Qualitative and Quantitative Estimations

Understanding different testing methods used commonly in a clinical biochemistry laboratory.

1. Qualitative & Quantitative Estimations Karthik G Kamath K Associate Professor Department of Biochemistry 1

2. Specific Learning Objectives Describe end point reactions. Divide end point reactions into two types Describe qualitative & quantitative tests. Divide quantitative tests into two sub types Describe mono & multi step methods. Describe rate reactions. Divide rate reactions into two types. Describe visible kinetic methods. Describe ultraviolet kinetic methods. 2

3. End Point Reactions Methods carried out under specific conditions & maintaining specific temp, pH, & reaction period At the end, reaction b/w analyte & specific reagent results in a colored compound, turbidity or certain components (NAD, NADH) Concentration of analyte can be determined by intensity of end product They can be divided into Qualitative & Quantitative tests 3

4. Qualitative Tests Detects presence / absence of a specific analyte E.g. Benedict’s test, Fouchet’s test 4

5. Quantitative Tests Detects the exact concentration of the specific analyte in a given sample Can be of 2 types Multi-step methods Mono-step methods 5

6. Multi-step Methods Procedure is followed through several steps by using many reagents Most methods are time-consuming & non-specific E.g. Blood glucose – Folin Wu, Uric acid – Henry-Caraway’s 6

7. Mono-step Methods Comprises of only one-step In the specified amount of reagent, a specified quantity of specimen is added & mixed The intensity of the end product produced is measured photometrically E.g. GOD-POD, Total protein & albumin - Biuret etc. 7

8. Rate of Reaction Methods When a reagent reacts with a specific analyte The initial rate of reaction is directly proportional to the concentration of the analyte under study Rate of reaction can be continuously monitored for up to 2 to 3 mins under defined & controlled conditions Reaction conditions are carefully selected to maintain optimum concentration of reagents & substrates with optimum pH & temp 8

9. For the initial rate of reaction determination, a specialized photometer/spectrophotometer is required With a thermocuvette & a lamp that can emit UV radiation along with a visible range Determination involves measurement of the amount of change in OD produced in a defined time interval All determinations are kinetic measurements 9

10. In most methods, enzyme- catalyzed reactions are used Determination of rate of reaction under defined & controlled conditions provide a specific & sensitive method for measurement of enzymes 10

11. Rate of Reaction – Visible Kinetic Methods Colored complex is formed Initial rate of formation of colored compound is directly proportional to the specimen analyte Change in absorbance per min (▲A/min) is determined That is directly proportional to the enzyme concentration 11

12. Rate of Reaction – U-V Kinetic Methods Similar to visible kinetic method But, formation of colored complex does not take place End products NAD, NADH, NADP, and NADPH absorb UV light (340nm) at variable proportions 12

13. References Praful B Godkar , Darshan P Godkar ; Textbook of Medical Laboratory Technology – Clinical Laboratory Science & Molecular Diagnosis; Third Edition; 2016; 62-71 13