Validation of Analytical Methods PDF

Validation of Analytical Methods Dr. Mohamed.A.Mahdi  Method Validation It should be provided by the following: 1. accuracy. 2. Precision. 3. Sensitivity. 4. Specificity. 5. Linearity.  Sensitivity Ability to exclude false negative. Is to measure the...

Validation of Analytical Methods Dr. Mohamed.A.Mahdi  Method Validation It should be provided by the following: 1. accuracy. 2. Precision. 3. Sensitivity. 4. Specificity. 5. Linearity.  Sensitivity Ability to exclude false negative. Is to measure the incidence of positive results in patients known to have a condition, that is ‘true positive’ (TP). A sensitivity of 90% implies that only 90% of people known to have the disease would be diagnosed as having it on the basis of that test alone. 10% would be ‘false negative’ (FN).  Specificity Ability to exclude false positive. Is to measure the incidence of negative results in persons known to be free of a disease, that is ‘true negative’ (TN). A Specificity of 90% implies that only 10% of disease- free people would be classified as having the disease on the basis of the test result. They would have ‘false positive’ (FP) result.  Calculation Sensitivity &Specificity are calculated as: Specificity = TN x100 all without disease (FP + TN) Sensitivity = TP x100 all with disease (TP + FN)  Examples Specificity is ability to correctly identify individuals without disease. 1000 people tested: 1. 875 positive tests (275 false positive). 2. 125 negative tests (25 false negative). Specificity = TN x100 all without disease (FP + TN) = 100 x100 275 + 100 = 0.27 x 100 = 27%.  Sensitivity is ability to correctly identify individuals with disease. 1000 people tested: 1. 875 positive tests (275 false positive). 2. 125 negative tests (25 false negative). Sensitivity = TP x100 all with disease (TP + FN) = 600 x100 600 + 25 = 0.96 x 100 = 96%.  Positive Predictive Value (PPV): Explain the test result if +ve, that is means patients have a disease. PPV %= True positive x 100 Total positive Negative Predictive Value (PNV): Explain the test result if -ve, that is means patients have not a disease. PNV %= True Negative x 100 Total Negative  Criteria for Control Sera 1. Available in sufficiency quantity. 2. Stable-available in convenient vial volumes. 3. Vary minimally in concentration and composition from vial to vial. 4. Closely resemble the specimen in both its physical and chemical characteristics. 5. Safe to use. 6. Not expensive. 7. For hematology analyzer control need to have same consistency and color in human serum.  Interpretation of quality control results monthly Usually internal quality control result assessed based on accuracy and precision. For precision the laboratory is precise when CV% (Coefficient Variation) is less than 5 %.  For accuracy the mean always used to calculate (z-score) which indicate the internal accuracy of the laboratory performance. Z-score = measured mean – target mean standard deviation  Z-score result categories into four stages of accuracy : a. 0-0.5 excellent b. 0.5-1 satisfied c. 1-1.5 acceptable d. 1.5 -2 poor  Routine Quality Assurance Should be performed every 3 weeks as: 1. Calibration of instruments. 2. Checking the validity of reagents. 3. Checking the validity of STDs. 4. Checking the validity of anticoagulants. 5. Training of the lab staff periodically. 6. Checking the control materials. 7. Using control test daily with in each batch of specimens. 8. Using external quality control every 1 to 3 months. 9. Checking the statistical program of control system.  Measurement of Accuracy of Method Achieved by many ways include :- 1. Comparison :- by compare the method of the test with a reference method which define as a method with negligible in accuracy ,is the best comparative method that can be employed but it may be laborious ,complicated and time consuming.  Beside that most laboratories are not staffed and equipped to perform reference methods ;for all that ,the accuracy of the method is measured using other method with known bias. 2.Using control sera set :- by way described in internal Q.C. 3. Recovery :- show whether a method measures all of the analyte or only part of it. Recovery define as a percentage different from the value of 100%. - Method :- a test sample is prepared by adding small a mount of concentration analyte in diluents to the patient sample.  