1. Method Selection and Evaluation Method Selection and Evaluation D. Kefaya EL- Sayed Mohamed Prof. Of Clinical Pathology (Clinical Chemistry), Mansoura University

2. Before a new test or methodogy is introduced into the laboratory. both managerial and techinal information must be compiled and carefully considered. The information should be collected from many different sources,including manufacturer sales representatives,colleagues,scientific presentations,and the scientific literature.

5. Linear range:

6. O.D O.D 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 200 400 600 800 0.0 200 400 600 800 Glucose concentration (mg/dl)

9. Observed value True value RE SE TE µ X

11. Inaccuracy:  The difference between a measured value and its true value.  Is due to the presence of systematic analytical error (S.E.)  S.E. can be constant or proportional.

12. Test Values Test Values Constant E Ideal Proprtional E 0 30 Reference Values

13. Inaccuracy  Can be estimated from three studies: recovery. recovery. interference. interference. a comparison-of-methods study a comparison-of-methods study

15. Example of a recovery study Sample preparation Sample 1:2.0 ml serum + 0.1 ml H2O Sample 2:2.0 ml serum +0.1 ml 20 mg /dl calcium standard Sample 3:2.0 ml serum +0.1 ml 50 mg /dl calcium standard Concentration Concentration Calcium measured added Recovered Recovery Calcium measured added Recovered Recovery Sample 1 7.50mg/dl Sample 2 8.35mg/dl 0.95mg/dl 0.85mg/dl 89% Sample 3 9.79mg/dl 2.38mg/dl 2.29mg/dl 96%

16. Calculation of recovery Concentration added = standard concentration x Ml standard Ml standard + ml serum Ml standard + ml serum = 20 x 0.1 = 20x 1 = 20 = 0.95 (Sample2) = 20 x 0.1 = 20x 1 = 20 = 0.95 (Sample2) 2+0.1 2.1x10 21 2+0.1 2.1x10 21 conc. recovered = conc. ( diluted test) – conc. ( baseline) = 8.35-7.5=0.85 = 8.35-7.5=0.85 recovery = Conc. Recovered = x100% recovery = Conc. Recovered = x100% Conc. added Conc. added = 0.85 x 100 = 89 % ( sample 2) = 0.85 x 100 = 89 % ( sample 2) 0.95 0.95

17. ( b )Interference:  The interference experiment is used to measure systematic errors caused by substances other than the analyte

18. Interference: Interference:  An interfering material can cause systematic errors in one of two ways : The material itself may react with the analytical reagents. The material itself may react with the analytical reagents. Or it may alter the reaction between the analyte and the analytical reagents Or it may alter the reaction between the analyte and the analytical reagents

19. Interference: Interference:  The interference likes recovery except that the substance suspected of interference is added to the patient sample.  The concentration of the potentially interfering material should be in the maximally elevated range.  If an effect is observed its concentration should be lowered to discover the concentration at which test resultes are frist invalidated

20. Example of an Interference study Sample preparation Sample 1 : 1.0 ml serum + 0.1 ml H2O (base line) Sample 2 : 1.0 ml serum + 0.1 ml of 10 mg/dl magnesium standard Sample 3 : 1.0 ml serum + 0.1 ml of 20 mg/dl magnesium standard Calcium Measured Magnesium added Interference Calcium Measured Magnesium added Interference Sample 1 9.80mg/dl Sample 2 10.53 mg/dl 0.91mg/dl 0.73mg/dl Sample 2 11.48 mg/dl 1.81mg/dl 1.68mg/dl

21. Calculation of interference Concentration added = standard concentration x = 10x 0.1 = 10 = 0..91mg/dl (sample2) = 10x 0.1 = 10 = 0..91mg/dl (sample2) 1.1 11 1.1 11 Interference = conc. ( diluted ) – conc. ( baseline) = 10.53 – 9.8 =0.73 (sample2) = 10.53 – 9.8 =0.73 (sample2) ml standard ml standard + ml serum

22.  Common interferences (eg., hemoglobin, lipids,bilirubin,anticoagulants preservatives, and so on)also should be tested.  Glick and Ryder have presented " interferographs " for various chemistry instruments – these are graphs relating analyte concentration measured versus interferent concentration

23. ( C ) comparison – of methouds Experiment:  The best comparative method that can be used is the reference method, which is a method with negligible inaccuracy in comparison with its imprecision.

24.  Reference methods may be laborious and time- consuming Because most laboratories are not staffed and equipped to perform reference methods, the results of the test method are usually compared with those of the method routinely in use.

