Comparative Evaluation of a Partial Thromboplastin Reagent Containing a Non-settling,
Particulate Activator
ARTHUR L. BABSON, PH.D., AND SUSAN R. BABSON, M.S.
Warner-Lambert Research Institute, Morris Plains, New Jersey 07950
ABSTRACT
Babson, Arthur L., and Babson, Susan R.: Comparative evaluation of a
partial thromboplastin reagent containing a non-settling particulate activator. Am. J. Clin. Pathol. 62: 856-860, 1974. Five commercial reagents
for determining the activated partial thromboplastin time were compared on
an instrument with photometric clot detection. The activating agents
employed in these reagents are ellagic acid, celite, kaolin and a new agent,
colloidal silica. T h e reagent containing colloidal silica was the most sensitive
to low levels of heparin. It also was the only reagent with substantial
buffering capacity, and in precision on repetitive tests it was equal to or
better than any of the other reagents evaluated. Although there were no
striking differences among the reagents in sensitivities to moderate deficiencies in individual coagulation factors, the colloidal silica reagent was most
sensitive to factor V and the reagents containing ellagic acid were
significantly less sensitive to factors X and XII. (Key words: Partial
thromboplastin; A P T T reagent; Particulate activator; Heparin sensitivity;
Factor sensitivity; Precision; Buffering Capacity.)
thromboplastin
time (APTT) is a sensitive test for alterations in the intrinsic coagulation pathway.
Its two main uses are in screening patients
for coagulation defects and in monitoring
patients treated with heparin. Recent reports have shown significant variation
among various commercial APTT reagents in sensitivities to in-vitro heparin 6
and levels of factors VIII, IX, XI and
XII. 5 The activators in these reagents
were celite, kaolin, and ellagic acid, and
the data in both studies were obtained on
a Fibrometer.
Coagulation instruments incorporating
photometric sensing of the end point are
increasing in use. Recently, an APTT
T H E ACTIVATED PARTIAL
Received January 14, 1974; revised J u n e 11, 1974;
accepted for publication J u n e 11, 1974.
Address reprint requests to Dr. Babson.
856
reagent with a new activator, colloidal
silica, has become available. This activator
is particulate but does not settle on standing as do celite and kaolin. The present
study compares the performance of this
reagent with those of a number of major
commercial APTT reagents on an instrument employing photoelectric end
point detection. In addition to sensitivity
to heparin and the stage 1 coagulation
factors, we have compared the A P T T
reagents for precision and buffering
capacity.
Materials and Methods
Normal Plasma
Blood from 2 5 - 3 0 healthy donors was
collected in one tenth volume of 3.8%
sodium citrate. After centrifugation, the
December
1974
ACTIVATED PARTIAL THROMBOPLASTIN REAGENT
857
plasma was pooled and recentrifuged. All
plasma samples were kept on wet ice
during the course of the experiments.
Several different normal pools were used
in the various experiments.
Heparinized Plasma
Lithium heparin (Calbiochem., San
Diego, Calif. 92112), 148 units per mg.,
was accurately weighed out and diluted in
water to a concentration of 20 units per
ml. Small volumes of this solution were
added to normal plasma to give final
concentrations of 0.05, 0.1, 0.2 and 0.3
units per ml.
Factor-deficient plasma
Commercially available congenital
factor-deficient plasmas (General Diagnostics, Morris Plains, New Jersey 07950)
were mixed with normal plasma to give
20% and 40% concentrations of specific
factors.
Heparin Concentration units/ml
FIG. 1. Sensitivities of five commercial activated
partial thromboplastin reagents to heparin added to
the same pooled, citrated plasma.
APTT Reagents
All reagents were obtained commercially. Table 1 describes the reagents used.
