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Physicists have a long tradition of building their own
equipment, and are often fascinated by its
mechanics. Biologists fascination is primarily with
the mechanics of nature and, for many, the
machines themselves are simply tools
complicated black boxes that produce the results
they need. It doesnt help that the tools biologists
are using may have been designed by physicists,
and that the two groups tend to use different jargon.

Nature 2007; 447: 116

Quantitative analytical methods have become more
reliable and more standardized.
Emphasis moved away from methods development to
the selection and evaluation of those commercial
available methods that suit a particular laboratory best.
Commercial kit methods are ready for implementation in
the laboratory, often in a closed analytical system on a
dedicated instrument.
Furthermore, method evaluation is a costly exercise in
terms of reagents, specimens, and labour and time of
the professionals doing the evaluating.
If not done properly it wastes laboratory revenue and
time, if the method is accepted might lead to errors in
medical decisions based on results the method
generates on patient samples.
Generally what happens is that
laboratories are most concerned with
getting the methods up and running that
there is little time, or thought given, to
selection and evaluation studies.
The most common scenario is the
implementation of readily available commercial
kit methods, often in a closed analytical system
on a dedicated instrument.

When a new clinical analyzer is included in the

overall evaluation process, various instrumental
parameters also require evaluation. Information
on most of these parameters should be available
from the instrument manufacturer, who should
also be able to furnish information on what user
studies to conduct in estimating these
parameters for an individual analyzer.
Establish need

Method selection

Definition of quality



Submission of Quality control
specimen practices

Reasons for Selecting a New Method

improve accuracy and / or precision over

existing methods
to reduce reagent cost
to reduce labour cost
new analyzer or instrument
to measure a new analyte
Evaluation of need
Application characteristics
Method characteristics
Analytical performance characteristics
Scopes of Method Evaluation

Evaluation is the determination of the analytical performance

characteristics of a new method.

Validation is confirmation by examination and provision of

objective evidence that the particular requirements for a specific
intended use can be consistently fulfilled.

Verification is confirmation by examination of objective evidence

that specified requirements have been fulfilled.

Demonstration is a minimum evaluation for a laboratory to use

to show that it is able to obtain expected results by following the
manufacturers instructions. This is appropriate for test systems
whose performance characteristics have been well studied and
Method Evaluation and Validation
Main purpose is error assessment.
To demonstrate that prior to reporting patient
test results, it can obtain the performance
specifications for accuracy, precision, and
reportable range of patient test results,
comparable to those established by the
The laboratory must also verify that the
manufacturers reference range is appropriate
for laboratorys population.
An Overview of Qualitative Terms and Quantitative Measures Related
to Method Performance
Qualitative Concept Quantitative Measure

Closeness of agreement of mean value
A measure of the systematic error
with true value

Imprecision (sd)
Repeatability (within run)
A measure of the dispersion of random
Intermediate precision (long term)
Reproducibility (interlaboratory)

Accuracy Error of measurement

Closeness of agreement of a single Comprises both random and systematic
measurement with true value influences
Total Analytical error TEA.




Constant and proportional errors.
Analytical Sensitivity
Several terms describe the different aspects of the
minimum analytical sensitivity of a method.
Limit of absence (LoA) is the lowest concentration of
analyte that the method can differentiate from zero.
Limit of detection (LoD) is the minimum concentration
of analyte whose presence can be quantitatively
detected under defined conditions.
Functional sensitivity or limit of quantification (LoQ)
is the minimum concentration of analyte whose presence
can be quantitatively measured reliably under defined
The concentration at which the CV = 20%.
Illustration of different aspects of analytical sensitivity
or detection limits.
Random Analytical Error (RE)
Factors contributing to random analytical error (RE) are those
that affect the reproducibility of measurement. These include:
instability of the instrument,
variations in the temperature,
variations in the reagents and calibrators (and calibration-
curve stability),
variability in handling techniques such as pipetting, mixing,
and timing, and
variability in operators.
These factors superimpose their effects on each other at
different times. Some cause rapid fluctuations, and others
occur over a longer time. Thus RE has different components
of variation that are related to the actual laboratory setting.
Random Analytical Error (RE)

Within-run component of variation (wr)

Within-day, between-run variation (br)

Between-day component of variation (bd)

Within-run component of variation (wr)

is caused by specific steps in the procedure:

1. sampling

2. pipetting precision

3. short-term variations in temperature and

4. stability of the instrument.

