Salma O.M.Abdalla. et al. / Journal of Science / Vol 5 / Issue 1 / 2015 / 44-47.

e ISSN 2277 - 3290

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Journal of Science Medicine

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COMPARISON BETWEEN THE PERFORMANCE OF LOCALLY

PREPARED THROMBOPLASTIN AND A COMMERCIAL BRAND
Salma O.M. Abdalla1, Shamseldein M. Ahamed1, Ahmed A. Mohamedani2,
GadAllah Modawe3*
1
Faculty of Medical Laboratory Science, Department of Clinical Chemistry, University of Gezira, Wad Medani, Sudan,
2
Department of Pathology, Faculty of medicine, University of Gezira, Wad Medani, Sudan.
3
Faculty of Medicine, Department of Biochemistry, Omdurman Islamic University, Omdurman,Sudan.

ABSTRACT
Prothrombin Time (PT) is the test which measures the extrinsic pathway of coagulation in the presence of an
optimal concentration of tissue extract from thromboplastin. The quality of the later is the whole mark the sensitivity of the
test and the international normalize ratio (INR) is being created to make inter laboratory comparison between PTs. To prepare
different brain extracts from rabbits, sheep and test their performance compared to commercial brand.Thromboplastins were
prepared using standard manual method from brains of rabbits and sheep for preparation: single rabbit brain (SRB), two
mixed rabbit brains (TMRB), freeze dried single rabbit brain (FDSRB) and single sheep brain (SSB). The study involved 100
citrated venous blood specimen. Plasma was separated immediately and was tested. The data was analyzed statistically by
using SPSS program version- II to compare between the means of locally prepared reagents and the commercial brand. The
result of PT showed significant differences when compared with the commercial brand (P. Value 000). The international
sensitivity (ISI) compared to the (1.52) with ISI of the commercial brand. The best one of the locally prepared thromboplastin
was (FSRB) with an ISI 1.76. The sensitivity tests for (SRB), (TMRB), (FSRB) and (SSB) were respective 75%, 70%, 80%,
31%. The raw materials for local thromboplastins are available, cheap and easy to be extracted and prepared, but should be
corrected by ISI, the recommended reference preparation is the freeze dry one.

Keywords: Thromboplastin, Prothrombin Time, rabbit brains, Sudan.

INTRODUCTION
The central event in the coagulation pathway is
the production of thrombin, which acts upon fibrinogen to
produce fibrin clot. This clot is further strengthened by
the cross linking action of factor XIII which itself is
activated by thrombin. The two commonly used reagents
in coagulation tests; Activated Partial Thromboplastin
Time (APTT) and Prothrombin Time (PT), have been
used historically to define the two pathways of
coagulation ; the intrinsic and extrinsic pathways,
respectively [1-2].
Source of the prepared thromboplastin mainly as
brain, placenta, liver and tissue. Because different
thromboplastin preparation have different sensitivity ,the
WHO establish the international normalize ratio (INR)
[3].
The international committee on thrombosis and
haemestasis for reporting the results of blood coagulation
test. All results are standardized using the international
sensitivity index for particular thromboplastin reagent and
utilized to perform the test [4]
These are used for thromboplastin test to monitor
anticoagulant such as Worfarin.
INR is useful in monitoring the degree of oral
anticoagulants of some agents such as Warfarin. In
healthy people, the INR is about 1.0, for a patient on
anticoagulant the INR typically should be 2-4, and for
patients with mechanical heart values [4-5].
The INR is calculated from the PT and is

