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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.
INTRODUCTION
The central event in the coagulation pathway is [3].
the production of thrombin, which acts upon fibrinogen to The international committee on thrombosis and
produce fibrin clot. This clot is further strengthened by haemestasis for reporting the results of blood coagulation
the cross linking action of factor XIII which itself is test. All results are standardized using the international
activated by thrombin. The two commonly used reagents sensitivity index for particular thromboplastin reagent and
in coagulation tests; Activated Partial Thromboplastin utilized to perform the test [4]
Time (APTT) and Prothrombin Time (PT), have been These are used for thromboplastin test to monitor
used historically to define the two pathways of anticoagulant such as Worfarin.
coagulation ; the intrinsic and extrinsic pathways, INR is useful in monitoring the degree of oral
respectively [1-2]. anticoagulants of some agents such as Warfarin. In
Source of the prepared thromboplastin mainly as healthy people, the INR is about 1.0, for a patient on
brain, placenta, liver and tissue. Because different anticoagulant the INR typically should be 2-4, and for
thromboplastin preparation have different sensitivity ,the patients with mechanical heart values [4-5].
WHO establish the international normalize ratio (INR) The INR is calculated from the PT and is
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Salma O.M.Abdalla. et al. / Journal of Science / Vol 5 / Issue 1 / 2015 / 44-47.
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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 Adcock, DM and Johnston M (2012) evaluated
to the complete dryness by freezing process with high the International Normalized Ratio of Prothrombin Time
freezing temperature -60C. following multiple depletion of coagulation factors, using
When we compared freeze dried single rabbit rabbit brain and human placenta. Correlation of INR
brain with single rabbit brain without freezing, and to calculated by two different sensitivities ,one is to
emphasize this we referred to the sensitivity ratio, for absorbed plasma ,indicate that discrepancy of sensitivities
freeze dried single rabbit brain which was 80%, while was emphasized as large slopes of regression line and
single rabbit brain was 70 %. Table (2) . intercepts with rabbit thromboplastines.
The advantages of the freeze dried preparation is Chantarangkul V et al., (2006) reported about
that it keeps the powder preparation with its same natural international collaborative study for the calibration of a
characteristic with complete dryness, and it can be stored proposed international standard for thromboplastin, rabbit
in refrigerator for a long time as a powder. brain. The assigned International Sensitivity Index value
The sensitivity of all locally prepared extractions was (1.15). This is reasonable and it could have been better
was less than 100 %,this may be due to fact that, the if facilities for preparation of the reagents were optimum.
purification of these extractions was done manually by Van den Besselaar AMHP, (2010) said that, most
hand, in a non strictly sterilized condition, compared with commercially available PT reagents contain crude tissue
commercial reagent which is prepared in sterile factor extracted from natural sources, e.g., rabbit brain,
conditions and by a special machine. rabbit brain/lung mixtures, human placenta or bovine
The freeze-dried powder can be stored in brain. The crude extracts, because they are natural
refrigerator for a long time because it was prepared under products, often lack lot to lot uniformity. For example,
special sterilization condition. This ensured that the rabbit brain thromboplastins show seasonal variability.
commercial reagents can be stored for a long time in Another problem with natural extracts is the presence of
refrigerator but the locally prepared cannot be stored for a contaminants. For example, human tissue factor may be a
long time. source of HIV or other human viral diseases and many
Similar results were obtained by others previous natural- sourced thromboplastins also contain other
researchers: extraneous coagulation factors which can detrimentally
Study done by Poller L (2012) concluded found affect the PT value [11].
a discrepant sensitivity to factor V, VII, and X levels of There for good preparation operated procedure
two thromboplastin reagents (Commercial reagent and must be followed.
local reagent of rabbit brain). It showed a significant BudnitzDS, Said that, if verification fails, then
variability in INR value even under optimal conditions of the laboratory must not report patient results until the
INR calibration. correct ISI is determined. The laboratory must establish
Our results were corrected by different lSI levels. that: instrument(s) was (were) working properly; reagents
We found that the freeze-dried single rabbit brain results were reconstituted correct; mean normalize
were close to the commercial reagent results, and these prthrombintime(MNPT)was determined accurately
results agree with Poller L study 2012 [8]. (geometric mean); and no clerical or mathematical errors
Van den Besselaar AMHP et al., (2012); were made. If these are correct then proceed to local
reported on Prothrombin Time and its standardization calibration of the ISI [12].
using International Normalized Ratio (INR) based on Poller L,(2012) reported that, all currently
International Sensitivity Index (lSI) that is calibrated by available PT tests utilize a PT reagent containing crude
International Reference Preparation (IRP) for tissue factor extracted from natural sources. It is
thromboplastin.Discrepancy of sensitivities to plasma important a PT reagent to have the following
absorbed multiple coagulation factors and plasma from characteristics: sensitive to abnormal samples, a well
patients, was seen with rabbit brain thromboplastin, but defined normal PT value for normal plasma, give accurate
not with human origins. Discrepancy of sensitivities and reproducible results, have lot-to-lot consistency, must
observed in rabbit thromboplastin was emphasized when be stable for storage in the freeze-dried state and must be
converted to INR value. stable after reconstitution.(13).
Table 1. Comparison between Prothrombin Times of locally prepared reagents and a commercial brand
(N= 100 for each)
Locally prepared n = 100 Commercial brand
Variable P. Value
Mean S.D (Seconds) n = 100 Mean S.D (Seconds)
Single rabbit brain 16.326.02 17.85.81 0.00
Two mixed rabbit brains 31.821.38 19.58.23 0.00
Freeze dried single rabbit brain 26.114.21 18.87.69 0.00
Single sheep brain 68.427.8 24.513.57 0.00
n = Number; S.D = Standard deviation; Mean /second unit time.
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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)
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