Comparison
of Activated
plastin
Partial
T h r o m b o -
Reagents
CAROL SIBLEY, B.S., MT(ASCP), JACK W. SINGER,
M.D.,
AND Ross J. WOOD, P H . D .
Center for Disease Control, Health Services and Mental Health Administration,
Public Health Service, Department of Health, Education, and Welfare,
Atlanta, Georgia 30333
ABSTRACT
Sibley, Carol, Singer, Jack W., and Wood, Ross J.: Comparison of activated
partial thromboplastin reagents. Am. J. Clin. Pathol. 59: 581-586, 1973.
Five commercial reagents and one noncommercial reagent for determining
activated partial thromboplastin times were tested for reproducibility of
results and ability to detect abnormalities of the intrinsic coagulation system.
The sensitivity of the reagents to factors VIII and IX varied markedly; two
reagents were so insensitive to factor IX that results obtained were misleading.
Reproducibility of results with all reagents tested was satisfactory, although
wide inter-reagent variability in results was noted.
IN JULY 1971, the Center for Disease Control (CDC) conducted a survey of all laboratories enrolled in the CDC prothrombin
time proficiency testing program. The five
partial thromboplastin reagents most commonly used by these laboratories, as well
as a "homemade" reagent, are the subject
of the following study. There is almost no
information in the literature on the reproducibility or variability of results with
commercial partial thromboplastin reagents or on the sensitivity of these reagents to the clotting factors they purport
to measure.
In this study, the variability, reproducibility, and general range of activated partial thromboplastin times (APTT) for a
normal and a grossly abnormal plasma
were determined for each reagent. The
sensitivity of each reagent to factors VIII,
IX, XI, and XII was measured and compared.
Materials and Methods
Fibrometer® system consisting of two
Fibrometers with 0.3 ml. heads, a thermal
prep-block, and a single pipetting gun was
used to perform the activated partial
thromboplastin times. A plasma sample
with a prolonged A P T T was tested with
each of our Fibrometers, and the two instruments which showed the best precision
were used throughout the study.
One lot of each of five commercial activated partial thromboplastin (APT) reagents t was used throughout the study. A
» BBL Division of BioQuest, Cockeysville, Maryland.
f APT I = Activated Cephaloplastin, Dade Reagents, Division of American Hospital Supply Corporation, Miami, Florida. APT II = Activated
Thrombofax, Ortho Diagnostics, Raritan, New Jersey. APT III = Fibrolet, BBL, Division of BioQuest,
Cockeysville, Maryland. APT IV = Partial Thromboplastin Time Test—Hyland, Division of Travenol
Laboratories, Inc., Costa Mesa, California. APT V
= Platelin Plus Activator, General Diagnostics, Division of Warner-Chilcott, Morris Plains, New
Jersey.
Use of trade names is for identification only and
Received May 3, 1972; received revised manu- does not constitute endorsement by the Public
script June 12, 1972; accepted for publication June Health Service or by the U. S. Department of
Health, Education, and Welfare.
30, 1972.
581
582
SIBLEY ET AL.
A.J.C.P.— Vol. 59
The normal and abnormal plasma pools
were assayed for factors I, II, and X as
previously described.2 Factors V and VII
were assayed by a substitute prothrombin
Factor
Normal Pool
Abnormal Pool
method which used factor V-deficient and
factor VH-deficient plasma, respectively, as
I
275 mg. per 100 ml. 172 mg. per 100 ml.
the substrate. Factors VIII, IX, XI, and
II
100% activity
19% activity
XII were assayed by the method of Simone
V
94% activity
72% activity
VII
92% activity
7% activity
and colleagues.8 The vial-to-vial variability
VIII
128%, activity
82% activity
of each pool was tested prior to the actual
IX
171%, activity
5% activity
study. The coefficients of variation were
X
100% activity
14% activity
XI
100%, activity
39%, activity
3.5% and 5.2% for the normal and abnorXII
119%, activity
79%, activity
mal pools, respectively. The stability of the
plasma samples was tested by performing
new vial of reagent was opened each day replicate APTT's with APT VI at zero and
and was kept on melting ice until tested. 5 hr. The plasma samples were kept on
Celitet and inosithin§ were used to pre- melting ice during the time lapse. The repare the "homemade" reagent fl by the rults obtained after 5 hr. were within two
method of Abildgaard and co-workers 1 as standard deviations of the earlier results.
modified by Simone and colleagues.3 The The actual testing required less than 3 hr.
working reagent was made each day of each day. Immediately prior to testing each
testing and was kept at room temperature. day, five vials of the normal plasma were
The order in which the reagents were reconstituted and pooled, as were five vials
tested was randomized each day.
of the deficient plasma. The plasma pools
The strength of the calcium chloride were kept on ice throughout the testing.
