validation of laboratory methods with important guidelines

1. VALIDATION OF LABORATORY TEST
DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY
Presenter: Daniel Mwandu
Facilitator: Dr. Doreen Komori
Date: 22th Feb, 2024
Venue: Microbiology Lab

2. Presentation Outline
• Introduction
• Important of methods validation
• Pre- validation
• Methods validation parameters
• Post validation monitoring

3. INTRODUCTION
• Validation is the process of testing a
measurement procedures to asses it performance
if inline with acceptance criteria.
• Laboratory method validation is a process by
which a method is determined to meet the
requirement for the intended use.
• It ensure accuracy, precision and reliable results
• It help to determine the magnitude of error.
• It ensure good customers services

5. ISO 15189:2022 , Clause 7.3.3 The laboratory
shall validate the following
• Non standard methods
• Laboratory designed/ developed methods
• Standard methods used outside their intended.
• Validated methods subsequently modified

6. Reason for establishment of new methods
• Clinical need of new analyte
• Improve diagnosis and treatment due to
short TAT.
• Improve accuracy and precision
• Reduce cost

7. • Clause 6.4.3. state that “ the laboratory shall
verify the equipment's/methods to conforms the
specified acceptability criteria.
• Clause 6.4.4. state that “each equipment/ test
method should be used as specified by
manufacturer, unless validated by laboratory.
• Clause 7.3.2.verification
• Clause 7.3.3 validation

8. 3/4/2024 8
Performance characteristics
VALIDATION
Performed for inhouse/modified
methods
It normally it done by manufacturer
to establish the acceptance criteria
VERIFICATION
Confirm the performance claims
by manufacturer

9. But I’m using an FDA approved kit
3/4/2024 9
FDA
APPROVED
Conduct verification only
Perform validation if some of the
parameters were not validated
Non -APPROVED
Conduct validation and
verification

10. Tests to validate
• Waived test(simple test approved by FDA) i.e.
UPT – No need of validation
• Non-waived test(complex test) validation is
needed i.e. Stain techniques.
• Unmodified FDA approved- no need of validation
• Modified /non-FDA approved need validation

11. Important of methods validation
• It ensure accuracy and reliable results
• Understanding of uncertainty in the
methods.
• Provide confidence in the method
through measuring robustness and
ruggedness

12. Pre- validation
Issue to consider prior validation.
Methods purpose and scope
Regulatory guidelines eg SOPs
Method development
Parameter acceptance criteria
A calibrated equipment to be validated.
Materials: eg reagents, reference standard

13. Methods validation parameters
Measurement
type
Description
01 Trueness Reference intervals
02 Accuracy measure the closeness of the test results to the
true value
03 Precision Repeatability, measure degree of agreement
04 Linearity
(Reportable
range)
Ability to produce results, that are directly
proportional to the analyte concentration within a
specified range
05 Uncertainty Measure Standard deviation.
06 Sensitivity Detection
07 Specificity Ensure selectivity, without interference

14. Methods validation parameters
Measurement
type
Formula
01 Trueness Reference interval
02 Accuracy (TP+TN )/ (TP+TN+FP +FN)x100%
03 Precision (TP )/ (TP + FP)x100%
04 Linearity Y= mX + C.
05 Uncertainty(MoU) CV=SD/x
06 Sensitivity TP/(TP+FN) X100%
07 Specificity TN/(TN+FP) X100%

15. Trueness, Accuracy, and precision

16. Precision
• To perform precision test is running 20 of one
sample depending on your schedule,
• Analysis and interpret statics measure including
mean, SD,CV, 2SD range and SD goal
• The precision test will pass if the calculated SD
is below the SD goal

17. Accuracy
• It need 40 different samples
• Sample are tested in comparison of new method
with existing accuracy method(gold standard)
 Measure the statics(Linearity) includes
 Correlation coefficient(R) greater 0.9
 Slope for 95% CI is 1.000
 Intercept for 95% CI is 0.000

18. Reportable range/linearity
• Assess the lowest and highest reliable results,
• Used especially when there is two calibrators
• Run 5 sample with known concertation in
triplicate
• Assess Linearity statics, then point out error
rate, and evaluate if allowable
• Error allowable is are proximity to reportable
range

19. Sensitivity and Specificity
• Done by comparing new method with gold
standard method.
• It involved running greater 100 sample in each
methods.
• Obtained results should be tabulated in two way
table
• Then, sensitivity and specificity will be calculated

20. Examples
• A validation of a new rapid syphilis test kit, YYY was
conducted at your laboratory. 500 specimen known to
positive were tested and 479 results came out positive
and others negative. A total of 506 known negative
sample were tested and a total of 490 results were
negative and others were positive.
• The acceptability criteria for both Diagnostic sensitivity
and diagnostic specificity is set at 98%.
• Use a contingency table to calculate the validation
results.
• What would be your decision after obtaining the
validation results?

21. GOLD STD TEST
NEW TEST POSITIVE NEGATIVE TOTAL
POSITIVE 479 16 495
NEGATIVE 21 490 511
TOTAL 500 506 1006

22. • Sensitivity=TP/D+X100%=95.8%
• Specificity=TN/D-X100%=3.2%

23. MoU
• It a systematically assess the factors influencing
the measurement
• It measure the actually parameter that should be
included with any measurement results obtained
• It measure existing doubt(degree from margin)
• It tell us about the quality of testing methods
• Errors are not the same as uncertainty

24. Post validation monitoring
• To provide validation report which should
includes performance specifications, results
obtained, and final statement(performance was
achieved or not)
• Proficiency Testing eg EQA
• System Performance Monitoring eg monitoring
of deviation like LJ charts.

25. summary

26. References
• ISO 15189:2022
• Analytical performance in accordance with CLSI guidelines,
Update of 2017.
• ICH Q2(R2) Validation Of Analytical Procedures Scientific
Guideline, December 2023.
• Published Articles.