Prothrombin Time SOP

INR SOP.
Doc: 303 Rev 0
1 PURPOSE
1.1 To describe the manual method for determining the PT-INR on citrated
plasma (blue tube).
2 SCOPE
2.1 All staff who perform the manual INR method.
3 PROCEDURE
The Prothrombin time measures the clotting time of plasma in the presence of a
concentration of tissue extract, Ca++, Mg++ and clotting factors that indicates the
overall efficiency of the extrinsic pathway of the coagulation system.The
Prothrombin time measures changes in factors II, VII and X, three of the principal
clotting factors depressed by Coumarin drugs. The PT also reflects changes in factor
V which is not reduced by oral anticoagulants. The test depends on the activation of
factor X in the presence of factor VII by tissue factor and the bypassing of the intrinsic
clotting pathway.
A prolonged result can be due to congenital (inherited) or acquired factor deficiency

(Factors I,II,V,VII & X), DIC, the presence of inhibitors, liver disease, Vitamin K
deficiency, anticoagulant therapy such as Warfarin (Coumarin) and to a lesser degree
heparin, Ratex ingestion, an old specimen, a specimen with the incorrect citrate
anticoagulant/blood ratio (Low volume) or a clotted specimen.
3.1 Preparation of Specimens, Reagents and Controls
3.1.1 Specimen Requirements
a) Blood must be collected in a 3,2% (105 mm/L or 0.105M)

trisodium citrate (blue) tube which is commercially available.
The proportion of blood to anticoagulant must be 9:1.
b) Care must be taken when collecting blood for coagulation tests
that there is no undue venous stasis or frothing which may
cause platelet activation.
c) Run test as soon as possible after collection but not later than
24hrs after collection. Storage at 4°C is not recommended due
to the activation of factor VII that affects some samples. In the
presence of high ambient temperatures a maximum of 4 hours
storage at room temperature is recommended and storage at
4°C in these circumstances would be more advantageous and
outweigh risks of cold activation.
Do not perform the PI/INR if:

 The blood is clotted
 The blood is haemolysed
 The blood sample tube volume is insufficient (not filled to the
recommended level).There is excess citrate in that the ratio
of anticoagulant to blood is incorrect.
 The blood is older than 24 hours. In exceptional cases, like
specimens delivered by overnight transport, the tests can be
done and resulted with an appropriate comment.
Page 1 of 3
INR SOP. Doc: 303 Rev 0
3.1.2 Reagents Required
a) A Thromboplastin Reagent that is insensitive to therapeutic

levels of heparin.
b) Check package insert for directions for reconstitution
procedures, storage of reagent and stability after reconstitution.
3.1.3 Controls
a) COMMERCIAL CONTROLS – are available that will cover the

clinical significant range. Prepare reagents as per the
manufacturer’s instructions. The value of this control should
range between 10 and 14 seconds.
b) MNPT (mean Prothrombin Time) – this value is the mean
value of the pooled plasma done on each new lot number of PT
reagent. MNPT values are used to calculate the INR.
3.2 Equipment Required
3.2.1 Glass test tubes of a standard size (10 to 12 mm in diameter x 75mm

in length).
3.2.2 100 l automated pipette with appropriate tips.200 l automated
pipette with appropriate tips.
3.2.3 2 or 3 Stopwatches.
3.2.4 A 37°C waterbath with a tolerance of no more than ± 0.5°C
3.3 Test Procedure
3.3.1 Check waterbath temperature (37ºC ± 0,5ºC is acceptable) and

document on the temperature chart (once a day) and on the worksheet
for every batch of tests performed.
3.3.2 Write the patient name and lab number on the worksheet.
3.3.3 All tests and controls must be performed in duplicate.
3.3.4 To two test tubes add 100µl control.
3.3.5 Incubate the tubes at 37 C in water bath for one (1) to two (2) minutes
(maximum five (5) minutes).
3.3.6 To these tubes, swiftly add 200µl of pre warmed (37°C) Innovin and
immediately start the stopwatch.
3.3.7 Thereafter gently rock the tube by tilting at regular intervals (tilts
through 90° every ± 2 to 3 seconds is recommended), submerging the
bottom of the tube in the water bath on every down stroke. Check for
clot formation on the up stroke. Stop the stopwatch when the clot is
detected.
3.3.9 Record all times on the work sheet and take the average reading of the
duplicate results. The mean of the patient’s results and the MNPT
value for the specific reagent, will be used in the INR calculation. Initial
the coagulation worksheet. Repeat the test if there is a difference of
more than 10% between the dublicate tests.
3.3.10 Report PT up to 120seconds (2 minutes), thereafter it is reported as:
Failed to clot within 120 seconds.
Page 2 of 3
INR SOP. Doc: 303 Rev 0
3.4 Calculations
When calculating the INR or Prothrombin Index (PI) it is important to

remember to use the MNPT value.
The INR (International Normalised Ratio) is calculated as follows:
First calculate the Prothrombin Ratio as follows:

Patient Prothrombin Time = Prothrombin Ratio (PR)
MNPT
INR = PR ISI
The ISI value: is supplied by the manufacturer of the thromboplastin and is

specific for each batch of thromboplastin.
3.5 ISI is the international sensitivity index of the reagent/instrument combination.
INR Normal range: 0,9 to 1,.3

Therapeutic (Warfarin Therapy) range: 2,0 to 4.0
Calculating the Protrombin Index (PI)
PI (%) = MNPT x 100
Patient Time 1
Normal Range: 80% to 100%

Therapeutic Range: 25% To 50%
All results must be telephoned to the ward/Dr who must immediately be

notified if the specimen is clotted, insufficient or haemolysed.
3.6 Precautions
With a prolonged PT or when failed to clot within 120 seconds, always check
for:
3.6.1 A clotted specimen. Remix specimen and check for small clots using
an orange stick.
3.6.2 That water-bath temperature is correct. (Temperature affects the
speed of clotting).
3.6.3 That tubes and pipette tips are clean.
3.6.4 That expired reagents were not used.
3.6.5 There is not excess citrate in that the ratio of anticoagulant to blood is
incorrect (check that Venoject tube is filled to recommended level).
3.6.6 An unduly high PCV, so that there is less plasma than normal (under
citrated).
4 REFERENCES
4.1 Practical Haematology, Sir John V. Dacie, S.M. Lewis. Eighth edition, 1995,
p307.
Page 3 of 3

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INR SOP.

Doc: 303 Rev 0

2.1 All staff who perform the manual INR method.

A prolonged result can be due to congenital (inherited) or acquired factor deficiency

3.1 Preparation of Specimens, Reagents and Controls

3.1.1 Specimen Requirements

a) Blood must be collected in a 3,2% (105 mm/L or 0.105M)

Do not perform the PI/INR if:

3.1.2 Reagents Required

a) A Thromboplastin Reagent that is insensitive to therapeutic

a) COMMERCIAL CONTROLS – are available that will cover the

3.2 Equipment Required

3.2.1 Glass test tubes of a standard size (10 to 12 mm in diameter x 75mm

3.3 Test Procedure

3.3.1 Check waterbath temperature (37ºC ± 0,5ºC is acceptable) and

When calculating the INR or Prothrombin Index (PI) it is important to

First calculate the Prothrombin Ratio as follows:

The ISI value: is supplied by the manufacturer of the thromboplastin and is

3.5 ISI is the international sensitivity index of the reagent/instrument combination.

INR Normal range: 0,9 to 1,.3

Normal Range: 80% to 100%

All results must be telephoned to the ward/Dr who must immediately be

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