varied according to each manufacturer's diOn five nonconsecutive days, five replirections. In the procedure used with APT cates each of the normal and deficient plasVI, 0.025 M calcium chloride was used. In mas were tested with each reagent. The
all procedures, the calcium chloride re- following procedure was used for each of
mained in the heating block at 37 C.
the reagents. After 0.1 ml. of activated
The plasma samples were prepared by partial thromboplastin reagent had been
the method of Zucker and associates4 ex- incubated for 2 to 3 min. at 37 C., 0.1 ml.
cept that 0.6 Gm. of buffer || was used per of plasma was added to the reagent and
100 ml. of plasma instead of the original thoroughly mixed by drawing the mixture
1.19 Gm. The normal and abnormal plasma up into the pipette tip and expelling it
samples tested consisted of two separate 10 times. The mixture was incubated exlarge pools obtained from normal male actly 4 min.; then 0.1 ml. of warmed calblood donors. However, the abnormal cium chloride was added and the automatic
plasma had been adsorbed with Alhydrox # timer was started.
to remove some of the clotting factors. The
Assays for factors VIII, IX, XT, and XII
plasma pools were dispensed in 1 ml. ali- were performed by two technologists, each
quots, lyophilized, and stored at 4 C.
using a Fibrometer system as described
above with 0.4 ml. heads. A different factor
% Kilter-Cel, Johns-Manville, Jolict, Illinois.
was assayed each day with the six reagents.
§ Associated Concentrates, Woodsidc, Long IsThe str&ngth of calcium chloride recomland, New York.
H APT VI—Celite-inosithin.
mended by the manufacturers was used
II N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
arid (HEPES), Calbiochem, Los Angeles, California. with each of the five reagents. A strength
of 0.035 M was used with APT VI. The
# Cutter Laboratories, Berkeley, California.
Table 1. Concentrations of the Various
Coagulation Factors in the Normal
and Abnormal Plasma Pools
April 1973
583
ACTIVATED PARTIAL THROMBOPLASTIN REAGENTS
Table 2. Results Obtained with the Normal Plasma Pool
Reagent
Mean*
(See.)
AIT 1
APT II
APT III
APT IV
AIT V
APT VI
39.17
35.88
33.01
35.74
49.11
38.76
Standard Deviation Standard Deviation
of Replicate Assays of Single Assays
in the Same Day on Different Davs
(Sec.)
(Sec.)
2.02
1.44
0.35
1.09
1.31
1.96
2.02
1.46
0.41
1.50
2.10
1.96
F Ratio
between Days
0.71
1.0S
2.56
5.49f
8.85f
0.70
* The mean of the pool for each reagent was obtained from the results of all 5 days of testing.
t The F Ratio is statistically significant at the 1% level and indicates that results were not the same for all days'.
Table 3. Results Obtained with the Abnormal Plasma Pool
Reagent
Mean*
(Sec.)
APT I
APT It
APT 111
APT IV
AIT V
APT vr.
69.40
81.56
109.72
143.44
90.79
160.58
Standard Deviation Standard Deviation
of Replicate \ssavs of Single Assays
in the Same Day on Different Davs
(Sec.)
(Sec.)
3.72
5.45
3.77
11.04
3.13
S.60
5.12
5.45
4.24
14.50
4.55
9.04
!•' Katio
between Days
5.48f
0.46
2.32
4.63f
6.5Sf
1.52
* The mean of tlie pool for each reagent was obtained from the results of all 5 days of testing.
f The K Ratio is statistically significant at the 1% level and indicates that results were not the same for all days.
calcium chloride was maintained at 37 C.
during the testing with all procedures.
All plasma samples were prepared according to the method of Zucker and associates/ modified as previously described.
For each factor assayed, a normal plasma
pool and a plasma deficient in that specific
factor were tested. The substrate used in
each assay was the plasma of a person severely deficient in that particular factor.
All plasma specimens were reconstituted
immediately prior to testing. All reconstituted and diluted plasmas were kept on
melting ice until tested.
A substitute A P T T was performed by
using the reagents according to the manufacturer's directions. APT VI was used according to the method of Simone and coworkers.3 In each procedure, the reaction
mixture contained 0.1 ml. of activated partial thromboplastin reagent, substrate, and
diluted plasma. Depending on the reagent
being used, the mixture was incubated for
3 to 5 min. at 37 C ; then 0.1 ml. of calcium chloride was added and the automatic
timer was activated.
The normal reference plasma was diluted from 1:5 through 1:80, with the 1:10
dilution representing 100% activity. Each
dilution was tested in duplicate. The normal plasma samples were diluted 1:10 and
1:20 and the factor-deficient plasma samples diluted 1:2 and 1:5 were tested in
duplicate. For each dilution, the per cent
activity was interpolated from the reference
curve, and the results were averaged to
obtain the mean per cent activity for each
sample.
