International Sensitivity Index (ISI) value & Calibration of Test Thromboplastin Reagent

International Sensitivity Index (ISI)

• Thromboplastins are derived from different sources (human, rabbit etc.) for use in Prothrombin Test (PT).
• The PT carried out with each of these Thromboplastins gives different results → Different sensitivities to recognize clotting factor deficiency or coagulation system.
• This difference in sensitivity of Thromboplastins to recognize the status of coagulation system under investigation is called Sensitivity Index.   
• International WHO Reference Thromboplastin has been assigned International Sensitivity Index value of 1.0 
• All other reference thromboplastins are calibrated to this above mentioned WHO Reference Thromboplastin & an ISI value is assigned to that particular thromboplastin in comparison.  
• These Reference Thromboplastins (calibrated against WHO Reference Thromboplastin) are in turn used to calibrate a Test Thromboplastin (any Thromboplastin made in lab or available commercially). 
• Special Note: A test thromboplastin must be calibrated against a reference thromboplastin from the same species. So, Rabbit Thromboplastin is calibrated against Rabbit Reference Thromboplastin. 

Method of Calibrating a Test Thromboplastin

• Step 1 
• Get Citrated Platelet Poor Plasma (PPP) from 20 healthy donors & from 60 patients stabilized on Tablet Warfarin for ≥6 weeks. 
• Step 2 
• Perform Prothrombin Test (PT) using Reference Thromboplastin on above mentioned 80 (20 + 60) samples in duplicate & take mean PT value for each 
• Total tests with reference thromboplastin → 80 x 2 = 160
• The maximum allowable difference between the two reading on same sample during duplicate testing is only 10%
• If difference is >10% → Repeat PT on that sample in duplicate again
• Step 3
• Perform PT using Test Thromboplastin on above mentioned 80 (20 + 60) samples in duplicate & take mean PT value for each
• Total tests with test thromboplastin → 80 x 2 = 160 
• The maximum allowable difference between the two reading on same sample during duplicate testing is only 10%
• If difference is >10% → Repeat PT on that sample in duplicate again
• Step 4
• The means of each pair of readings for Reference Thromboplastin (Step 2) & Test Thromboplastin (Step 3) are plotted on a Log-Log Graph:
• Y-axis → Mean PT readings for Reference Thromboplastin 
• X-axis → Mean PT readings for Test Thromboplastin 
• Step 5
• Draw the Line of Best Fit (on computer, simple linear regression may be applied instead – sufficiently accurate)
• Step 6
• Mark points on the best fit line as under: 
• Point A → Just below the lowest recorded PT
• Point B → Just above the longest recorded PT
• Draw a horizontal line from Point A parallel to X-axis 
• Draw a vertical line parallel to Y-axis
• Mark the point where the two lines intersect as Point C
• Step 7 
• Measure the lengths of the lines accurately in mm (millimeters)
• Calculate the Slope as under: 
• Slope = Vertical line meeting Point C / Horizontal line meeting Point C 
• Slope =  (Point B to Point C (mm))/(Point A to C (mm))
• Example: if B – C = 55 mm & A – C = 35 mm 
• Slope = 55/35 = 1.57 
• Step 8 
• Multiply the Slope value with the ISI of Reference Plasma 
• Example: 
• If the ISI of Reference Plasma is 1.1 
• ISI of Test Thromboplastin = 1.1 x 1.57 = 1.74 
• Step 9 
• Calculate the International Normalized Ratio (INR) from the above calculated ISI value
• INR is the ratio of Patient’s PT compared to Geometric Mean Normal PT (GMNPT) that is corrected for the sensitivity of the Thromboplastin used 
• GMNPT → Geometric Logarithmic Mean of Normal PT that is determined for each batch of Thromboplastin by testing 20 normal PPP samples 
• PT Ratio = (PT of the Patient)/GMNPT
• INR =[PT Ratio]^ISI