Corresponding Author:-Gad Allah Modawe Email:-gadobio77@hotmail.com

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Salma O.M.Abdalla. et al. / Journal of Science / Vol 5 / Issue 1 / 2015 / 44-47.
intended to allow valid comparison of results regardless
of the type of PT reagent used among different
laboratories.
ISI
INR = ( patient PT
Mean normal PT [6]
The international sensitivity index (ISI) is a
measure of the sensitivity of a particular PT reagent.
Different PT reagents have different sensitivities to factor
deficiencies. The ISI for each reagent is determined by the
manufacturer.(7) the objective of this study was To
prepare different brain extracts from rabbits ,sheep and
test their performance compared to commercial brand .
MATERIAL AND METHODS
This is a descriptive, analytical experimental
study, conducted in the University of Gezira medical
laboratory, Wad Medani City, Gezira State; Central of the
Sudan .
Blood samples were collected from 100 subjects
who were referred to the haematology department for PT
investigation. 4.5 ml of venous blood was taken in 0.5 ml
of tri-sodium citrate anticoagulant. The plasma was
separated immediately into plain containers.
Thromboplastin preparation
From single rabbit brain, two mixed rabbit
brains and single sheep brain.
The preparation involved three step.
Acetone Dried Brain Powder
Steps followed
The membranes of freshly collected brain
were stripped and washed free from blood.
The brain placed in about three times its
volume cold acetone.
The brain was macerated twice for 3 minutes
and then filtered through an absorbent on a
funnel.
The acetone extraction was repeated five
times after the two extractions by increasing the time
of exposure to acetone to 20 min for each subsequent
extraction.
The acetone-dried brain was spread on a piece
of paper and allowed to dry [1,7].
Preparation of liquid suspensions
Steps followed
0.9g of NaCl was mixed with 0.9g of phenol,
and then dissolved in 100 ml of distilled
water.
3.6g of the acetone-dried brain was added to
the mixture (phenol-saline solution).
The mixture solution was allowed to stand for
4-5hours at room temperature. The solution was mixed
every 30 minutes.
The solution was then kept in refrigerator at
4C for 24 hours before use.
3- freeze drying
After preparation of acetone dried brain
powder, the powder was kept inrefrigerator at 4 C
for 24 hours, then it was transferred to a freeze drying
machine at - 60 C for24 hours (dry ice), this machine
produces a completely dry product keeping the same
original volume and natural characteristic. {Freeze
drying was done only for SRB}.
Calibration of thromboptastins
The results of Prothrombin Time were plotted
on log-log graph paper, the results of the reference
preparation on the vertical axis (Y) and the locally
prepared reagents on the horizontal axis (X). The
logarithms of the PT were plotted on arithmetic graph
paper, the relationship between the two
thromboplastins was determined by the slope of the
line [8].
Calculation of International Sensitivity Index (ISI)
The PT of the local thromboplastin was
plotted on the horizontal axis (x) and with the
reference thromboplastin on the vertical axis (y) on
double log graph paper, the best-fit line was drawn by
naked eye and the slope was obtained as follows:
Points (a) and (b) are marked on the line just
below the shortest recorded PT and above the longest
recorded PT.
(c) is a point where a horizontal line through (a)
and vertical line through (b) meet.
The calculation
The lSI of the test: thromboplastin =Slope (b) x lSI of
reference thromboplastin [9]
Calculation of INR
Then PT was estimated manually at 37 C
water bath, time expressed in seconds, and then INR
was calculated [10-11].
INR = PT ratio obtained by the test thromboplastin to
the power of the lSI of the test reagent.
RESULTS AND DISCUSSION
The results of the Prothrombin Time for locally
prepared reagents showed different values, when
compared with the commercial reagent with significant
variation (P. value 000) - table (1) but when comparing
by lSI: the lSI of freeze dried single rabbit brain was
found to be (1.76) - table 2 - which is close to that of the
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Salma O.M.Abdalla. et al. / Journal of Science / Vol 5 / Issue 1 / 2015 / 44-47.
lSI of the commercial reagent (1.52), and this may be due
to the complete dryness by freezing process with high
freezing temperature -60C.
When we compared freeze dried single rabbit
brain with single rabbit brain without freezing, and to
emphasize this we referred to the sensitivity ratio, for
freeze dried single rabbit brain which was 80%, while
single rabbit brain was 70 %. Table (2) .
The advantages of the freeze dried preparation is
that it keeps the powder preparation with its same natural
characteristic with complete dryness, and it can be stored
in refrigerator for a long time as a powder.
The sensitivity of all locally prepared extractions
was less than 100 %,this may be due to fact that, the
purification of these extractions was done manually by
hand, in a non strictly sterilized condition, compared with
commercial reagent which is prepared in sterile
conditions and by a special machine.
The freeze-dried powder can be stored in
