Method Selection and
Evaluation
D. Kefaya EL- Sayed Mohamed
Prof. Of Clinical Pathology (Clinical
Chemistry), Mansoura University
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.
Linear range:
O.D
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
200
400
600
800
Glucose concentration (mg/dl)
Observed value
True value
RE
X
µ
SE
TE
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 .
Test Values

30
Constant E
Ideal
Proprtional E
0
30
Reference Values
Inaccuracy
 Can be estimated from three studies:

recovery .

interference .

a comparison-of-methods study
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
Calcium measured
Sample 1
7.50mg/dl
Sample 2
8.35mg/dl
Sample 3
9.79mg/dl
added
0.95mg/dl
2.38mg/dl
Recovered Recovery
0.85mg/dl
2.29mg/dl
89%
96%
Calculation of recovery
Concentration added = standard concentration x Ml standard
Ml standard + ml serum
= 20 x 0.1 = 20x 1 = 20 = 0.95 (Sample2)
2+0.1
2.1x10 21
conc. recovered = conc. ( diluted test) – conc. ( baseline)
= 8.35-7.5=0.85
recovery = Conc. Recovered
= x100%
Conc. added
= 0.85 x 100 = 89 % ( sample 2)
0.95
( b )Interference:

The interference experiment is used to measure
systematic errors caused by substances other
than the analyte
Interference:



An interfering material can cause systematic
errors in one of two ways :
The material itself may react with the
analytical reagents.
Or it may alter the reaction between the
analyte and the analytical reagents

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
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
Sample 1
Sample 2
Sample 2
Calcium Measured
9.80mg/dl
10.53 mg/dl
11.48 mg/dl
Magnesium added
0.91mg/dl
1.81mg/dl
Interference
0.73mg/dl
1.68mg/dl
Calculation of interference
Concentration added = standard concentration x
ml standard
ml standard + ml serum
= 10x 0.1 = 10 = 0..91mg/dl (sample2)
1.1
11
Interference = conc. ( diluted ) – conc. ( baseline)
= 10.53 – 9.8 =0.73 (sample2)

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
( 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 .

Reference methods may be laborious and timeconsuming 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.




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
represent many different pathologic
conditions.
Duplicate analyses of each sample by each
method.

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.
Test method

Regression line
Reference
Example of Method selection and
evaluation
(Glucose in Serum)

Analytical Needs :-
Rapid procedure is needed at time out side the
regular working hours.


Sample Volume of 0.2 ml or less

Analytical range of 0.0 to 500.0 mg/dl.

Turn arround time 30 min.or less is needed

Quality goals:-
The medical decision levels of interest are:

50 (hypoglycemia)

200 mg/dl (hyperglycemia)

The decision level for screening is 140mg/dl is not needed.
Precision goals are
Total error goals (TEA) are
1.5 mg/dl at 50 mg/dl
5.0mg/dl at 200 mg/dl
6.0 mg/dl at 50.0mg/dl
20.0mg/dl at 200.0 mg/dl

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.



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 .

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

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
O.D
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
200
400
600
800
Glucose concentration (mg/dl)

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
Recovery:-
Glucese
solution
10,000mg/dl
0.0ul
100.0ul
400.0ul
Nacl
0.15mol/l
400.0ul
300.0ul
0.0ul
Total
61mg/dl
171mg/dl
10.0mL
10.0mL
10.0mL
a
a
a
b
b
b

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

Recovery % =

Recovered glucose
Glucose added
X100
Proportional error = 100- recovery%
Recovery:-
Glucose added
ml/10ml
mg/dl
Glucose
found
0.0
100
400
0
100
400
0.0
100
400
0
100
400
61
159
457
171
268
562
Pooled sera
A
A
A
B
B
B
Glucose
Recovered
mg/dl
--159-61=98
369
--97
391
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


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.

The difference must be < TEA.
Interference:
INTERFERENCE
METHOD FOR GLUCOSE
Type
( Serum)
Concentration
Comparative
(mg/dl)
Test
Normal
---
112±4.4
113±5.6
Bil.(Icteric)
6.5mg/dl
98±5.3
97±6.4
Hb.(Hemo.)
96mg/dl
122±3.2
120±7.6
Turbid
144±5.5
140±7.9
A660 =0.45