Within-day, between-run variation (br)

is caused by:
1. instability of calibration curve
2. differences in recalibration that occur
throughout the day,
3. longer term variations in the instrument,
4. small changes in the condition of the
calibrator and reagents,
5. changes in the condition of the laboratory
during the day, and
6. fatigue of the laboratory staff.
Between-day component of variation (bd)
is caused by:
1. daily variations in the instrument,
2. changes in calibrators and reagents
(especially if new vials are opened each day),
3. changes in staff from day to day.
4. Although not a true random component of
variation, any drift in the stability of the
calibration curve over time greatly affects the
bd as well.
Total Variance of a Method (t2)

t2 = wr2 + br2 + bd2

RE = t
Familiarization with the method

It is essential that operators of the method become

thoroughly familiar with the details of the method and
instrument operation before the collection of any data
that will be used to characterize the methods
May include training by the manufacturer.
It should be of sufficient duration that, at its completion,
the operators can perform all aspects of the method or
instrument operation comfortably.
Experiments for Estimating Analytical Errors
The importance of daily examination and plotting of
comparison-of-method data cannot be over emphasized,
and the data must be carefully examined for outliers.

Definition of an outlier from a regression line:

| yi Yi| > 4sx,y

Outlier specimens must be detected immediately and
reanalyzed by both methods so that the data can correct
or confirm the outlier.
An example evaluation study: Cholesterol in serum.

Step 1: Analytical needs

Rapid procedure with a turnaround time of 30 min
suitable for lipid clinic requirement. Short turnaround
time means that patients do not have to come back for
treatment based on lipid-profile results.
A sample volume of 200 L.
Analytical range of 0 to 20 mmol/L.
High through-put.
Analytical goals
An example evaluation study: Cholesterol in serum.
Analytical Goals

Analyte Acceptable Decision level Allowable Maximum Medically based

performance XC error sd maximum sd
criteria (CLIA 88) (CLIA 88) (Fraser)
(CLIA 88) (CV%) (CV%)
Albumin 10% 35 g/L 3.5 0.9 (2.6%) 0.5 (1.43%)
Cholesterol 10% 5.2 mmol/L 0.52 0.13 (2.5%) 0.14 (2.7%)
88 mol/L 26 7.0 (8%) 1.8 (2.0%)
Creatinine 15%
265 mol/L 40 9.7 (3.7%) 6.2 (2.3%)
0.08 (2.9%)
2.75 mmol/L 0.33 0.06 (2.2%)
0.18 (2.6%)
Glucose 10% 6.9 mmol/L 0.69 0.15 (2.2%)
11.0 mmol/L 1.10 0.24 (2.2%)
Hb A1C 7.0% 0.35% 0.14%
3.0 mmol/L 0.50 0.12 (4%) 0.07 (2.33%)
K 0.5 mmol/L
6.0 mmol/L 0.50 0.12 (2%) 0.14 (2.33%)
ALP 30% 150 U/L 45 11 (7.3%) 5.1 (3.4%)
CK 30% 200 U/L 60 15 (7.5%) 40 (20%)
An example evaluation study: Cholesterol in serum.

Step 2: Quality goals

Medical decision (XC) levels of interest for cholesterol analysis
are taken as 4.5 mmol/L; levels below this indicate low risk of
CVD, and 6.0 mmol/L; high risk, levels above this should be
actively treated with cholesterol lowering drugs, respectively.

Precision goals for cholesterol are defined to be 0.12 mmol/L at

4.5 mmol/L and 0.15 mmol/L at 6.0 mmol/L (2.5%).

Total error goals (TEA) are 0.45 mmol/L at 4.5 mmol/L and 0.60
mmol/L at 6.0 mmol/L (10%).
Total Analytical error. (TEA)

For Cholesterol

10% = 2.5% + 7.5%

RE = 2.5%

SE = 7.5%

TEA = 10%
An example evaluation study: Cholesterol in serum.

Step 3: Method selection

Existing laboratory analyzer Beckman-Coulter LX20


Cholesterol kit specifically designed for this analyzer.

Senior operator who is familiar with this particular analyzer

and is available to do the evaluation.
An example evaluation study: Cholesterol in serum.

Step 4: Test material selection

Synchron 1: mean [cholesterol] 2.71 mmol/L,
Synchron 2: mean [cholesterol] 4.19 mmol/L, and
Synchron 3: mean [cholesterol] 5.82 mmol/L.