APTT Assays
All assays were p e r f o r m e d on a
Coag-A-Mate/Single Channel (General
Diagnostics, Morris Plains, New Jersey
07950). T h e circular test trays containing
0.1 ml. of A P T T reagent were warmed to
37 C. At accurately timed intervals, 0.1 ml.
of plasma was forcibly added to provide
mixing with the A P T T reagent. T h e
p l a s m a - A P T T reagent mixtures were left
Table 1. A P T T Reagents Used in This Study
Designation
for
This Study
Activator
Automated APTT*
A
Colloidal silica
5 min.
25 mM
Platelin plus activator*
B
Celite
5 min.
25 mM
Activated Thrombofax
Reagentf
C
Ellagic acid
3 - 5 min.
20 mM
Partial thromboplastin
time testt
D
Kaolin
3 min.
30 mM
Activated Cephaloplastin§
E
Ellagic acid
2 min.
20 mM
Reagent
* General Diagnostics. Division of Warner-Lambert Company. Morris Plains. N.J. 07950.
t Ortho Diagnostics. Raritan, N.J. 08869.
t Hyland, Div. Travenol Laboratories, Inc., Costa Mesa, Calif. 92626.
§ Dade. Division American Hospital Supply Corporation, Miami. Fla. 33152.
Recommended
Contact Time
Calcium
Chloride
Concentration
858
A.J.C.P. —Vol. 62
BABSON AND BABSON
Table 2. T h e Sensitivity of A P T T
Reagents to Moderate
In dividual Factor
Deficiencies
little as 0.05 units of heparin per ml.
Reagents D and E were the most insensitive to heparin. Reagents B and C were
intermediate.
Factor Concentration
100%
A
B
C
D
E
35.5*
39.0
43.2
42.2
35.0
39.7
41.1
43.8
44.3
37.7
(4.2)*
(2.1)
(0.6)
(2.1)
(2.7)
47.7 (12.2)*
44.8 (5.8)
49.8 (6.6)
50.5 (8.3)
45.1 (10.1)
Factor VIII
A
B
C
D
E
34.1
39.7
45.0
45.0
33.9
39.8
47.7
50.3
50.2
40.0
(5.7)
(8.0)
(5.3)
(5.2)
(6.1)
44.1
50.1
56.7
55.3
45.3
(10.4)
(10.4)
(12.7)
(10.3)
(11.4)
Factor IX
A
B
C
D
E
34.8
43.2
46.9
45.0
39.8
40.2
46.5
51.5
50.7
45.6
(5.7)
(3.3)
(4.6)
(5.7)
(5.8)
52.3
57.4
63.0
54.7
56.5
(17.5)
(14.3)
(16.1)
(9.7)
(16.7)
Factor X
A
B
c
D
E
31.2
36.5
38.5
38.6
24.6
44.4 (13.2)
50.4 (13.9)
47.1 (8.6)
51.5 (12.9)
32.5 (7.9)
56.6
63.0
53.1
63.0
46.7
(25.4)
(23.5)
(14.6)
(24.4)
(12.1)
A
B
C
D
E
35.4
43.1
39.7
42.2
35.3
52.0
59.9
58.9
57.5
54.5
(16.6)
(16.8)
(19.2)
(15.3)
(19.2)
68.0
76.0
75.9
75.7
69.6
(34.6)
(33.9)
(36.2)
(33.5)
(34.3)
A
B
C
D
E
31.2
36.5
38.5
38.6
24.6
42.6 (11.4)
49.1 (12.6)
43.4 (4.9)
50.9 (12.3)
28.9 (4.3)
52.7
64.7
48.4
59.8
35.4
(21.5)
(28.2)
(9.9)
(21.2)
(10.8)
Factor V
Factor XI
Factor XII
40%
Specific Factor Sensitivity
Reagen I
20%
* Figures in parentheses show the increase in APTT over 100%
normal plasma. All numbers are the average APTT in seconds.
undisturbed at 37 C according to the
contact time recommended by each reagent manufacturer (see Table 1). T h e test
was initiated with 0.1 ml. of the specified
calcium chloride solution delivered by the
Coag-A-Mate. All data reported are averages of duplicate or triplicate determinations.
