فحص الـ Prothrombin Time

Laboratory Evaluation of Coagulation

Coagulation screening tests screen for the integrity of the intrinsic and extrinsic coagulation pathway’s. These tests depends on testing the time required for platelet poor plasma (PPP) to clot after the addition of laboratory reagents, our end point is the formation of soluble fibrin clot (i.e. after the action of thrombin on fibrinogen, forming fibrin monomers which then polymerize, primary polymerization, without the action of factor XIIIa). These tests are called prothrombin time (PT) which test extrinsic pathway integrity, and activated partial thromboplastin time (APTT) which test intrinsic pathway integrity. Prolonged times for these tests are associated with factor deficiency, presence of inhibitors, and liver diseases. To control and monitor anticoagulation therapy PT and APTT are usually performed. Also, preoperatively these tests are routinely performed, to see if the patient has tendency to bleed during the surgical operation or not, when these tests show prolonged times; usually operations are postponed, until these prolonged tests are corrected.

Prothrombin Time (PT, Protime[/CENTER)]

Prothrombin time (PT) test the efficiency of extrinsic coagulation
pathway, i.e. factors: VII, X, V, II, and fibrinogen.
The procedure is simple, a commercial tissue extract (thromboplastin, which consists of phospholipids and tissue factor) is added to the patient PPP (platelet poor plasma), then the mixture is re-calcified (the term recalcified is used since when we collect the blood specimen in 3.2% sodium citrate we remove endogenous calcium from the sample, when we are adding calcium we are actually recalcifying the sample, Ok), and a stop watch is started, the time required for fibrin polymerization formation is measured.
The commercial tissue thromboplastin is usually extracted and prepared from animal tissue (e.g. rabbit brain or kidney), but these extracts and preparations have different and low sensitivities to coagulation factor deficiency, also they show lot-to-lot variability. Seasonal variability is also a recognized variability in these extracts. Their sensitivities to the presence of heparin also vary. They are usually sensitive to only 30-40% factor deficiency, if the factor activity is more than 30-40% activity, then PT time will be normal, and deficiency will not be detected, only detected by specific factor assays. Human origin thromboplastins are not used because of the potential infection by hepatitis and HIV viruses. But there are now available in the market thromboplastins which are called lipidated human recombinant thromboplastins which are safe and more sensitive to factor deficiency, and have reduced sensitivity to heparin (this reduced sensitivity to heparin is required especially when both heparin and warfarin are given together to hypercoaguable (thrombophilic) patients, and also recombinant thromboplastin has reduced turn around times(TAT). Recombinant thromboplastin usually does not show lot-to-lot variability, with no seasonal variability. These reagents are much better, safe, and are more recommended than animal thromboplastin extracts.Prothrombin time is usually requested for the following reasons:-
1] Suspected cases of factor deficiency.
2] Monitoring anticoagulation therapy.
3] Preoperative procedure.
4] Circulating anticoagulants.
5] Liver disease.

PT Reporting
:

Historically, PT is reported as a ratio, in which you divide the
patient PT time on the normal plasma PT time (Patient PT over Normal plasma PT), as you run patient sample for PT you should run simultaneously a normal control sample. Normal control is usually purchased commercially, together with abnormal plasma that could be high or low control plasmas; alternatively normal control could be prepared in home (i.e. in your laboratory) by pooling 20 normal plasmas and then testing the pooled plasma for PT time, then you divide the pooled plasma into aliquots, which are then freezed at temp. below 18C, for subsequent daily use.

Patient PT time

[center]PT Ratio

=
Normal Control PT time

Now, this PT ratio is not recommended, why?: because different laboratories and countries use different thromboplastins from different manufacturers and different lot #, with different sensitivities and characteristics, and we have stated earlier, that human origin thromboplastin is not used because of their potential viral infections. WHO, supply manufacturers with reference human thromboplastin to calibrate their thromboplastin extracts, so these commercial animal origin thromboplastins are calibrated against this WHO human thromboplastin, this calibration will yield a calibration factor or correction factor or index, this factor is called ISI (International Sensitivity Index), each manufacturer will supply us with this calibration index factor. So, our reporting system has been modified to:-

ISI

Patient PT time

INR=

Control PT time

It is actually a modified PT ratio, the difference that we also raise it to the power of the ISI value. This ratio is called INR (International Normalized Ratio); I.e. INR is different from the conventional PT ratio, by only raising it to the power of ISI.
As ISI is near 1.0 it will better (WHO human thromboplastin has an ISI of 1.0, so when your commercial thromboplastin has an ISI of 1.0, so as if you are using the WHO reference thromboplastin), it is recommended now that the ISI should not be more than 1.5, and better be between 1-1.2, and this largely should be taken in consideration when purchasing commercial thromboplastins. The lipidated recombinant human thromboplastin has a reduced ISI (about 1-1.2), and being from human origin, and noninfectious, contributes to consider it the best available thromboplastin in the market.
Some physicians accept more what is called PT % activity:-

Normal Control PT time
% activity = x 100
Patient PT time

The best PT result expression for patients on oral anticoagulation is

by the use of INR. It is wrong and totally not recommended to use %activity in expressing PT results, and unfortunately it is still widely used by lazy physicians nowadays.

Normal Range:
PT time is measured in seconds, normal range depends largely on the type of commercial thromboplastin used by your lab.
Procedure:
1- Pipette 0.1 ml of patient PPP into a test tube.
2- Add to the tube 0.1 ml of the prewarmed (37ºC) commercial thromboplastin, incubate for 3 minutes (or as stated by the manufacturer).
3- Add 0.1 ml of CaCl2 (0.025 M), and start a stop watch.
4- Tilt the tube first after 5 seconds, then every 1-2 seconds, until fibrin clot is formed.
5- Record the time needed for fibrin clot formation in seconds.
Notes:
1- The PT procedure is performed at 37ºC in a water bath.
2- Usually PT for the patient is done in duplicates or even triplicates and the average is calculated, which will then be used for your calculations and computations.
3- Compute the INR, and report it together with the patient PT time and the control PT time.
Example of Reporting PT Results:
- ISI for the reagent thromboplastin = 1.3
- Patient PT time = 14 seconds
- Normal Control PT time = 12 seconds

1.3
14 1.3
INR = = 1.16 =
12

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