Pooled patient serum two levels A and B matrix closest to

real patient serum.

20 Patient serum samples to be run in parallel with existing

laboratory method.
An example evaluation study: Cholesterol in serum.

Step 5: Within-run imprecision

Performed by analyzing 6 aliquots of Synchron 1, 2, and
3 and Pool A and B within a run.

Mean (mmol/L) sd (mmol/L) RE %

Synchron 1 2.69 0.028 1.04

Synchron 2 4.21 0.042 1.00
Synchron 3 5.80 0.073 1.26
Pool A 4.89 0.057 1.17
Pool B 6.54 0.109 1.67
An example evaluation study: Cholesterol in serum.

Step 5a: Within-run imprecision

Testing for acceptable performance
RE against Maximum allowable CV%

CLIA 88: 2.5% > synchron 1: 1.04% < Fraser: 2.7%

CLIA 88: 2.5% > synchron 2: 1.00% < Fraser: 2.7%
CLIA 88: 2.5% > synchron 3: 1. 26% < Fraser: 2.7%
CLIA 88: 2.5% > pool A: 1.17% < Fraser: 2.7%
CLIA 88: 2.5% > pool B: 1.67% < Fraser: 2.7%

proceed with step 5b

An example evaluation study: Cholesterol in serum
Step 5b: Within-run imprecision
Testing for acceptable performance
RE against TEA

If 4 x RE > TEA reject method

If 4 x RE < TEA proceed with step 6

With the TEA = 10% for cholesterol, the within-run imprecision

of synchron 1, 2, 3 and pool A and B each passes the test.

Proceed to step 6.
An example evaluation study: Cholesterol in serum.

Step 6: Between-run (day-to-day) precision

Performed by analyzing aliquots of pool A and B for 20 days

Mean sd
RE % 4 x RE%
(mmol/L) (mmol/L)

Pool A 4.93 0.098 1.99 < 2.5 7.96 < 10

Pool B 6.49 0.135 2.08 < 2.5 8.32 < 10

An example evaluation study: Cholesterol in serum
Step 7: SD has confidence intervals
Factors for computing one-sided confidence intervals
for standard deviation.

Degrees of
A0.05 A0.95
freedom (N 1)

1 0.5103 15.947
5 0.6721 2.089
10 0.7391 1.593
15 0.7747 1.437
20 0.7979 1.358
An example evaluation study: Cholesterol in serum
Step 7: Confidence-interval estimate of random error REU
and REL ; N = 20

Mean sd sdU= sdL= REU= REL=

(mmol/L) (mmol/L) sd x A.95 sd x A.05 4 x sdU 4 x sdL
4.93 0.098 0.133 0.078 0.532 0.312

6.49 0.135 0.183 0.108 0.732 0.432

REU pool A > 0.493 and REU pool B > 0.649

An example evaluation study: Cholesterol in serum
Step 8: Validation of linearity or reportable range
Obtained pool C by combining all serum samples with
[cholesterol] > 15 mmol/L.
Prepared the following samples:

Sample 1 Special prepared with [cholesterol] 0

Sample 2 3 parts sample 1 + 1 part pool A

Sample 3 Pool A

Sample 4 Pool B

Sample 5 2 parts sample 1 + 2 parts pool C

Sample 6 Pool C
An example evaluation study: Cholesterol in serum
Step 8: Validation of linearity or reportable range
Pools analyzed by Kendal-Abell method (reference method)


Pool A 4.88

Pool B 6.52

Pool C 16.7
An example evaluation study: Cholesterol in serum
Step 8: Validation of linearity or reportable range
Samples 1, 2, 3, 4, 5, and 6 were analyzed in triplicate in a single run in
random order.

Theoretical (X) Mean (Y) Bias (%)

Sample 1 0 0.035 +0.035 (N/A)

Sample 2 1.22 1.967 -0.024 (-2.0)

Sample 3 4.88 4.846 -0.034 (-0.7)

Sample 4 6.52 6.47 -0.05 (-0.77)

Sample 5 8.09 7.99 -0.1 (-1.24)

Sample 6 16.7 16.35 -0.35 (-2.1)

Reportable Range of Serum-[cholesterol]

Method (Y) [cholesterol] mmol/L

Y = 0.9565 X + 0.3125
R = 0.9989


0 5 10 15 20
Theoretical (X) [cholesterol] mmol/L
An example evaluation study: Cholesterol in serum

Step 9: Estimation of SE from the linearity study which is a

comparison of the method against reference method.
The following statistics were obtained by linear
regression analysis:

Y = 0.956 X + 0.313 mmol/L SY,X = 0.294

Mean X = 6.235 Mean Y = 6.276

Bias = | mean Y mean X| = 0.041 mmol/L

This is the estimate of SE at the mean of the data.