Results
Table 1 lists the concentrations of the
various coagulation factors in the normal
and abnormal pools.
584
SIBLEY ET AL.
A.J.C.P.— Vol 59
Factor VIII Assay
APT 1
APT II
APT IV
APT V
APT III
APT VI
300200
370-
20-
20 30 40 50
% Activity
1
200 300
Factor IX Assay
APT I
APT IV
APT II
APTV
APT III
APT VI
Fics. 1 to 4. Comparison of the computed line
o£ best fit of the factor
assays obtained with each
activated partial thromboplastin. The slope is a
measure of the sensitivity
of a reagent to a specific
300
200
^ so'^- ^ i - r c c - - ^ — — — — _ _
i 60" ^ ^ r , s 2 S s ^ ^:;;- — ^ ^ t r r r r ~ ^
i 50-• SsS5aSf~ *~'~
30-
20 30 40 50
% Activity
It should be noted from the data presented in Tables 2 and 3 that the A P T T
results varied greatly with the different activated partial thromboplastin reagents, especially in die abnormal range. The range
between normal and abnormal was very
small with APT I, II, and V.
The within-day and between-days standard deviations of the APTT's were computed for both the normal and abnormal
plasmas. The results are shown in Tables
I
200 300
2 and 3. The between-days F ratio was
computed by dividing the between-days
mean square by the within-day mean
square. I n most instances, this indicated
that the results were not significantly different when within-run and between-run
values were compared. However, the results were statistically significant at the 1%
level for A P T I in the abnormal range
and for APT IV and APT V in both the
normal and abnormal ranges.
April 1973
585
ACTIVATED PARTIAL THROMBOPLASTIN REAGENTS
Factor XI Assay
factor. (Abscissa: X = log
of per cent activity; Ordinate: Y = log of time in
sec.) Note that all of the
reagents were relatively
sensitive to factors XI and
XII and less sensitive to
factors VIII and IX.
APT 1
APT IV
APT II
APT V
APT III
APT VI
30 10 50
% Activity
.
200 300
Factor XII Assay
APT 1
..
..
APT IV
APT II
APTV
APT III
APT VI
300
200
100
20 30 40 50
% Activity
A comparison of the computed line of
best fit (in which the abscissa is X = log
of per cent activity and the ordinate is Y =
log of time in sec.) of the factor assays obtained with each APT is shown in Figures
1 to 4. Inasmuch as sensitivity increases
proportionately with the slope, the slope
is a measure of the sensitivity of a reagent
to a specific factor. Figures 1 to 4 indicate
that all of the reagents were relatively sensitive to factors XI and XII and less sensi-
200 300
tive to factors VIII and IX. APT's I and
V appeared to be practically insensitive to
factor IX. They also had the least ability
to differentiate the normal and abnormal
sample in the A P T T . This may have been
due to the low factor IX activity of the
artificially depleted pool.
Discussion
At the present time, there appears to be
no activated partial thromboplastin re-
586
SIIU.EY irr A i..
agent that gives reproducible results and
is equally sensitive to all of the factors of
the intrinsic clotting system.
From the data obtained in this study,
APT reagents II, III, and VI seem to have
the best reproducibility of the reagents
tested. However, the observed day-to-day
variability is probably not clinically important for any of the reagents except APT
IV. The lack of reproducibility of APT IV
may be due to the use of kaolin as the
activator. It has been reported that celite
gives a better endpoint in the Fibrometer
apparatus than the granular clot formed
with kaolin. 3 The capacity of APT reagents I, II, and V to detect a combined
II, IX, and X deficiency would appear to
be limited since the range between normal
and abnormal pools is so small.
All of the activated partial thromboplastin reagents differed in their sensitivity to
factors VIII, IX, XI, and XII. According
A.J.C.P.—Vol. 59
to our data, APT IV was the most sensitive
and APT V was the least sensitive to factors VIII and IX. APT V was so insensitive
to factor IX that results would be clinically
misleading, except in severe deficiency
states. APT II was the most sensitive to
factors XI and XII, and APT's I and 111
were the least sensitive to factors XI and
XII, respectively. All reagents performed
satisfactorily in assays for factors XI and
XII.
References
1. Abildgaard CF, Cornet JA, Johnson H, et al:
Screening tests for disorders of thromboplastin
formation. Pcdiatr Clin N Am 9:819-832, 1962
2. Goldenfarb PB, Cathey MH, Zucker S, et al:
Changes in the hemostatic mechanism after
myocardial infarction. Circulation 43:538-546,
1971
3. Simone JV, Vanderheiden J, Abildgaard CF: A
semiautomatic one stage factor VIII assay with
a commercially prepared standard. J Lab Clin
Med 69:706-712, 1967
4. Zucker S, Cathey MH, West B: Preparation of
quality control specimens for coagulation. Am
J Clin Pathol 53:924-927, 1970