Results
Table 2 summarizes the data on
factor-deficient plasmas. There were no
marked differences among the various
reagents in responses to m o d e r a t e
deficiencies in factors VIII, IX and XI.
Reagent A was more sensitive to factor V
deficiency and reagents C and E were
least sensitive to factors X and XII. Factor
XI deficiencies prolonged the A P T T the
most and factor V deficiencies the least.
Precision
The standard deviations of 12 consecutive APTT's of a normal plasma and a
plasma with 15% factor VIII were determined for each reagent. T h e means and
standard deviations are shown in Table 3.
Buffering Capacity
Figure 2 shows the effect of adding
50-/xl. increments of 0.1 N NaOH or 0.1
N HC1 to 2 ml. of A P T T reagent on the
pH of the mixture. Reagent E had no
buffering at all except for that provided
by the ellagic acid itself. The pK of ellagic
acid is too high to protect againt pH shifts
in the physiologic range. Reagents B, C
and D had minimal buffering to added
base but very little to acid. Reagent A had
substantial buffering to both acid and
base.
Table 3. The Precision of A P T T
Reagents on 12 Consecutive
Tests
Reagent
Heparin Sensitivity
Figure 1 shows the sensitivities of the
various reagents to in-vitro heparin. Reagent A was the most sensitive, showing a
marked increase in the A P T T with as
A
B
C
D
E
(
Normal Plasma
32.9
35.0
33.8
39.4
26.5
±
±
±
±
±
0.7
1.0
0.7
3.4
1.2
sec.
sec.
sec.
sec.
sec.
15% Factor VIII
52.8 ± 0.6 sec.
66.5 ± 2.4 sec.
61.4 ± 1.7 sec.
*
56.0 ± 1 . 1 sec.
Insufficient data because of failures in detection of end point.
December
1974
859
ACTIVATED PARTIAL THROMBOPLASTIN REAGENT
FIG. 2. Titration curves for five
commercial activated partial thromboplastins. T h e reagents showing the least
change in pH with the addition of acid
or base have the greatest buffering
capacities.
15
10
1
Acid Added meq/L
Zucker et al.1 have shown that the pH of
fresh citrated plasma in unstoppered
tubes can increase from 7.1 to 8.5 in four
hours, owing to loss of carbon dioxide,
and this change can significantly affect the
prothrombin time. T o see whether this
pH change would affect the APTT's with
buffered and unbuffered reagents, normal plasma with and without added heparin was adjusted to pH's of 7.1, 7.7, and
8.5 by blowing carbon dioxide or air over
the surface of the plasma. Table 4 shows
the APTT's of these plasmas as measured
by reagents A and E. The pH of the
sample had no effect on the A P T T with
reagent A but profoundly influenced the
A P T T as measured by reagent E. With
this reagent a change in the pH of the
plasma as little as 0.8 pH units caused as
much as a 22.5 second difference in the
APTT.
Discussion
Heparin is generally considered to be
the drug of choice in the treatment of
0
i
i
i
5
10
15
Bate Added meq/L
20
venous thrombosis and pulmonary embolism. Recent evidence indicates that the
efficacy of heparin treatment can be monitored by the activated partial thromboplastin time. 1 There is also a growing
interest in the use of low-dose heparin
treatment for the prevention of venous
thrombosis. 2,4 It is clear that monitoring
heparin, either in therapeutic doses for
the treatment of established venous
thromboembolism or in low doses for the
prophylaxis of venous thrombosis, requires a test which is sensitive to the
effects of heparin. Only reagent A was
found to be adequately sensitive to 0.05
units of heparin per ml. However, the
sensitivities of the other reagents were
considerably greater than reported by
Soloway et al.6 They presumably studied
many of the same reagents that we did,
but found that five of six reagents gave
APTT's of less than 70 seconds at a
heparin concentration of 0.7 units per ml.
