07/04/2013
Clinical Chemistry Methodologies:
Generally, three methods are followed for the estimation of various analytes in clinical chemistry.
They are:
1. End Point method or end poind chemisty
2. Fixed time method or fixed time chemistry
3. Kinetic method or Kinetic Chemistry
1. End Point Method:
This method is employed for the estimation of analytes, which would be completely consumed in the reaction. The end point for a particular analyte is normally acheived within 5 to 15 minutes at 37 C. the coloured complex/ non coloured complex thus formed at the end of reaction period is read for its absorbance using a aspectro-photometer or photometer.
This method is standardized to consume the amount of analyte equivalent to the linearity level mentioned for individual chemistries, within the stipulated reaction time, as per the protocol outlined.
In the end point methods the absorbance, at a specific wavelength, of the complex being formed is measured. This absorbance increases against the reagent blank absorbance, over a period of time. This increases in absorbance continues till it reaches a stable value, marking the end point of the reaction being reached. No further change in absorbance would occur, even if the conversion of the anaylte being measured.
Fixed Type Method:
The reagents employing this methodology are based on the principle of the difference in absorbance between an initial value and final value, during a specified time interval. The time interval is optimized in such a way to minimize the interferences with the test analytes from other analytes in the sera or sample. The assumption is that a constant amount of product is produced during the entire time period.
Generally these tests are performed with a pre-incubation of the sample with the reagent, During this, any substance other than the test analyte, present in the sample, which could react with the reagent system would react completely, such that they do not interfre with the time interval is standardized and fixed to a minute or two, to give accurate and precise results for the analyte being estimated.
Fixed time chemistries could be of two types:
a. Increasing Type;
This could also be called as Fixed time chemistry progressing in positive direction. In this method, the final absorbance of the reacton is always higher than the initial absorbance. The difference between the final and initial absorbance, called as delta absorbance is always positive.
b. Decreasing Type;
This could be also called as fixed time chemisty progressing in negative direction. In this method, the initial absorbance of the reaction is always higher than the final absorbance. The difference between the final and initial absorbances, called as delta absorbance is always negative.
3. Kinetic Method
The reagents employing this method are based on the principle of measurement of the difference in absorbance beween two points over a period of specified time during the progress of the reaction. The assumption is that a constant amount of product is produced during the time interval being monitered. Usually, the reaction time is short to avoid any danger of enzyme degradation.
These tests are performed with a preincubation, during which any substances in the sample, which might interfere with the test would have fully reacted with reagent system, so as not to interfere with the actual assay.
In kinetic test procedures, the difference in absorbance between tow points, taken during the linear stage of progression of the test is taken into consideration, to yield the delta absorbance. This time interval could be typically like 1 minute or 20 seconds. The delta absorbance obtained would be multiplied by an appropriate factor for calculations, to give the quantity of the analyte being tested in the sample. The delta absorbance, that are consistent over a period of time are taken for calculation. These tests are standardized to give a linear delta absorbance over a period of time and upto the specific linearity mentioned for each analyte.
Kinetic Chemistries could be classified as:
a. Increasing Type: In this type, the reaction proceeds in positive direction, where in, the initial absorbance is always lower than the latter absorbance.
b. Decreasing Type: This type of chemistry is also called as the negative direction type. In this method, the difference between a latter point of absorbance and an initial point, over a specified period of time is always negative.
Dr -mayada zahw