refrigerator for a long time because it was prepared under
special sterilization condition. This ensured that the
commercial reagents can be stored for a long time in
refrigerator but the locally prepared cannot be stored for a
long time.
Similar results were obtained by others previous
researchers:
Study done by Poller L (2012) concluded found
a discrepant sensitivity to factor V, VII, and X levels of
two thromboplastin reagents (Commercial reagent and
local reagent of rabbit brain). It showed a significant
variability in INR value even under optimal conditions of
INR calibration.
Our results were corrected by different lSI levels.
We found that the freeze-dried single rabbit brain results
were close to the commercial reagent results, and these
results agree with Poller L study 2012 [8].
Van den Besselaar AMHP et al., (2012);
reported on Prothrombin Time and its standardization
using International Normalized Ratio (INR) based on
International Sensitivity Index (lSI) that is calibrated by
International Reference Preparation (IRP) for
thromboplastin.Discrepancy of sensitivities to plasma
absorbed multiple coagulation factors and plasma from
patients, was seen with rabbit brain thromboplastin, but
not with human origins. Discrepancy of sensitivities
observed in rabbit thromboplastin was emphasized when
converted to INR value.
Table 1. Comparison between Prothrombin Times of locally prepared reagents and a commercial brand
(N= 100 for each)
Locally prepared n = 100
Variable
Mean S.D (Seconds)
Single rabbit brain 16.326.02
Two mixed rabbit brains 31.821.38
Freeze dried single rabbit brain 26.114.21
Single sheep brain 68.427.8
n = Number; S.D = Standard deviation; Mean /second unit time.
Adcock, DM and Johnston M (2012) evaluated
the International Normalized Ratio of Prothrombin Time
following multiple depletion of coagulation factors, using
rabbit brain and human placenta. Correlation of INR
calculated by two different sensitivities ,one is to
absorbed plasma ,indicate that discrepancy of sensitivities
was emphasized as large slopes of regression line and
intercepts with rabbit thromboplastines.
Chantarangkul V et al., (2006) reported about
international collaborative study for the calibration of a
proposed international standard for thromboplastin, rabbit
brain. The assigned International Sensitivity Index value
was (1.15). This is reasonable and it could have been better
if facilities for preparation of the reagents were optimum.
Van den Besselaar AMHP, (2010) said that, most
commercially available PT reagents contain crude tissue
factor extracted from natural sources, e.g., rabbit brain,
rabbit brain/lung mixtures, human placenta or bovine
brain. The crude extracts, because they are natural
products, often lack lot to lot uniformity. For example,
rabbit brain thromboplastins show seasonal variability.
Another problem with natural extracts is the presence of
contaminants. For example, human tissue factor may be a
source of HIV or other human viral diseases and many
natural- sourced thromboplastins also contain other
extraneous coagulation factors which can detrimentally
affect the PT value [11].
There for good preparation operated procedure
must be followed.
BudnitzDS, Said that, if verification fails, then
the laboratory must not report patient results until the
correct ISI is determined. The laboratory must establish
that: instrument(s) was (were) working properly; reagents
were reconstituted correct; mean normalize
prthrombintime(MNPT)was determined accurately
(geometric mean); and no clerical or mathematical errors
were made. If these are correct then proceed to local
calibration of the ISI [12].
Poller L,(2012) reported that, all currently
available PT tests utilize a PT reagent containing crude
tissue factor extracted from natural sources. It is
important a PT reagent to have the following
characteristics: sensitive to abnormal samples, a well
defined normal PT value for normal plasma, give accurate
and reproducible results, have lot-to-lot consistency, must
be stable for storage in the freeze-dried state and must be
stable after reconstitution.(13).
Commercial brand
P. Value
n = 100 Mean S.D (Seconds)
17.85.81 0.00
19.58.23 0.00
18.87.69 0.00
24.513.57 0.00
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Salma O.M.Abdalla. et al. / Journal of Science / Vol 5 / Issue 1 / 2015 / 44-47.

Table 2. lSI for locally prepared reagents corrected and compared with commercialreagent. as well as sensitivity in
brackets.
Variable Single rabbit Two mixed Freeze dried Single sheep
brain Rabbit brains single rabbit brain
brain
Locally 1.84 (75%) 1.94 (70%) 1.76 (80%) 2.78 (31%)
prepared .
reagents
(lSI)
Commercial 1.52 1.52 1.52 1.52
Brand
(lSI)

CONCLUSION accuracy is needed to further reduce clinical problems

The INR is an important clinical tool for associated with anticoagulation. To this end, the
monitoring Warfarin derivatives therapy. The introduction development of local reagent accuracy through the use of
of the INR has added several orders of magnitude of local ISI verification and ISI calibration adds even greater
accuracy to anticoagulant monitoring. Still, more confidence to the correct reporting of INR values.

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