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- THIRUMURUGAN @ MURALI.P.
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.
Method selection
Definition of quality
goal
Method
Method
development
evaluation
Implementation
Routine
Submission of Quality control
analysis
specimen practices
Result
report
Reasons for Selecting a New Method
Trueness
Bias
Closeness of agreement of mean value
A measure of the systematic error
with true value
Precision
Imprecision (sd)
Repeatability (within run)
A measure of the dispersion of random
Intermediate precision (long term)
errors
Reproducibility (interlaboratory)
TEA = RE + SE
RE
SE
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
conditions.
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)
Components
1. sampling
2. pipetting precision
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),
and
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)
RE = t
Familiarization with the method
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
TEA = RE + SE
RE = 2.5%
SE = 7.5%
TEA = 10%
An example evaluation study: Cholesterol in serum.
Proceed to step 6.
An example evaluation study: Cholesterol in serum.
Mean sd
RE % 4 x RE%
(mmol/L) (mmol/L)
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
Pool
6.49 0.135 0.183 0.108 0.732 0.432
B
Sample 3 Pool A
Sample 4 Pool B
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)
[cholesterol]
mmol/L
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.
20
Method (Y) [cholesterol] mmol/L
Y = 0.9565 X + 0.3125
R = 0.9989
15
10
0
0 5 10 15 20
Theoretical (X) [cholesterol] mmol/L
An example evaluation study: Cholesterol in serum
XC1 XC2
XC1
XC2
Use of method decision chart.
A method with:
14
12
Test Method mmol/L
y = 1.0032x - 0.0233
10
R = 0.999
8
0
0 2 4 6 8 10 12 14
Comparative Method mmol/L
Bland - Altman Difference Plot
3
2
% Difference
1
0
-1 0 5 10 15
-2
-3
[Cholesterol] mmol/L
Interpretation of comparison of
methods study.