Method Selection and Method Selection and EvaluationEvaluation

Method Selection and Method Selection and EvaluationEvaluation

D. Kefaya EL- Sayed MohamedD. Kefaya EL- Sayed Mohamed

Prof. Of Clinical Pathology (Clinical Prof. Of Clinical Pathology (Clinical Chemistry), Mansoura UniversityChemistry), Mansoura University

Before a new test or methodogy is introduced into Before a new test or methodogy is introduced into the laboratory.the laboratory.

both managerial and techinal information must be both managerial and techinal information must be compiled and carefully considered. compiled and carefully considered.

The information should be collected from many The information should be collected from many different sources,including manufacturer different sources,including manufacturer

sales representatives,colleagues,scientific sales representatives,colleagues,scientific presentations,and the scientific literature.presentations,and the scientific literature.

Linear range:

O.DO.D 1.11.1 1.01.0 0.9 0.9 0.80.8 0.70.7 0.60.6 0.50.5 0.40.4 0.30.3 0.20.2 0.1 0.1 0.0 200 400 600 8000.0 200 400 600 800

Glucose concentration (mg/dl)Glucose concentration (mg/dl)

Observed value True valueObserved value True value

RE

SE

TE

µ X

InaccuracyInaccuracy:: The difference between a measured value and The difference between a measured value and

its true value. its true value. Is due to the presence of systematic analytical Is due to the presence of systematic analytical

error (S.E.)error (S.E.) S.E.S.E. can be constant or proportional . can be constant or proportional .

Test ValuesTest Values

Constant E

Ideal

Proprtional E

0

30

30

Reference Values

InaccuracyInaccuracy Can be estimated from three studies:Can be estimated from three studies: recovery .recovery . interference .interference . a comparison-of-methods studya comparison-of-methods study

Example of a recovery studyExample of a recovery studySample preparationSample preparationSample 1:2.0 ml serum + 0.1 ml H2OSample 1:2.0 ml serum + 0.1 ml H2OSample 2:2.0 ml serum +0.1 ml 20 mg /dl calcium standardSample 2:2.0 ml serum +0.1 ml 20 mg /dl calcium standardSample 3:2.0 ml serum +0.1 ml 50 mg /dl calcium standardSample 3:2.0 ml serum +0.1 ml 50 mg /dl calcium standard ConcentrationConcentration

Calcium measured added Recovered RecoveryCalcium measured added Recovered RecoverySample 1 7.50mg/dlSample 1 7.50mg/dlSample 2 8.35mg/dl 0.95mg/dl 0.85mg/dl 89%Sample 2 8.35mg/dl 0.95mg/dl 0.85mg/dl 89%Sample 3 9.79mg/dl 2.38mg/dl 2.29mg/dl 96%Sample 3 9.79mg/dl 2.38mg/dl 2.29mg/dl 96%

Calculation of recoveryCalculation of recoveryConcentration added = standard concentration x Ml standardConcentration added = standard concentration x Ml standard

Ml standard + ml serumMl standard + ml serum = = 2020 x 0.1 = 20x 1 = 20 = 0.95 (Sample2)x 0.1 = 20x 1 = 20 = 0.95 (Sample2)

2+0.12+0.1 2.1x10 212.1x10 21conc. recovered = conc. ( diluted test) – conc. ( baseline)conc. recovered = conc. ( diluted test) – conc. ( baseline)

= = 8.35-7.58.35-7.5==0.850.85 recovery = Conc. Recovered = x100%recovery = Conc. Recovered = x100%

Conc. addedConc. added

= = 0.850.85 x 100 = 89 % ( sample 2)x 100 = 89 % ( sample 2) 0.950.95

( (bb) ) InterferenceInterference::

The interference experiment is used to The interference experiment is used to measure systematic errors caused by measure systematic errors caused by substances other than the analyte substances other than the analyte

Interference:Interference: An interfering material can cause systematic An interfering material can cause systematic

errors in one of two ways :errors in one of two ways : The material itself may react with the The material itself may react with the

analytical reagents.analytical reagents. Or it may alter the reaction between the Or it may alter the reaction between the

analyte and the analytical reagentsanalyte and the analytical reagents

Interference:Interference: The interference likes recovery except that the The interference likes recovery except that the

substance suspected of interference is added to the substance suspected of interference is added to the patient sample.patient sample.

