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Method Validation 10 03

This document discusses method validation for a new chemistry analyzer. It explains that method validation involves assessing analytical errors through experiments to determine imprecision, bias, linearity, interferences, and detection limits. The results are compared to clinical laboratory improvement amendments (CLIA) quality requirements to determine if the new method is acceptable. Key aspects of method validation include replication studies to assess imprecision, method comparison studies to detect bias, and interference, recovery, and detection limit experiments.

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CARDENAS GONZALES LUIS ALBERTO
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96 views30 pages

Method Validation 10 03

This document discusses method validation for a new chemistry analyzer. It explains that method validation involves assessing analytical errors through experiments to determine imprecision, bias, linearity, interferences, and detection limits. The results are compared to clinical laboratory improvement amendments (CLIA) quality requirements to determine if the new method is acceptable. Key aspects of method validation include replication studies to assess imprecision, method comparison studies to detect bias, and interference, recovery, and detection limit experiments.

Uploaded by

CARDENAS GONZALES LUIS ALBERTO
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Method validation

Yash Pal Agrawal


10/15/03
Further Reading: Basic method validation: Training in analytical
quality management for healthcare laboratories by James O
Westgard (copy available at the VA)
Scenario
• As lab.director you are testing a new
chemistry analyzer. You have done all the
tests, statistics etc which the med.tech gives
you in a neat 100 page folder.
• Is the new analyzer (method) acceptable ?
How to know that the new
method/analyzer is acceptable ?
• Answer: ERROR ASSESSMENT.
• Amount of error won’t affect interpretation
of the test result and compromise patient
care
Correlation between 2 methods
• Cholesterol assays
R=0.999
• Intercept 15 mg/dL
• Information about the

New Method
size of the analytical
error is more useful

Old method
ERROR ASSESSMENT
• What kind of analytical errors may occur?
• What expts. provide data about those errors
• How to perform those experiments ?
• How much data to collect to assess errors ?
• What statistics provides best estimates of
errors ?
• What size errors are allowable ?
Method Validation Regulations
http://cms.hhs.gov/clia/default.asp
• Waived tests:Follow manufacturer’s instr.
• Unmodified mod-high complexity:
– 1. Replication -Æ imprecision (Random error)
– 2. Comparison of methods -Æ Bias (Systematic
Error)
– 3. Linearity -Æ reportable range
– 4. Reference range: own or text
Method Validation Regulations
http://cms.hhs.gov/clia/default.asp
• Modified moderate-high complexity:
– Previous 4 requirements PLUS
– 5. Detection Limit: -Æ analytical sensitivity
– 6. Interference:Æ constant interferences
– 7. Recovery: Æ proportional interferences
CLIA quality requirements
• Absolute concentration limits: target value
± 1 mg/dL for Ca++
• As a %: target value ± 10 % for albumin
• As a distribution of a survey group: Target
value ± 3SD for TSH
Linearity/Reportable range
• Lowest and highest
test results that can be
reported
• Draw line manually or
by linear regression
• Check 4-5 levels in
triplicates
Types of analytical errors
• 1. Random error or imprecision
• 2. Systematic error or inaccuracy
– Constant error
– Proportional error
• 3. Total error: Random + Systematic
1. Random Error (imprecision)
Observed
• Replicate measurements mean
on single sample
• Error which is positive or
negative
• Direction/magnitude
cannot be predicted
• SD and CV of a set of
results express the random
error
235 265
2. Systematic error or inaccuracy
True value
• Systematic error
always in one
direction-Æ causes
test results to be high
or low
• How high or low =
Bias
235 265
• Two types: Constant
or proportional error Bias
3. Total Error
True value
Observed value
• RE +SE=Total Error
• A single measurement
can be in error by the
expected bias + 2SD
(random error)
235 265

Bias RE

Total Error
Errors
Do replicates
Interference (within/between run)
study
Proportional
Constant RE SE
SE
New Method

RE

Recovery
study
Old method
Determination of Random error
• Test results on 20 samples of the same material
• Time factor: within run, within day, between days
• Sample Matrix (blood, urine, CSF): Standards,
control, pools
• Test at medical decision levels
• Short term: sw-run or sw-day < 0.25 Tea
• Long term: S tot < 0.33 Tea
• E.g. ALT target value ± 20 %
Determination of Bias/inaccuracy
• Comparison of methods experiment
• Minimum 40 patient samples that cover
reportable range
• Preferably duplicates, time period factors
• Sample stability
• Plot as “difference plot” if 1:1 agreement
expected or as “comparison plot” if not
Determination of Bias/inaccuracy
• Which statistics to use:
– Linear regression : when wide analytical range.
E.g. glucose
– T-test: narrow analytical range, e.g. K
Determination of Bias/inaccuracy
• Linear Regression:
– Yc = a + bXc, slope=b, intercept = a
– Y= 2.0 + 1.03X, thus Y value for a decision
value of Chol at 200 mg/dL is = 208
– The systematic error at level of 200 mg/dL is (2
+ 1.03 * 200)=208, minus 200 =8
Difference Plot
Difference (Test-Comparative method)

Zero
line

Comparative method result


Determination of Bias/inaccuracy
• Paired t-test. Also provides SD of the
differences as a t-value (i.e. is there a
difference or bias between the 2 methods)
• E.g. n=40, Na = 141 mmol/L and 138.5
mmol/L by other method. Bias = 2.5
mmol/L
Determination of Interference
Add interference
• To estimate constant Add water
systematic error
• Interferences such as
bilirubin, hemolysis,
lipemia
water
Analyte, e.g. glucose
6 glucose _ 6 glucose
Interference, e.g. lipemia
Zero bias
Determination of Recovery
• Used less frequently Add analyte Add water
• To estimate
proportional SE
• Caused by a substance
in sample matrix that
reacts with analyte and
competes with
analytical reagents (10 G-6G/4G) * 100 = 100 %
recovery
Determination of Detection limit
Blank Spiked
• To estimate the lowest sample sample
conc. of analyte that
can be measured
• Blank solution= zero
conc. of analyte
• Spiked solution = low
conc. of analyte
Types of detection limits
LLD BLD FS
• Lower limit of
detection, LLD
• Biologic limit of
detection, BLD
• Functional sensitivity,
FS
Measurement value
Zero or
blank Spiked samples
Determination of LLD
LLD BLD FS
• Lower limit of
detection
• Mean of the blank +
2 SD
• LLD=mean blk + Zs blk
where Z= 2 Measurement value
Zero or
blank Spiked samples
Determination of BLD
LLD BLD FS
• Biologic limit of
detection, BLD
• LLD + 2 SD of a
spiked sample whose
conc. Approximates
the detection limit
Measurement value
• Represents real world
Zero or
blank Spiked samples
Determination of FS
LLD BLD FS
• Functional Sensitivity,
FS
• Mean conc. of spiked
sample whose cv is 20
%
• Many samples need to
Measurement value
be run
Zero or
blank Spiked samples
Transference of reference
intervals
• IFCC gives formal protocols, n=120 for
each sub group
• From literature: Divine judgement
• Verified with 20 samples, if 2 or fewer
results fall outside the reported limits
• Verify with 40-60 samples: compare mean
and SD with reported ref. values
• Calculation from comparative method
Method validation keys
• Linearity/reportable range
• Replication experiment
• Comparison of methods experiment
• Interference, recovery and detection limits
• Method performance compared with CLIA
recommendations
Further reading
• Basic method validation: Training in
analytical quality management for
healthcare laboratories

By James O Westgard

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