Another sample is prepared from the patient specimen by dilute it with the same volume ,but containing only a diluents.both diluted specimen are then analyzed by the test method.the amount recovered is the difference between the measured value of the2 samples. Ex :- specimen for blood glucose. -patient specimen (without adding analyte )=100mg\dl. -patient specimen (with 50 mg\dl glucose added)=145mg\dl Recovery = 45 x100 =90% 50 The concentration recovery=conc. (diluted test )- original conc. Recovery(%) =conc. Recovery x100 conc. added 4.Interferance method :- - Used to measure systematic error caused by substances other than the analyte which cause these errors either by react with analytical reagent or alter the reaction between the analyte and analytical reagent. This method is similar to recovery except that the substance suspected of interference is added to the patient sample. - Common interferences (hemoglobin (haemolysis)- bilirubin –lipids-anticoagulants –preservatives ,etc). Also turbidity. - Interference minimized by using blank solution or what is called (test blank)to correct for other substance in the sample by adding the sample to the blank solution but without analytical reagent (reactive ingredient).  Standards (STD) - STD solutions contains all chemicals and test substance with certain known concentration ,there are different type of STD used:- 1/ Primary STD:- with the substance or chemicals that is of the highest purity and can be measured directly to produce substance of exact known concentration.  Characteristics :- a. Has high M.W. b. Stable. c. Easily dissolved in water. d. Don’t contain interfering substances. 2- Secondary STD:- is define as substance of low purity whose concentration is determined by comparison to primary STD. 3- Internal STD :- substance not normally present in sample or STD used.added in known a mount.it is useful to correct inaccuracy. 4- Arbitary STD :- contain un known concentration of analyte. Using in case of immunoglobulin assay.  Normal or Reference values - The amount of substance present in body fluid or excretions in healthy individual. The results are affected by both biological and laboratory factors. - Biological factors :- make differences in test result among healthy people.include :- 1. Age :- for example :higher alkaline phosphates activity in growing children compared with adults. 2. Sex :- e.g. higher values of plasma urea in men compared with women during the reproductive phase of life. 3. Diet &nutritional state :- many tests affected by diet. 4. Muscular activity :- e.g. concentration of plasma creatinine rises following exercise. 5. posture :- e.g. plasma protein levels are lower in samples collected from patients when they are lying down. 6. Time of the day :- e.g. T3,T4 highly concentration at morning. 7. Genetic factor. 8. Also weight –phase of menstrual cycle – emotional state –geographical location- climate or intrinsic haemostatic variation can affect normal values (Race & rural or city life).  Laboratory factors :- 1. Type of sample :- e.g. plasma glucose is 12- 13 % higher than in whole blood. 2. Test method :- Normal value vary from one method to another because some methods are more specific than others. 3. Lab accuracy.  Laboratory factors :- 1. Type of sample :- e.g. plasma glucose is 12- 13 % higher than in whole blood. 2. Test method :- Normal value vary from one method to another because some methods are more specific than others. 3. Lab accuracy.  Method Description Analytical method :- is a set of instructions that describe the procedure – equipments and materials necessary to obtain results. - To describe any method ,the description must include all the following:- 1.Principle :- the base of reaction happened or the relation ship between reactive ingredient in analytical reagent and substance (analyte)to be measure in the sample. 2. Specification of reagent and instruments being used. 3. Calibration of STD:- necessary to establish whether the beer-lambert law and formula can be applied (i.e whether the absorbance being measured increases in a linear way with its concentration and to what concentration the method is linear. 4. Procedure of a test. 5.Requirements of specimen :- because some methods affected by different factors so you have to consider the collection time- anticoagulant- preservatives- temperature. 6.The steps of reaction. 7. Calculation. 8. Analytical range. 9. Safety precaution.  How to Introduce a New Method 1. Check the linearity :- analysis of set of serial dilution of STD to define over what range the O.D and concentration linear. 2. Check the precision :- repeat runs to check that results is reproducable from batch to batch or from day to day. 3. Analysis of normal subject to confirm the normal values. This involves performing the previous method a long side the new one and noting whether both agree as to which result are normal and which are abnormal. 4.Check recovery of the method. 5. Comparison between the results from an old and new method. - This done on the same patient sample. - The analysis by both methods. - should be performed on the same day. - Preferably with in 4 hours. - It recommended that at least 40 samples and preferably 100, be run by both methods. - After all that it is essential to warn clinical staff. - When the method become routine, it must renew of STD periodically to a void changes on staying and compare new STD with an old one. In enzyme assay, consider that tests are affected by the change of condition. (Temp -Time –Light – Rays and so on).