25.  Westgard et al27 and the NCCLS35 recommend that at least 40 sample and preferably 100 samples, be run by botli methods. span the clinical range span the clinical range represent many different pathologic conditions. represent many different pathologic conditions. Duplicate analyses of each sample by each method. Duplicate analyses of each sample by each method.

26.  If 40 specimens are compared, two to five patient specimens should be analyzed daily for a minimum of 8 days.  If 100 specimens are compared the comparison study should be carried out during the 20-day replication study.

27. Test method Test method Reference Regression line

28. Example of Method selection and evaluation (Glucose in Serum)  Analytical Needs :- Rapid procedure is needed at time out side the regular working hours. Rapid procedure is needed at time out side the regular working hours. Sample Volume of 0.2 ml or less Sample Volume of 0.2 ml or less Analytical range of 0.0 to 500.0 mg/dl. Analytical range of 0.0 to 500.0 mg/dl. Turn arround time 30 min.or less is needed Turn arround time 30 min.or less is needed

29.  Quality goals:- The medical decision levels of interest are: 50 (hypoglycemia) 50 (hypoglycemia) 200 mg/dl (hyperglycemia) 200 mg/dl (hyperglycemia) The decision level for screening is 140mg/dl is not needed. The decision level for screening is 140mg/dl is not needed. Precision goals are 1.5 mg/dl at 50 mg/dl 5.0mg/dl at 200 mg/dl 5.0mg/dl at 200 mg/dl Total error goals (TEA) are 6.0 mg/dl at 50.0mg/dl 20.0mg/dl at 200.0 mg/dl 20.0mg/dl at 200.0 mg/dl

30.  Method selection :-  In kit form  Can be set up on an existing laboratory instrument  Requirements:-  Primary reference solutions are prepared for calibration  Control materials  Randomly selected clinical specimen.

31.  Within – Run Precision :  Analyze 20 aliquots of low abnormal control mean ±SD 56.5 mg/dl ± 0.7  Analyze 20 aliquots of moderatly high control mean ± SD 182.6 ±2.1 mg/dl  SDs are less than the allowable error in non automated procedure.

32.  Day to Day precision:  Two Q.C pools are analyzed for 20 days mean ± SD  SD :1.5 mg /50 mg glucose 5.0 mg/200mg glucose 5.0 mg/200mg glucose

33.  Analytical Range :-  Glucose solutions prepared from a stock glucose reference solution of 1000mg/dl  Duplicate analyses  The plot shows excellent linearity to 600mg/dl. which meets the specification for linearity to 500mg/dl.  The absorbance at zero glucose concentration is the reagent blank value

34. O.D O.D 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 200 400 600 800 0.0 200 400 600 800 Glucose concentration (mg/dl)

35.  Recovery:-  Two pooled sera are perpared as baseline specimens 61mg/dl,171mg/dl  concentrated glucose solution is prepared (10,000mg/dl)  two different amounts of glucose are added to 9.6 ml of pooled sera

36. 171mg/dl61mg/dlTotal Nacl 0.15mol/l Glucese solution 10,000mg/dl bbbbbb aaaaaa 10.0mL 400.0ul 300.0ul 0.0ul 100.0ul 400.0ul Recovery:-

37.  Quadruplicate assays of A and B are performed and take the mean  Subtract the original (basal) glucose levels (61,171) from the mean of each quadruplicate Recovered glucose Recovered glucose  Recovery % =  Proportional error = 100- recovery% Recovered glucose Glucose added X100

38. Glucose Recovered mg/dl Glucose found Glucose added Pooled sera mg/dlml/10ml --- 159-61=98 369 --- 97 391 61 159 457 171 268 562 0.0 100 400 0 100 400 0.0 100 400 0 100 400 AAABBBAAABBB Recovery:-

39.  Interference:  The potential interference of visible abnormal specimens is tested by: A analyzing a series of icteric, turbid,and hemolyzed sera using the new method and an established method know to be free of such interferenc

40.  The difference ( ) the two methods are 1 – 4 mg which is less than the allowable TEA. B add a small amount of highly concentrated solutions to pooled sera e.g. Ascorbic acid, sod. Salicylate, Trisodium citrate, Heparin, disod. EDTA. B add a small amount of highly concentrated solutions to pooled sera e.g. Ascorbic acid, sod. Salicylate, Trisodium citrate, Heparin, disod. EDTA.  The difference must be < TEA.

41. METHOD FOR GLUCOSEINTERFERENCE Test Comparative (mg/dl) Concentration Type ( Serum) 113±5.6 97±6.4 120±7.6 140±7.9 112±4.4 98±5.3 122±3.2 144±5.5 --- 6.5mg/dl 96mg/dl Turbid Normal Bil.(Icteric) Hb.(Hemo.) A 660 =0.45 Interference: