This document outlines the key steps in method validation for clinical chemistry laboratories. It defines method validation and discusses when methods should be validated. The key aspects that must be validated include calibration, precision, accuracy, linearity, limit of detection, analytical range, sensitivity, specificity, ruggedness and robustness. Thorough documentation of the validation study is also required. Validating a method involves experimentally testing these parameters and documenting the results to provide objective evidence that the method is suitable for its intended use.

1. METHOD
VALIDATION
BY:-
DR. HINAL SHAH

2. INTRODUCTION
 Method selection and validation – Key steps in the process of
implementing new methods in the clinical laboratory.
 In Clinical Chemistry Laboratory- influenced strongly by guidelines.

3.  The Clinical & Laboratory Standards Institute (formerly National Committee
for Clinical Laboratory Standards) and International Organization for
Standardization have published a series of protocols and documents to be
followed by CCL and manufacturers for method validation.
 Also, laboratory accreditation requirements have to be met.

4. DEFINITIONS
 “The suite of procedures to which an analytical method is subjected to provide
objective evidence that the method, if used in the manner specified, will
produce results that conform to the statement of the method validation
parameters”
 “The process used to confirm that the analytical procedure employed for a
specific test is suitable for its intended use”
 ISO/IEC 17025 (ISO/IEC 2005, section 5.4) states that method validation is
“confirmation by examination and provision of objective evidence that the
particular requirements for a specified intended use are fulfilled”

5. When Should Methods Be Validated?
 No method should be used without validation
 ISO/IEC 17025 encourages the use of methods published in international,
regional, or national standards and implies that if these methods are used
without deviation, then the validation requirements have been satisfied.
 What does need to be validated are-i.
Nonstandard methods, designed and developed by individual laboratories
ii. Standard methods used outside their scope
iii. Amplifications and modifications of standard methods.

6. Method selection/
development and
validation cycle

7. VERIFICATION
 Synonymous with Single Laboratory Validation
 To be done when a laboratory uses a method for the first time (with
standard materials)
 Also done when a particular aspect of the method or its implementation is
changed, for example-a)
New analyst
b) New equipment or equipment part
c) New batch of reagent
d) Changes in the laboratory premises

8.  Minimum verification is to analyze a material before and after the change
and check for consistency of the results, both in terms of mean and
standard deviation.
 Basic experiments that should be performed to verify the use of a standard
method before first use in a laboratory:
 Bias/recovery
 Precision
 Measurement uncertainty
 Calibration model (linearity)
 Limit of detection

9. METHOD SELECTION- CRITERIA
1. Medical Need
2. Analytical Performance
3. Practical Criteria- depend on whether it is a Laboratory Developed
Test or a Commercial kit method.

10. Method Validation-
Requirements
I. Calibration
II. Precision
III. Accuracy and Trueness
IV. Linearity
V. Limit of Detection (LOD)
VI. Analytical/Measuring Range
VII. Analytical Sensitivity
VIII. Analytical Specificity and Interference
IX. Ruggedness and Robustness
X. Documentation

11. I. CALIBRATION
o Established by measurement of samples with known quantities of the analyte
(calibrators). [Only certified reference materials with known traceability should
be used]
o Done by- Chemical standards or samples with known quantities of analyte
present in the typical matrix.
o Calibration functions- may be linear or curved.
o If the assumed calibration function does not correctly reflect the true
relationship between instrument response & analyte concentration, a systemic
error or bias is likely to be associated with the analytical method.

12. II. PRECISION
 Precision has been defined as the closeness of agreement between
independent results of measurements obtained under stipulated
conditions.
 Depends on – the stability of the instrument response for a given quantity
of analyte.
 The degree of precision is usually expressed on the basis of statistical
measures of imprecision, such as Standard Deviation(SD) OR Coefficient
of Variation(CV)

13.  If calibration function is linear and imprecision remains same over
analytical measurement range, SD also tends to be constant over
analytical measurement range. If imprecision increases proportionally, SD
also tends to increase proportionally to the concentration and relative
imprecision(i.e. CV) remains constant.
 Imprecision of measurements is solely related to the random error.
 Precision is specified as follows:-

14. 1) Repeatability: Corresponds to with-in run precision. It is closeness of
agreement between results of successive measurements carried out
under the same conditions.
2) Reproducibility: Closeness of agreement between results of
measurements performed under changed conditions of measurements.
2 specifications of reproducibility –
 Total or between-run precision (intermediate precision):- its method of
assessment depends on usage of method and variation in
analyst/equipment.
 Inter-laboratory precision.

15.  An important aspect of a full method validation is estimating bias
components attributable to the method itself and to the laboratory carrying
out the analysis. It can be evaluated by inter-laboratory studies.

16. III. TRUENESS & ACCURACY
 Trueness:-
• Closeness of agreement between the mean value (obtained from a large
series of results of measurements) & the true value.
• Bias – Difference between the mean and the true value.
Inversely related to the trueness.
• Qualitative term.
• True value – in practice is the accepted reference value.
• Evaluated by comparison of measurements by a given routine method & a
reference measurement procedure.

17.  Accuracy :-
• Closeness of agreement between the result of a measurement and true
value.
• Qualitative term.
• Influenced by- BIAS and IMPRECISION. Reflects total error.
• Total error= bias + 2SD
Concepts of recovery, drift and carryover are related to trueness.

18. RECOVERY
 It means - the fraction of a test material in a matrix that can be quantified.
target conc. – conc. of blank
 % recovery= -------------------------------------------- X 100
measured conc.
 A recovery close to 100% is a prerequisite for a high degree of trueness,
but does not ensure unbiased results.

19. DRIFT & CARRYOVER
 DRIFT- caused by instrument instability over time, so that calibration
becomes biased.
 CARRYOVER- must be close to ‘Zero’ to ensure unbiased results.

21. IV. LINEARITY
 It refers to the relationship between measured and expected values over
the analytical measurement range.
 Linearity of the analytical procedure is its ability to obtain test results which
are directly proportional to the concentration of analyte in sample.
 Most of the times, a dilution series of a sample is examined. E.g., linearity
of glucose estimation is found by measuring serial dilutions of a stock
solution of 1000mg/dl concentration.

22. A response curve of an electrochemical measurement of copper with concentration of analyte
y is the measured current, x is the concentration of copper

23. V. LIMIT OF DETECTION
 Defined as the lowest value that exceeds the measurements of a blank
sample.
 It is the “minimum detectable net concentration”.
 It presumably is the smallest concentration of analyte that can be
determined to be actually present, even if the quantification has large
uncertainty
 “Limit of detection”  a clearly defined cut off above which the analyte is
measured and below which it is not

24. VI. ANALYTICAL / MEASURING
RANGE
 It is the analyte concentration range over which measurements are within
the declared tolerances for imprecision and bias of the method.
 This range extends from the lowest concentration (i.e. lower limit of
quantification {LloQ}) to the highest concentration (i.e. upper limit of
quantification {UloQ}) for which the performance specifications are fulfilled.

25. VII. ANALYTICAL SENSITIVITY
 Defined as the ability of an analytical method to assess small differences in
the concentration of the analyte.
 The smaller the random variation of the instrument response and the
steeper the slope of the calibration function at a given point, the better is
the ability to distinguish small differences in analyte concentration.
 It depends on the PRECISION of the method.

26. VIII. ANALYTICAL SPECIFICITY &
INTERFERENCE
 Analytical specificity is the ability of an assay procedure to determine the
concentration of the target analyte without influence of potentially interfering
substances or factors in the sample matrix.
 Interfering substances/factors:-
i. Hyperlipemia
ii. Hemolysis
iii. Bilirubin
iv. Antibodies
v. Degradation products of the analyte
vi. Exogenous substances
vii. Anticoagulants

27.  Interferences from hyperlipemia, hemolysis and bilirubin – concentration
dependent &can be quantified as a function of the concentration of the
interfering compound.
 CLSI has published a detailed protocol for evaluation of interference.

28. IX. RUGGEDNESS & ROBUSTNESS
 A method is classed as rugged if its results remain sufficiently unaffected
as designated environmental and operational conditions change.
 Practicality of the method ultimately depends on how rugged it is.
 A robustness study addresses two areas of concern: the need for a
method to be able to yield acceptable results under the normal variation of
conditions expected during routine operation, and the portability of the
method between laboratories with changes in instrumentation, people, and
chemical reagents.

29. X. DOCUMENTATION
o It is the most important step in method validation.
o ‘If you have not documented, you have not done’
o Proper documentation is proof of validity.
o Method validation is only as good as its documentation, which describes
the method, the parameters investigated during the method validation
study, the results of the study, and the Method Validation conclusions
concerning the validation.

30. Reasons for Documentation
1. to allow a user of the method to understand the scope of validation and
how to perform experiments within the scope of the validation
2. the method is not validated until it is documented. No number of
experiments is of any use if they are not documented for future users and
clients to consult
As the documentation will become part of the laboratory’s quality
assurance system, proper documentary traceability (as opposed to
metrological traceability of a result) must be maintained, with revision
versions and appropriate authorizations for updates.

31. When all the above steps are
complete, the method is said to be
validated and can be now put into use.

32. References
1. Tietz Textbook of Clinical Chemistry and Molecular Diagnosis; Edition;
Chapter 2; pg
2. Pictures and graphs from m.authorstream.com