T h e reason for this discrepancy is not
readily apparent, but it may be due in
860
BABSON AND BABSON
Table 4. Effects of Plasma pH on
Apparent A P T T as Measured
by Strongly Buffered (A)
and Unbuffered (E)
Reagents
Norni: :il I'lasma
pH 7.1
|)ll 7.7
pH 8.5
Heparini/.ed Plasma
Reagent A
Reagent K
Reagent A
Reagent E
33.9 sec.
33.9 sec.
33.1 sec.
34.8 sec.
26.9 sec.
38.1 sec.
55.6 sec.
56.1 sec.
56.1 sec.
43.9 sec.
37.6 sec.
60.1 sec.
part to the different instruments used for
clot detection. Our results are more in
line with data found by Hirsh and Gallus
using A P T T reagents prepared in their
laboratory. 3
O u r data on specific factor deficiencies
are also somewhat at variance with those
reported by Sibley et al.5 They found our
reagent D more sensitive to factors VIII
and IX and our reagent B less sensitive
than three other reagents. We found no
marked differences among the various
reagents in sensitivities to those two factors. They also found our reagent C most
sensitive to factor XI and XII levels, while
we observed no increased sensitivity to
factor XI with this reagent and decreased
sensitivity to factor XII. These authors
used a four-part system as employed in
factor assays to quantitate specific factor
deficiencies. The A P T T test is employed
considerably more widely as a screening
test for stage 1 coagulation defects. Our
experiments represented this use.
The ability to detect a factor deficiency
would be influenced by the precision of
the method as well as the sensitivity to
that factor. Sibley et al.5 found a marked
lack of reproducibility with our reagent D
on the Fibrometer. We also observed the
least precision with this reagent, probably
resulting from fluctuations in optical density as the kaolin rapidly settles out.
A.J.C.P.—Vol.
62
Recent lots of reagent D received since
this work was completed contain a notice
proscribing its use on instruments with
photoelectric end point detection. We
found the best precision with reagent A.
However, the precision with reagents B, C
and E was quite satisfactory.
T h e importance of pH control in
biological systems is not adequately recognized by many workers in blood coagulation. This is in spite of the fact that
enzymologists have long realized the need
for pH control, and blood coagulation is
considered to be a series of enzymatic
reactions. In many coagulation tests, including the A P T T and the prothrombin
time, plasma comprises one third of the
reaction mixture. T h e p H of plasma is
variable and will change rapidly if the
sample is not protected against loss of
carbon dioxide. The pH is best controlled
with adequate buffering in the reagent.
Only reagent A could be considered to
have substantial buffering capacity.
References
1. Basu D, Gallus A, Hirsh, et al: A prospective
study of the value of monitoring heparin
treatment with the activated partial thromboplastin time. N Engl J Med 287:324-327, 1972
2. Gallus AS, Hirsh J, Tuttle RJ, et al: Small
subcutaneous doses of heparin in prevention
of venous thrombosis. N Engl J Med
288:545-551, 1973
3. Hirsh J, Gallus, AS: T h e activated partial
thromboplastin time. N Engl J Med 288:1410,
1973
4. Kakkar VV, Corrigan T, Spindler J, et al:
Efficacy of low doses of heparin in prevention
of deep-vein thrombosis after major surgery:
A double-blind, randomized trial. Lancet
2:101-106, 1972
5. Sibley C, Singer JW, Wood RJ: Comparison of
activated partial thromboplastin reagents. Am
J Clin Pathol 59:581-586, 1973
6. Soloway HB, Cornett BM, Grayson JW: Comparison of various activated partial thromboplastin reagents in the laboratory control of
heparin therapy. Am J Clin Pathol 5 9 : 5 8 7 590, 1973
7. Zucker S, Cathey MH, West B: Preparation of
quality control specimens for coagulation. Am
J Clin Pathol 53:924-927, 1970