An example evaluation study: Cholesterol in serum

Step 9: Point estimate of SE at medical decision levels (XC).

For XC = 4.5 mmol/L, YC = 4.615 mmol/L
SE1 = | YC XC | = 0.115 mmol/L
Because SE1 < TEA = 0.45 mmol/L,
SE1 is acceptable.

For XC = 6.0 mmol/L, YC = 6.049 mmol/L

SE2 = | YC XC | = 0.049 mmol/L
Because SE2 < TEA = 0.6 mmol/L,
SE2 is acceptable
An example evaluation study: Cholesterol in serum
Step 10: Point estimate of TE
Criteria for acceptable performance:
TEA > TE = 3 x sd + | YC XC |
For XC1 = 4.5 mmol/L, YC1 = 4.615 mmol/L and sd = 0.098
TE1 = 3 x 0.098 + 0.115 = 0.409 mmol/L < 0.45 mmol/L
Performance acceptable

For XC2 = 6.0 mmol/L, YC2 = 6.049 mmol/L and sd = 0.135

TE2 = 3 x 0.135 + 0.049 = 0.454 mmol/L < 0.6 mmol/L
Performance acceptable
An example evaluation study: Cholesterol in serum
Step 11: Medical decision chart


Level mmol/L 4.5 6.0

TEA mmol/L 0.45 0.60
SE mmol/L 0.115 0.049
RE mmol/L 0.098 0.135
RE as % of TEA 21.8 22.5
SE as % of TEA 25.6 8.2
Medical decision Chart



Use of method decision chart.
A method with:

1. Unacceptable performance does not meet the requirement for

quality, even when the method is working properly. Not acceptable
for routine operation.

2. Marginal performance provides the desired quality when everything

is working correctly. But, difficult to manage in routine operation,
requires total QC strategy, well-trained operators, aggressive
preventive maintenance, etc.

3. Good performance meets requirement for quality and can be well-

managed in routine service. Requires multirule procedure with 4-6
control measurements per run.

4. Six sigma or excellent performance is clearly acceptable and easy

to manage in routine service and can be controlled
A comparison of methods experiment is performed to estimate
inaccuracy or systematic error.

This performed by analyzing patient samples by the new method

(test method) and a comparative method, then estimate the
systematic errors (SE) on the basis of differences observed
between the methods.

The systematic differences at the critical medical decision

concentrations are the errors of interest.

When possible, a reference method should be chosen for the

comparative method.

Any differences between a test method and a reference method

are assigned to the test method.
Cholesterol Methods Comparison Plot. N = 20


Test Method mmol/L

y = 1.0032x - 0.0233
R = 0.999

0 2 4 6 8 10 12 14
Comparative Method mmol/L
Bland - Altman Difference Plot

% Difference

-1 0 5 10 15

[Cholesterol] mmol/L
Interpretation of comparison of
methods study.

The differences are relatively small, not more than

2.2% across the concentration range of 2.0
15.0 mmol/L.

The two methods have the same relative accuracy.

The can be substituted for the other.

Recommended Minimum Studies for comparison
of methods experiment.

1. Select 40 patient specimens to cover the full working

range of the method.
2. Analyze 8 specimens a day within 2 hours by the test
and comparative methods.
3. Graph results immediately on a difference plot and
inspect for discrepancies.
4. Reanalyze specimens that give discrepant results.
5. Continue the experiment for 5 days if no discrepant
results are observed.
Recommended Minimum Studies for comparison of
methods experiment.
6. Continue for another 5 days if discrepancies are
observed during the first 5 days.

7. Prepare a comparison plot of all the data to assess the

range, outliers, and linearity.

8. Calculate the correlation coefficient and if 0.99 or

greater, calculate simple linear regression statistics and
estimate the systematic error at medical decision

9. Use the medical decision chart to combine the estimates

of SE and RE and make judgment on the total error
observed for the method.
NATURE, 18 September 2003
Monkeys reject unequal pay.
- Sarah Brosnan and Frans de Waal
Working for peanuts.
- Paul Smaglik