PROTHROMBIN TIMEWHY USE LOW ISI (HIGH SENSITIVITY)

PROTHROMBIN TIME ‐DEFINITIONDEFINITION

The Prothrombin time  is the functional determination of the extrinsic

l i   hcoagulation pathway.

It is a widely used laboratory assay for the detection of inherited or acquired the detection of inherited or acquired coagulation defects related tothe extrinsic pathway.

TERMSTERMS

• INR – INTERNATIONAL NORMALIZED RATIO

• WHO – WORLD HEALTH ORGANISATION

• ISI – INTERNATIONAL SENSITIVITY INDEX

IRP  INTERNATIONAL REFERENCE PREPARATION• IRP – INTERNATIONAL REFERENCE PREPARATION

INR – INTERNATIONAL NORMALIZED RATIONORMALIZED RATIO

Ever wondered why you obtain different results forEver wondered why you obtain different results for

•• different company PT reagents ?different company PT reagents ?•• different lots of the same companies PT reagents ?different lots of the same companies PT reagents ?p gp g

PT REAGENTS ARE MADE FROM TISSUE FACTORSPT REAGENTS ARE MADE FROM TISSUE FACTORS

BEING BIOLOGICAL PRODUCTS IT EXHIBITS A LOT OF VARIABILITYBEING BIOLOGICAL PRODUCTS IT EXHIBITS A LOT OF VARIABILITYBEING BIOLOGICAL PRODUCTS IT EXHIBITS A LOT OF VARIABILITYBEING BIOLOGICAL PRODUCTS IT EXHIBITS A LOT OF VARIABILITY

WHO expert committee report on Biological Standardization, Report 34

ISI – INTERNATIONAL SENSITIVITY INDEX

ISI is a correction factor  developed to correlate ISI is a correction factor  developed to correlate the sensitivity of commercial Thromboplastins to the sensitivity of commercial Thromboplastins to the the 11stst IRP. (International Reference Preparation).IRP. (International Reference Preparation).

IRP – A very responsive batch of human brain extract was designated as the 1st International Reference Preparation (IRP). p ( )

ISI – International Sensitivity Index

The ISI of the 1st IRP was 1.0

Th INR t ll th th bi ti tThe INR actually compares the prothrombin ratio measurement to the 1st IRP.

The INR represents the prothrombin time that would have beenp pobtained if the IRP had been used as the reagent.

Responsive PT reagents have lower ISI values.

Analytical precision is improved with reagents with low ISI.

Reagents with low ISI discriminates normal and warfarintreated patients better.

LOW ISI VS HIGH ISI

LOW ISI HIGH ISI

C ll d   Hi h  i i i   C ll d   L   i i i  Called as High sensitivity assay

Called as Low sensitivity assay

The reagent is highlysensitive to even small 

The reagent is NOT sensitiveto changes in PTsensitive to even small 

changes in the PTto changes in PT

Accommodates Factor deficiency

DOES NOT accommodateFactor deficiencydeficiency Factor deficiency

Higher levels of precision Poor precision

Better global commutability  Poor Commutabilitywith PT assays 

Shift towards low ISI assays globally

Shift away from High ISI kits globally

SAMPLES AND THEIR ROLE

MOST OF THE PROBLEMSMOST OF THE PROBLEMS CAUSED IN PT TESTINGIS BECAUSE OF SAMPLING.

SAMPLES AND THEIR ROLESAMPLES AND THEIR ROLE

ANTICOAGULANTWh l bl d h ld b ll t d ith 3 2% S di Cit tWhole blood should be collected with 3.2% Sodium CitrateAs the anticoagulant.

DO NOT USE 3 8% SODIUM CITRATEDO NOT USE 3.8% SODIUM CITRATE.

Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing, Adcock et al., Am J Clin Path 1997 Jan; 107 (1):105-10

SAMPLES AND THEIR ROLESAMPLES AND THEIR ROLEPREPARATION OF SAMPLES

Collect whole blood 9 volumes with 1 volume of 3.2% Sodium Citrate

Collect samples in a plastic tube

DO NOT USE GLASS VESSELS

Mix whole blood and Sodium Citrate wellMix whole blood and Sodium Citrate well

Centrifuge at 2500 rpm for 15 minutes.

The tube now has a clear area of Citrated plasma and a sediment plug.

PERFORM TESTS WITHIN 1-2 HOURS AFTER SPINNING

Clinically relevant differences in PT and INR values related to Blood sample Collection in Plastic VS Glass TubesEberhard W. Fiebig et al; Am J Clin Path; 2005; 124(6) 902-909

SAMPLES AND THEIR ROLESAMPLES AND THEIR ROLE

STORAGE OF SAMPLES

Remove citrated plasma from the centrifuged sediment

Place the same in a plastic container

Refrigerate

DO NOT FREEZE. (freezing prolongs clotting time).

Stored samples must be mixed well before useStored samples must be mixed well before use.

