Week 11 Measurement Procedures and Calculations PDF

Clinical Chemistry I 0202304 Instructor: Mohammad QABAJAH E-mail: [email protected] Week 11 Measurement Procedures and Calculations 2 Objectives - Define terminologies associate with measurements in clinical chemistry - List fundament...

Clinical Chemistry I 0202304 Instructor: Mohammad QABAJAH E-mail: [email protected] Week 11 Measurement Procedures and Calculations 2 Objectives - Define terminologies associate with measurements in clinical chemistry - List fundamental solutions in spectrophotometric measurements - Describe assays techniques in clinical chemistry measurements - Discuss about calibration curve Definition of Terms - Blank: A solution used to correct for background interferences that may come either from the sample or the reagent alone. I. Reagent blank: - A solution used to avoid interferences expected to come with reagents. - Contains all elements of the solution including the principal regent, buffer solution and others except the patient sample. II. Sample blank: - A solution used to avoid interferences expect to come with samples. - Contains all elements of the solution including the patient sample, buffer solution and others except the principal regent. Definition of Terms - Standard: - Pure solution having known concentration of interested substance in a fixed value (Single number). - Used as a reference value to compare the concentration of standard solution. - Control: - Matrix solution having either normal or pathologic known concentration of interested substance in a defined range. - Used to control the over all activities of the measurement. - Normal and Pathologic (Depends on the analyte: Low, High or Both) Definition of Terms - Molar absorptivity: A constant for one molar solution of a given compound at a given wavelength and a 1-cm path length under prescribed conditions of solvent, temperature, pH. - Transmittance: A ratio of transmitted radiant energy to incident radiant energy. - Absorbance: Negative logarithm of transmittance. Quantitative Measurements - Necessary to standardize instrument before measurement of intensity. - The basic procedure involves setting the minimum & maximum conditions of transmission & adjusting the metering to give appropriate readings - Maximum transmittance: Blank sample is used & instrument is adjusted to give either a reading of 100% transmittance or zero absorbance. - Zero transmittance: All light to the detector is cut off & the meter is adjusted to give a transmittance reading of zero. 7  Methods for Calculations There are two methods for calculations of analyte concentration of an unknown solution: I. Comparative Methods - Ratio of known standard to unknown - Use of standard calibration curve II. Absolute Methods - Use of known molar extinction coefficient 01-Dec-24 8  Calibration Curve Calibration can be prepared by plotting absorbance on the Y-axis versus concentration on the X-axis coordinate graph paper for a series of standard solution of known concentration Application of calibration curve: I. Analysis of concentration II. Verifying the proper functioning of analytical instruments 01-Dec-24 9 Assay Techniques in Clinical Chemistry Basically there are different types of analytical techniques: I. End-point Assay II. Kinetic Assay: A. Fixed Time Kinetic B. Continuous Monitoring End-point Assay - Reagent mixed with sample, and Abs is measured after incubation for time interval. - The reaction take place (Different reaction types: Enzymatic, Binding). - Absorbance reading at any time after incubation, and during the result reading time - Analyte concentration calculated by using appropriate absolute or competitive method End-Point Example 12 Kinetic Assay - Kinetic assay is a method in which the amount of substrate consumption or product formation is measured between end of incubation period, and substrate depletion phase. - Increasing or Decreasing - Two types of kinetic: I. Fixed-Time Kinetic II. Continuous Monitoring Assay (3 data points at least) Kinetic Assay - Fixed-Time Assay: Substrate is added and Abs is measured after a predetermined incubation period, at fixed time intervals (Starting point and Ending point). - Continuous Monitoring Assay: Multiple absorbance readings are recorded continuously over the entire reading time interval. Kinetic Examples 15 See YOU Next Lecture