The concentration of the potentially interfering The concentration of the potentially interfering material should be in the maximally elevated range.material should be in the maximally elevated range.

If an effect is observed its concentration should be If an effect is observed its concentration should be

lowered to discover the concentration at which test lowered to discover the concentration at which test resultes are frist invalidatedresultes are frist invalidated

Example of an Interference studyExample of an Interference study

Sample preparationSample preparation

Sample 1 : 1.0 ml serum + 0.1 ml H2O (base line)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 standardSample 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 standardSample 3 : 1.0 ml serum + 0.1 ml of 20 mg/dl magnesium standard

Calcium Measured Magnesium added InterferenceCalcium Measured Magnesium added Interference

Sample 1 9.80mg/dlSample 1 9.80mg/dl

Sample 2 10.53 mg/dl 0.91mg/dl 0.73mg/dlSample 2 10.53 mg/dl 0.91mg/dl 0.73mg/dl

Sample 2 11.48 mg/dl 1.81mg/dl 1.68mg/dlSample 2 11.48 mg/dl 1.81mg/dl 1.68mg/dl

Calculation of interferenceCalculation of interferenceConcentration added = standard concentration xConcentration added = standard concentration x

= 10x 0.1 = 10 = 0..91mg/dl (sample2)= 10x 0.1 = 10 = 0..91mg/dl (sample2)

1.1 111.1 11

Interference = conc. ( diluted ) – conc. ( baseline)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

Common interferences (eg., hemoglobin, Common interferences (eg., hemoglobin, lipids,bilirubin,anticoagulants preservatives, lipids,bilirubin,anticoagulants preservatives, and so on)also should be tested.and so on)also should be tested.

Glick and Ryder have presented " Glick and Ryder have presented " interferographs " for various chemistry interferographs " for various chemistry instruments – these are graphs relating analyte instruments – these are graphs relating analyte concentration measured versus interferent concentration measured versus interferent concentrationconcentration

( (CC ) ) comparison – of methouds comparison – of methouds ExperimentExperiment::

The best comparative method that can be used The best comparative method that can be used is the reference method , which is a method is the reference method , which is a method with negligible inaccuracy in comparison with with negligible inaccuracy in comparison with its imprecisionits imprecision . .

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

Westgard et al27 and the NCCLS35 Westgard et al27 and the NCCLS35 recommend that at least 40 sample and recommend that at least 40 sample and preferably 100 samples, be run by botli preferably 100 samples, be run by botli methods.methods.

span the clinical range span the clinical range represent many different pathologic represent many different pathologic

conditions. conditions. Duplicate analyses of each sample by each Duplicate analyses of each sample by each

method.method.

If 40 specimens are compared , two to five If 40 specimens are compared , two to five patient specimens should be analyzed daily for patient specimens should be analyzed daily for a minimum of 8 days.a minimum of 8 days.

If 100 specimens are compared the comparison If 100 specimens are compared the comparison study should be carried out during the 20-day study should be carried out during the 20-day replication study.replication study.

Test methodTest method

Reference

Regression line

Example of Method selection and Example of Method selection and evaluationevaluation

(Glucose in Serum) (Glucose in Serum)Analytical Needs :-Analytical Needs :-

Rapid procedure is needed at time out side the Rapid procedure is needed at time out side the regular working hours.regular working hours.

Sample Volume of 0.2 ml or lessSample 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 neededTurn arround time 30 min.or less is needed

Quality goals:-Quality goals:-

The medical decision levels of interest are: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/dlPrecision goals are 1.5 mg/dl at 50 mg/dl

5.0mg/dl at 200 mg/dl5.0mg/dl at 200 mg/dl

Total error goals (TEA) are 6.0 mg/dl at 50.0mg/dlTotal error goals (TEA) are 6.0 mg/dl at 50.0mg/dl

20.0mg/dl at 200.0 mg/dl20.0mg/dl at 200.0 mg/dl

Method selection :-Method selection :-

In kit formIn kit form Can be set up on an existing laboratory instrumentCan be set up on an existing laboratory instrument

Requirements:-Requirements:- Primary reference solutions are prepared for Primary reference solutions are prepared for

calibrationcalibration Control materialsControl materials Randomly selected clinical specimen.Randomly selected clinical specimen.