Effect of freezing method and storage at -20oC and -70oC on PT, aPTT and Plasma Fibrinogen levelsSonja Alesci et al; Thrombosis Research; Vol 124, Issue 1, May 2009.

INR – INTERNATIONAL NORMALIZED RATIONORMALIZED RATIO

WHO i t d d INR i  th   l  8 ’         f WHO introduced INR in the early 80’s as a means of Standardizing PT results.

ISI ISIISIISIINR = INR =  Patient PTPatient PT

Mean Normal PTMean Normal PT= (Prothrombin Ratio)= (Prothrombin Ratio)

ISIISI

WHO and International Committee on Thromobosis and Haemostasis – Expert committee reports

MEAN NORMAL PTO

FACTSFACTSEach lab must establish Mean Normal PTEach lab must establish Mean Normal PT for the reagent and the instrument used

Do not use Mean Normal PT derived from one company’ reagent with the other.

Procedures for Validation of INR and Local Calibration of PT/INR systems; Approved GuidelinesCLSI;Vol 25 No.23.

MEAN NORMAL PTO

FACTSFACTS

Each lab must establish Mean Normal PT for the reagent and the instrument used

Do not use Mean Normal PT derived from one company’ reagent with the other.

Procedures for Validation of INR and Local Calibration of PT/INR systems; Approved GuidelinesCLSI;Vol 25 No.23.

MEAN NORMAL PTMEASUREMENT

Collect 10 - 20 fresh samples (apparently normal)Collect 10 - 20 fresh samples (apparently normal)

Measure clotting times for PT using BIOREXFARS PT reagent

Assuming that the values obtained are:Assuming that the values obtained are:14 seconds, 12.1, 11.9, 16.2, 12, 12.5, 17.6, 12.3, 16.2, 17.5.

Choose 3 samples with the lowest clotting time – 12.1, 11.9, 12 secs

Pools these samples together.

Mix them well.

Measure clotting time for this pool using BIOREXFARS PT Reagent.

The value obtained is close to 12.0 seconds.

Label this pool 100% Sample.

A 100% sample denotes that all clotting factors are ok and 100% clotting has occurred.

MEAN NORMAL PTPreparation of 50% and 25% pools

50% pool is prepared by diluting 1 volume of 100% pool with 1 volume of physiological saline.

2 % l i d b dil i 1 l f 100% l25% pool is prepared by diluting 1 volume of 100% pool with 3 volumes of physiological saline.

Measure clotting times for the 50% and 25% pools withMeasure clotting times for the 50% and 25% pools withBIOREXFARS PT reagent.

Let us assume that the results were 14.8 seconds for 50%d 26 7 d f th 25% land 26.7 seconds for the 25% pool.

Construct a graph on a semi log graph paper using the Clotting timesobtained for the 100%, 50% and 25% pools.

MEAN NORMAL PT

1/Activity

25%

1/Activity

50%

Time in seconds

12 secs 14.8 secs 26.7 secs100%

25%

Competitor 1 BIOREXFARS

1/Activity

50%

Competitor 2

Typical graph with 100%, 50%And 25% pools

12 secs 14.8 secs 26.7 secs100%

Time in seconds

Typical graph of 3 different company’s PT rgts

READING THE INR TABLEPATIENT RESULTS

Let us assume that the Clotting time for a patient sample is 14.1 secs.

ISI VALUE OF THE KIT = 1.22

INR   ISI

S U OMEAN NORMAL PT = 12.0 SECS

INR =  Patient PT

Mean Normal PT= (Prothrombin Ratio)

INR = (14.1/12.0)1.22

(1 17)1.22(1.17)

INR = 1.26

READING THE INR TABLEMEAN PATIENT (MNP) AND PATIENT RESULTS % R INR

10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 100.0 1.00 1.00

9.0 9.5 9.9 10.4 10.8 11.3 11.7 12.2 12.6 13.1 13.5 111.9 0.90 0.88

9.5 10.0 10.5 10.9 11.4 11.9 12.4 12.8 13.3 13.8 14.3 98.9 0.95 0.94

ISI 1.22

10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 100.0 1.00 1.00

10.5 11.0 11.6 12.1 12.6 13.1 13.7 14.2 14.7 15.2 15.8 80.2 1.05 1.06

11.0 11.6 12.1 12.7 13.2 13.8 14.3 14.9 15.4 16.0 16.5 73.3 1.10 1.12

11.5 12.1 12.7 13.2 13.8 14.4 15.0 15.5 16.1 16.7 17.3 67.5 1.15 1.19

12.0 12.6 13.2 13.8 14.4 15.0 15.6 16.2 16.8 17.4 18.0 62.6 1.20 1.25

This row denotes the mean normal PTvaluesderived by running the pooled samples.            

