X is the arithemtic mean and t = tUa/2 is
the Student's t for n — 1 degrees of freedom
(n = number of replicates).10
The prothrombin times for commercial
control samples were within the manufacturers' stated ranges, as established by use
of the wire-loop technic (Verify tt) or the
Fibrometer (Fibro-Trol §§). The coefficient
of variation was less than 4% for each sample using the thermometric technic, which
%t Warner Chilcott, General Diagnostics Division,
Morris Plains, N. J.
§§ BioQuest, Division of Benton, Dickinson and
Co., Cockeysville, Md.
334
A.J.CP.—Vol.
BOSTICK AND CARR
60
+ 80
Fic. 4. Prothrombin
time recording of plasma
from a patient on anticoagulant therapy. A indicates the instant of addition of plasma to the
thromboplastin. B is the
sharp break in the curve
representing the clotting
end-point. Measurement
C, in time units, represents the prothrombin
time. T h e magnitude of
the end-point may be
measured as in D at a
predetermined time after
the end point (e.g., 20
sec).
>
£
+40
2
*.
+20
O
-20
TIME
Table 1. Accuracy and Precision of Thermometric Clot Detector Control Samples in
the Normal and Therapeutic Ranges
Control Sample
Manufacturer's
Specifications
Fibro-Trol-10 oxalate*
34 ± 3 sec.
Fibro-Trol-20 oxalate*
24 ± 3 sec.
Verify Citrate If
18 ± 2 sec.
Pooled normal plasma
—
Thermometric
End Point
31.9 ± 0.6
(n-S)
23.0 ± 0.8
(n=3)
17.5 ± 0.3
(n=4)
12.5 ± 0.4
(n = 6)
sec.
sec.
on anticoagulant therapy, were collected in
sodium citrate and the plasma separated.
Prothrombin times were determined by the
Fibrometer, and the remaining plasma
quickly frozen for later use with the thermometric technique. It should be noted
that the Fibrometer times were performed
with Ortho Brain Thromboplastin 1111 as
the thromboplastin reagent.
sec.
sec.
* Bio-Quest.
t Warner-Chilcott, General Diagnostics Division.
is comparable to that reported for similar
samples using the Fibrometer. 15
Correspondence between the Thermometric
Method and the Fibrometer
Because of its documented correspondence with manual prothrombin time measurements, 4 - 8 the Fibrometer f^f was selected as the instrumental reference technic. Fibrometer prothrombin times were
performed at Emory University Hospital
Coagulation Laboratory, Atlanta, Georgia.
T h e specimens of blood, all from patients
flfl BioQuest, Division of Benton, Dickinson and
Co., Cockeysville, Md.
20
22
24
26
28
30
PROTHROMBIN TIME (SEC). FIBROMETER
FIG. 5. Correlation of thermometric method with
Fibrometer method for determination of prothrombin time. Correlation coefficient is 0.98 for 24
samples.
|| || Ortho Diagnostics, Raritan, New Jersey.
September
1973
335
T H E R M O M E T R I C COAGULATION T I M E R
Figure 5 is the scatter diagram for 24 therapeutic range plasma prothrombin times
for the thermometric method versus the
Fibrometer. The solid line indicates the
locus of points if both methods were to give
identical results. The clashed lines represent
the 90% confidence interval about the leastsquares linear regression line (y = 0.94x +
1.5).11 The correlation coefficient is 0.98
(Student's t of 21, or p < 0.0005) for the 24
samples. Mean clotting times were 21.61
and 21.76 sec. for the Fibrometer and thermometric end-point methods, respectively.
Plasma Dilution
Curve
Many laboratories report coagulation
time measurements in terms of "per cent
of normal activity" as determined by comparison of the clotting time of the test sample versus that of a suitably diluted normal
sample. Therefore, we have investigated the
elfect of dilution on the end point as measured by the thermometric clot timer.
It is important to note that dilution of
the sample with 0.9% saline solution reduces the level of all coagulation factors.
On the other hand, it is known that barium
sulfate selectively absorbs factors II (prothrombin) and VII from plasma but does
not appreciably lower the concentration of
fibrinogen or factor V.9 Consequently, dilution of normal plasma with barium sulfateadsorbed plasma more closely mimics the
action of oral anticoagulant drugs (e.g.,
coumarins) which depress the synthesis of
factors II, VII, IX, and X. 6
Figure 6 illustrates thermometric prothrombin time recordings for a diluted
plasma specimen. If saline solution is used
as the diluent (Fig. 6b), the thermometric
prothrombin time end point may show a
considerable decrease in sharpness and magnitude when the concentration of plasma
is reduced to 15 to 30% of its normal value.
This is in contrast to the usually quite
sharp end point observed for an authentic
plasma of similar "activity" obtained from
a patient on anticoagulant therapy (Fig. 4).
L
._!
L
I
L
L
I
J
TIME
FIG. 6. Prothrombin time for a plasma specimen
diluted to 33% concentration: A, BaSO<-adsorbed
plasma as diluent; B, 0.9% saline solution as
diluent.
