PROTHROMBIN TIME
WHY USE LOW ISI (HIGH SENSITIVITY) PROTHROMBIN TIME ‐ DEFINITION
The Prothrombin time is the functional determination of the extrinsic coagulilation pathway.
It is a widely used laboratory assay for the detection of inherited or acquired coagulation defects related to the extrinsic pathway. TERMS
• INR – INTERNATIONAL NORMALIZED RATIO
• WHO –WORLD HEALTH ORGANISATION
• ISI – INTERNATIONAL SENSITIVITY INDEX
• IRP – INTERNATIONAL REFERENCE PREPARATION INR – INTERNATIONAL NORMALIZED RATIO
Ever wondered why you obtain different results for
• different company PT reagents ? • different lots of the same companies PT reagents ?
PT REAGENTS ARE MADE FROM TISSUE FACTORS
BEING 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 the sensitivity of commercial Thromboplastins to the 1st IRP. (International Reference Preparation).
IRP –A very responsive batch of human brain extract was designated as the 1st International Reference Preparation (()IRP). ISI – International Sensitivity Index
¾ The ISI of the 1st IRP was 1.0
¾ The INR act uall y compares th e proth rombi n rati o measurement to the 1st IRP.
¾ The INR repppresents the prothrombin time that would have been obtained if the IRP had been used as the reagent.
¾ Responsive PT reagents have lower ISI values.
¾ Analytical precision is improved with reagents with low ISI.
¾ Reagents with low ISI discriminates normal and warfarin treated patients better. LOW ISI VS HIGH ISI
LOW ISI HIGH ISI
CClldalled as High sensiiitivity CClldalled as Low sensiiiitivity assay assay The reagent is highly The reagent is NOT sensitive sensitive to even small to changes in PT changes in the PT Accommodates Factor DOES NOT accommodate deficiency Factor deficiency Higher levels of precision Poor precision
Better global commutability Poor Commutability with PT assays Shift towards low ISI assays Shift away from High ISI kits globally globally SAMPLES AND THEIR ROLE
MOST OF THE PROBLEMS CAUSED IN PT TESTING IS BECAUSE OF SAMPLING. SAMPLES AND THEIR ROLE
ANTICOAGULANT
Whol e bl ood sh ould b e coll ect ed with 3.2%S% So dium Cit rat e As the anticoagulant.
DO 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 ROLE
PREPARATION 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 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 Tubes Eberhard W. Fiebig et al; Am J Clin Path; 2005; 124(6) 902-909 SAMPLES AND THEIR ROLE
STORAGE OF SAMPLES
¾ Remove citrated plasma from the centrifuged sediment
¾ Place the same in a plastic container
¾ Refrigerate
DO NOT FREEZE. (freezing prolongs clotting time).
¾ Stored samples must be mixed well before use .
Effect of freezing method and storage at -20oC and -70oC on PT, aPTT and Plasma Fibrinogen levels Sonja Alesci et al; Thrombosis Research; Vol 124, Issue 1, May 2009. INR – INTERNATIONAL NORMALIZED RATIO
WHO itintrod uce d INR in the early 80’s as a means of Standardizing PT results.
ISI ISI INR = Patient PT = (Prothrombin Ratio) Mean Normal PT
WHO and International Committee on Thromobosis and Haemostasis – Expert committee reports MEAN NORMAL PT
FACTS ¾ Each lab must establish Mean Normal PT for the reagent and the instrument used
¾ 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 Guidelines CLSI;Vol 25 No.23. MEAN NORMAL PT
FACTS
¾ Each lab must establish Mean Normal PT for the reagent and the instrument used
¾ 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 Guidelines CLSI;Vol 25 No.23. MEAN NORMAL PT MEASUREMENT
¾ Collect 10 - 20 fresh samples (apparently normal)
¾ Measure clotting times for PT using BIOREXFARS PT reagent
¾ 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.
¾ Choose 3 samples with the lowest clotting time – 12.1, 11.9, 12 secs
¾ Pools these samples together.
¾ Mix them well.
¾ Measure clotting time for this pool using BIOREXFARS PT Reagent.
¾ The value obtained is close to 12.0 seconds.
¾ Label this pool 100% Sample.
A 100% sample denotes that all clotting factors are ok and 100% clotting has occurred. MEAN NORMAL PT
Preparation of 50% and 25% pools
¾ 50% pool is prepared by diluting 1 volume of 100% pool with 1 volume of physiological saline.
¾ 25%li% pool is prepared dbdili by diluting 1 volflume of 100%l% pool with 3 volumes of physiological saline.
¾ Measure clotting times for the 50%and% and 25% pools with BIOREXFARS PT reagent.
¾ Let us assume that the results were 14.8 seconds for 50% and 26.7 secondfds for th e 25%l% pool.
Construct a graph on a semi log graph paper using the Clotting times obtained for the 100%, 50% and 25% pools. MEAN NORMAL PT
25%
1/Activity
50%
100% 12 secs 14.8 secs 26.7 secs Competitor 1 BIOREX FARS Time in seconds 25% Competitor 2 Typical graph with 100%, 50% 1/Activity And 25% pools 50%
100% 12 secs 14.8 secs 26.7 secs
Time in seconds
Typical graph of 3 different company’s PT rgts READING THE INR TABLE
PATIENT RESULTS
Let us assume that the Clotting time for a patient sample is 14.1 secs.
ISSUOI VALUE OF THE KIT = 1.22 MEAN NORMAL PT = 12.0 SECS
ISI INR = Patient PT = (Prothrombin Ratio) Mean Normal PT
INR = (14.1/12.0)1.22
(1.17)1.22
INR = 1.26 READING THE INR TABLE
MEAN PATIENT (MNP) AND PATIENT RESULTS %RINR 100. 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 1.00 1.00 0 111. ISI 1.22 9.0 9.5 9.9 10.4 10.8 11.3 11.7 12.2 12.6 13.1 13.5 0.90 0.88 9 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 0.94 100. 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 1.00 1.00 0 10.5 11.0 11.6 12.1 12.6 13.1 13.7 14.2 14.7 15.2 15.8 80.2 1.05This 1.06 row denotes the Choose this column11.0 to 11.6 12.1 12.7 13.2 13.8 14.3 14.9 15.4 16.0 16.5 73.3 1.10mean 1.12 normal PTvalues correlate the patient11.5 12.1 12.7 13.2 13.8 14.4 15.0 15.5 16.1 16.7 17.3 67.5 1.15derived 1.19 by running the values for INR / Ratio12.0 12.6 13.2 13.8 14.4 15.0 15.6 16.2 16.8 17.4 18.0 62.6 1.20p 1.25ooled samples. or %. 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(eg: 1.31 12.0 secs) 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 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
¾ BY assaying known INR values which are traceable to WHO RBT 05.
¾ BY using reference or traceable samples and assaying them in duplicates and plotting them as logarithms. The ISI is then calculated using the slope of the line that is drawn as a best fit line between these sets of log values. SETTING ISI FOR PT REAGENTS
BIOREXFARSFARS METHOD FOR SETTING ISI
¾ BY assaying known INR values which are traceable to WHO RBT 05.
In BIOREXFARSFARS Diagnostics we use the AK Calibrant, which is 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 drawn From the seconds derived For 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 v alu e (seconds) to the X ax is. 6. Construct a line from this point (that meets the x axis) to to the Y2 axis. 7. 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 e MNPT val ues f or th e PT R eagent , cal cul at e the INR for the control. 4. Compare the INR values derived from the PT reagent with 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.