Choose this column to correlate the patient values for INR / Ratio 

12.5 13.1 13.8 14.4 15.0 15.6 16.3 16.9 17.5 18.1 18.8 58.3 1.25 1.31

13.0 13.7 14.3 15.0 15.6 16.3 16.9 17.6 18.2 18.9 19.5 54.6 1.30 1.38

13.5 14.2 14.9 15.5 16.2 16.9 17.6 18.2 18.9 19.6 20.3 51.3 1.35 1.44

14.0 14.7 15.4 16.1 16.8 17.5 18.2 18.9 19.6 20.3 21.0 48.4 1.40 1.51

14.5 15.2 16.0 16.7 17.4 18.1 18.9 19.6 20.3 21.0 21.8 45.8 1.45 1.57

p p(eg: 12.0 secs)

/or %.

15.0 15.8 16.5 17.3 18.0 18.8 19.5 20.3 21.0 21.8 22.5 43.4 1.50 1.64

15.5 16.3 17.1 17.8 18.6 19.4 20.2 20.9 21.7 22.5 23.3 41.3 1.55 1.71

16.0 16.8 17.6 18.4 19.2 20.0 20.8 21.6 22.4 23.2 24.0 39.4 1.60 1.77

16.5 17.3 18.2 19.0 19.8 20.6 21.5 22.3 23.1 23.9 24.8 37.7 1.65 1.84

17.0 17.9 18.7 19.6 20.4 21.3 22.1 23.0 23.8 24.7 25.5 36.1 1.70 1.91

17.5 18.4 19.3 20.1 21.0 21.9 22.8 23.6 24.5 25.4 26.3 34.6 1.75 1.98

18.0 18.9 19.8 20.7 21.6 22.5 23.4 24.3 25.2 26.1 27.0 33.3 1.80 2.05

18.5 19.4 20.4 21.3 22.2 23.1 24.1 25.0 25.9 26.8 27.8 32.0 1.85 2.12

19.0 20.0 20.9 21.9 22.8 23.8 24.7 25.7 26.6 27.6 28.5 30.8 1.90 2.19

19.5 20.5 21.5 22.4 23.4 24.4 25.4 26.3 27.3 28.3 29.3 29.8 1.95 2.26

20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0 28.8 2.00 2.33

20.5 21.5 22.6 23.6 24.6 25.6 26.7 27.7 28.7 29.7 30.8 27.8 2.05 2.40

21.0 22.1 23.1 24.2 25.2 26.3 27.3 28.4 29.4 30.5 31.5 26.9 2.10 2.47

21.5 22.6 23.7 24.7 25.8 26.9 28.3 29.0 30.1 31.2 32.3 26.1 2.15 2.54

SETTING ISI FOR PT REAGENTS

ISI FOR PT REAGENTS CAN BE SET IN TWO WAYS

BY assaying known INR values which are traceableBY assaying known INR values which are traceableto WHO RBT 05.

BY using reference or traceable samples and assayingthem in duplicates and plotting them as logarithms. The ISI is then calculated using the slope of the line thatis drawn as a best fit line between these sets of log values.is drawn as a best fit line between these sets of log values.

SETTING ISI FOR PT REAGENTS

BIOREXFARSFARS METHOD FOR SETTING ISI

BY assaying known INR values which are traceableto WHO RBT 05.

In BIOREXFARSFARS Diagnostics we use the AK Calibrant, whichis a commercially available calibration material for setting ISI values.

The advantages of using the AK Calibrant are:

-4 levels- Known INR values- Traceable to WHO RBT 05

HOW IS IT DONE?USE OF AK CALIBRANT

1. Reconstitute lyophilised material supplied.2. Ensure homogeneity.3. Choose the reagent to be ISI assigned.4. Incubate the PT reagent till it reaches 37oC.5. Assay all the 4 calibration material with the PT.

SECONDS

ISI VALUE

Calibration curve drawnCalibration curve drawn From the seconds derivedFor the lyophilised calibrators

Point 5 in the next slide

INR VALUE

HOW IS IT DONE?

CALIBRATION CURVE

1. Note down the seconds after assaying the cal samples.2. In the graph provided, plot the seconds vs the INR values.3. Draw a line thru the seconds that have been plotted.4. The line can be extended to the ISI area of the Y2 axis.5 Drop a line from the first al e (seconds) to the X a is5. Drop a line from the first value (seconds) to the X axis.6. Construct a line from this point (that meets the x axis) to

to the Y2 axis. 7 Read the ISI value off this line7. Read the ISI value off this line.

CHECKING THE ISI VALUE?

ISI VALUE CHECKS

1. Run controls with known INR values.2. Note the seconds for these controls.3 With th MNPT l f th PT R t l l t3. With the MNPT values for the PT Reagent, calculate

the INR for the control.4. Compare the INR values derived from the PT reagent

with the prescribed INR values of the controlwith the prescribed INR values of the control.5. The deviation should not be more than 10%.6. Run as many levels as possible, preferably normal

and elevated INRs.and elevated INRs.