When barium sulfate-adsorbed plasma is
used as the diluent in preparation of the
dilution curve, as in Figure 7, both normal
sharpness and magnitude are restored to
the thermometric end point (Fig. 6a).
Discussion
The objectives of automatic measurement
and recording of the clotting process include the exclusion of human error, saving
of time, acquisition of additional information not obtainable by other methods, and
the production of permanent records.13 The
thermometric method described satisfies
these objectives.
The addition of plasma to a thermally
incubated mixture of thromboplastin and
calcium, or the more convenient implementation of the addition of calcium solution to a plasma-thromboplastin mixture,
automatically marks the initiation of the
reaction. T h e end point, normally quite
sharp, is precisely found by extrapolation
with the device described here. Direct digital read-out of the end-point time could
be implemented by differentiation of the
output signal of the Wheatstone bridge.
The sharp signal spikes so produced (corresponding to points A and B in Fig. 4)
336
A.J.C.P.— Vol. 60
BOSTICK AND CARR
m 40
Fio. 7. "Prothrombin activity"
curve for pooled normal plasma,
diluted
with
BaSOj-adsoibed
plasma.
o 20
£
10-
10
20
30
40
50
60
70
80
90
100
% Concentration of Normal Plasma, Diluted with B0SO4-Absorbed Plasma
could be used to start and stop a precision
timer, thereby completely automating the
measurement step.
The graphical representation of the clotting process offers at least two possible
sources of information in addition to the
end-point time. First, the location of the
baseline is a direct check on the temperature of the test sample. The importance of
accurate temperature control has been
stressed in the literature; for example, a
deviation of 1 C. from the customary 37.5 C.
can prolong the prothrombin time by one
second.14 Also, the magnitude of the endpoint break my give information about the
biophysical properties of the clot.
The accuracy and precision of the thermometric method have been illustrated.
The agreement with the Fibrometer system
has been demonstrated in the determination of prothrombin time for patients on
oral anticoagulant therapy for ischemic
vascular disease.
Work is in progress to define more closely
the physicochemical processes responsible
for the end point, the optimum analytical
conditions, a more extensive comparison of
the technic with other existing technics, and
the application of the device to other coagulation tests such as activated partial
thromboplastin time.
References
1. Brittin GM, Biecher G: Instrumentation and
automation in clinical hematology, Progress in
Hematology. Volume VII. Edited by LM
Tocantins. New York, London, Grune and
Stratton, 1971, p p 326-329
2. Carr PW: Analytical and measurement aspects
of thermometric titrimetry, Critical Reviews
in Analytical Chemistry. Volume II. Edited
by L Meites. Cleveland, Chemical Rubber
Corporation, 1972, pp 491-557
3. Cawley LP, Eberhard L: A permanent record
for determinations of prothrombin time, using
the Spinco Analytrol as a recorder. Am J Clin
Pathol 37:219-226, 1962
4. Fcwell RG, Cundy A, Jenkins GC: T h e one
stage prothrombin test: A comparative study
of a mechanized prothrombin meter and
manual testing. Med Lab Tcchnol 29:147-151,
1972
5. Goldstein A, Aronow L, Klaman S: Principle of
Drug Action. New York, Harper and Row,
1968, pp 412-413
6. Jacobs AG, Freer JA: Use of a prothrombin
meter for Quick's one stage test. Br Med J
2:978-980, 1963
7. McCormick JB, Kopp JB: Semi-micro prothrombin time test chamber: A comparative
study of 750 duplicate tests with Sera-Tek
chambers, Mechrolab clot timer, and standard
Quick test tube methods. Tech Bull Regist
Med Technol 37:511-518, 1967
8. Miale JB: T h e fibrometer system for routine
coagulation tests: Prothrombin time and partial thromboplastin time, macro and micro.
Am J Clin Pathol 43:475^180, 1965
9. Miale JB: Laboratory Medicine, Hematology.
St. Louis, C. V. Mosby Co., 1967, pp 1010-1016
10. Natrella MG: Experimental Statistics. National
Bureau of Standards Handbook 91. Washington, U. S. Government Printing Office, 1966,
section 2-1.4.1
11. Ibid, section 5-4.1.2
12. Sibley C, Singer J W : Comparison of the Fibrometer System and the Bio/Data Coagulation
Analyzer. Am J Clin Pathol 57:369-372, 1972
13. von Kaulla KN: Quantitative methods for recording blood coagulation: Theoretical and
practical aspects, Progress in Hematology.
Volume III. Edited by LM Tocantins. New
York, London, Grune and Stratton, 1962, pp
218-243
14. Wehrmacher W H : Guarding the prothrombin.
Curr Med Digest 34:432-436, 1967
15. Woody GL, Brenner N, Foster RL: A comparison study of 175 duplicate determinations
using the Electra 500 and the standard fibrometers. Tech Bull Regist Med Technol 39:
112-115, 1970