Within – Run Precision :Within – Run Precision :

Analyze 20 aliquots of low abnormal control mean Analyze 20 aliquots of low abnormal control mean ±SD 56.5 mg/dl ± 0.7±SD 56.5 mg/dl ± 0.7

Analyze 20 aliquots of moderatly high control mean Analyze 20 aliquots of moderatly high control mean ± SD 182.6 ±2.1 mg/dl± SD 182.6 ±2.1 mg/dl

SDs are less than the allowable error in non SDs are less than the allowable error in non automated procedure .automated procedure .

Day to Day precision:Day to Day precision:

Two Q.C pools are analyzed for 20 days mean ± SDTwo Q.C pools are analyzed for 20 days mean ± SD

SD :1.5 mg /50 mg glucoseSD :1.5 mg /50 mg glucose

5.0 mg/200mg glucose5.0 mg/200mg glucose

Analytical Range :-Analytical Range :-

Glucose solutions prepared from a stock glucose Glucose solutions prepared from a stock glucose reference solution of 1000mg/dlreference solution of 1000mg/dl

Duplicate analysesDuplicate analyses

The plot shows excellent linearity to 600mg/dl. which The plot shows excellent linearity to 600mg/dl. which meets the specification for linearity to 500mg/dl.meets the specification for linearity to 500mg/dl.

The absorbance at zero glucose concentration is the The absorbance at zero glucose concentration is the reagent blank value reagent blank value

O.DO.D 1.11.1 1.01.0 0.9 0.9 0.80.8 0.70.7 0.60.6 0.50.5 0.40.4 0.30.3 0.20.2 0.1 0.1 0.0 200 400 600 8000.0 200 400 600 800

Glucose concentration (mg/dl)Glucose concentration (mg/dl)

Recovery:-Recovery:-

Two pooled sera are perpared as baseline specimens Two pooled sera are perpared as baseline specimens 61mg/dl ,171mg/dl61mg/dl ,171mg/dl

concentrated glucose solution is prepared (10,000mg/dl)concentrated glucose solution is prepared (10,000mg/dl)

two different amounts of glucose are added to 9.6 ml of two different amounts of glucose are added to 9.6 ml of pooled serapooled sera

Glucese solution

10,000mg/dl

Nacl 0.15mol/l

Total61mg/dl171mg/dl

0.0ul100.0ul400.0ul

400.0ul300.0ul

0.0ul

10.0mL10.0mL10.0mL

aaa

bbb

RecoveryRecovery-:-:

Quadruplicate assays of A and B are performed and Quadruplicate assays of A and B are performed and take the meantake the mean

Subtract the original (basal) glucose levels (61,171) Subtract the original (basal) glucose levels (61,171) from the mean of each quadruplicatefrom the mean of each quadruplicate

Recovered glucoseRecovered glucose

Recovery % =Recovery % =

Proportional error = 100- recovery%Proportional error = 100- recovery%

Recovered glucoseGlucose added X100

Pooled sera

Glucose added

Glucose found

Glucose Recovered

mg/dlml/10mlmg/dl

AAABBB

0.0100400

0100400

0.0100400

0100400

61159457171268562

---159-61=98

369---97

391

RecoveryRecovery-:-:

Interference:Interference: The potential interference of visible abnormal The potential interference of visible abnormal

specimens is tested byspecimens is tested by: :

AA analyzing a series of icteric , turbid,and analyzing a series of icteric , turbid,and hemolyzed sera using the new method and an hemolyzed sera using the new method and an established method know to be free of such established method know to be free of such interferenc interferenc

The difference ( ) the two methods are 1 – The difference ( ) the two methods are 1 – 4 mg which is less than the allowable TEA.4 mg which is less than the allowable TEA.

B add a small amount of highly concentrated B add a small amount of highly concentrated solutions to pooled sera e.g. Ascorbic acid , solutions to pooled sera e.g. Ascorbic acid , sod. Salicylate, Trisodium sod. Salicylate, Trisodium citrate, Heparin, disod. EDTA.citrate, Heparin, disod. EDTA.

The difference must be < TEA.The difference must be < TEA.

INTERFERENCEMETHOD FOR GLUCOSE

Type( Serum)

ConcentrationComparative

(mg/dl)Test

Normal

Bil.(Icteric)

Hb.(Hemo.)

A660 =0.45

---

6.5mg/dl

96mg/dl

Turbid

112±4.4

98±5.3

122±3.2

144±5.5

113±5.6

97±6.4

120±7.6

140±7.9

InterferenceInterference::