Validating New Laboratory Tests

1. Test/method — What analyte and platform are being validated? Default: not specified; flag [VERIFY].
2. Validation type — New test (full validation), new instrument for existing test (verification), or modified method? Default: verification of FDA-cleared method.
3. Regulatory classification — FDA-cleared/approved, laboratory-developed test (LDT), or modified FDA-cleared? Default: FDA-cleared.
4. Specimen types — What specimen types will be tested (serum, plasma, whole blood, urine, CSF, other)? Default: serum.
5. Comparator method — What is the reference or predicate method for comparison? Default: current laboratory method.
6. Clinical purpose — Screening, diagnosis, monitoring, or pharmacogenomics? Default: diagnosis.
7. Target go-live date — When must validation be complete? Default: 60 days.

Documents to Request

• Manufacturer's package insert with claimed performance specifications
• CLSI EP guidelines relevant to each validation study (EP05, EP06, EP09, EP15, EP28)
• Comparator method performance data (current precision, bias, AMR)
• Clinical Laboratory Improvement Advisory Committee (CLIAC) guidance for the analyte
• CAP checklist requirements for the specific test discipline
• Institutional validation protocol template
• Prior validation studies for the same analyte (if replacing an existing method)
• Proficiency testing survey results for the comparator method

Step 1: Precision Study (CLSI EP05-A3)

Evaluate the repeatability and within-laboratory precision of the new method:

Protocol:

• Minimum 2 levels of QC material or patient pools (normal and abnormal range)
• Run each level in duplicate, 2 runs per day, for a minimum of 20 days (CLSI EP05-A3 standard protocol)
• This yields at minimum 80 data points per level

Analysis:

Acceptance: Total imprecision (CV%) must be <= the manufacturer's stated precision or <= 1/4 of the total allowable error (TEa) for the analyte (per Westgard sigma-metric guidance).

Step 2: Accuracy / Method Comparison Study (CLSI EP09-A3)

Compare the new method to the reference or comparator method:

Protocol:

• Minimum 40 patient specimens spanning the clinically relevant concentration range
• Each specimen tested by both new and comparator method within 2 hours
• Include specimens at low, normal, and high concentrations
• Test each specimen once on each method (or in duplicate if precision is a concern)

Analysis:

• Deming or Passing-Bablok regression: Slope (target 0.9-1.1), intercept (target near 0), and correlation coefficient (r >= 0.975).
• Bland-Altman difference plot: Mean bias and 95% limits of agreement.
• Medical decision point bias: Calculate bias at each clinical decision point and compare to TEa.

Step 3: Analytical Measurement Range (AMR) and Linearity (CLSI EP06-A)

Verify the analytical measurement range (reportable without dilution):

Protocol:

• Prepare at minimum 5 concentration levels spanning the manufacturer's claimed AMR
• Use certified reference material, calibrator dilutions, or spiked patient pools
• Each level tested in triplicate (minimum) or quadruplicate
• Include concentrations at and beyond the claimed AMR boundaries

Analysis:

• Plot measured vs. expected values
• Calculate polynomial regression; compare first-order (linear) to higher-order models
• Non-linearity: If the difference between the linear and polynomial fit exceeds the TEa at any point, the AMR is narrowed to the linear range

Reportable Range: If the clinical range extends beyond the AMR, validate dilution protocols. Document dilution factor and recovery at each validated dilution step.

Step 4: Reference Range Verification (CLSI EP28-A3c)

Verify that the manufacturer's stated reference range is applicable to the laboratory's patient population:

Protocol — Verification (FDA-cleared tests):

• Test a minimum of 20 healthy individuals representative of the patient population (per CLSI EP28)
• If no more than 2 of 20 fall outside the manufacturer's claimed range, the reference range is verified
• If more than 2 of 20 fall outside, a full reference range study is required (120+ subjects per partition)

Protocol — Establishment (LDTs or when verification fails):

• Minimum 120 reference individuals per partition (e.g., male/female, pediatric/adult)
• Calculate 2.5th and 97.5th percentiles as reference limits using nonparametric methods
• Partition by age, sex, and other demographic variables as clinically appropriate

Step 5: Additional Validation Studies

Perform additional studies as applicable:

• Carryover study: Test high-low-low sequence to detect analyte carryover between specimens. Carryover < 1% of the high-concentration value is generally acceptable.
• Interference study (CLSI EP07): Test known interferents (hemolysis, lipemia, icterus, bilirubin, common drugs). Document interference thresholds.
• Limit of detection (LoD) / Limit of quantitation (LoQ): For assays where clinical decisions depend on low-level detection (troponin, tumor markers, drugs of abuse).
• Stability study: Specimen stability at room temperature, refrigerated, and frozen conditions.
• Specimen type comparison: If multiple specimen types are claimed (serum vs. plasma), perform a comparison study between them.

Step 6: Validation Report and Go-Live Approval

Assemble the complete validation report:

1. Study summary: Test name, platform, validation type, dates performed.
2. Precision results: CV% for each level with acceptance determination.
3. Accuracy results: Regression statistics, Bland-Altman analysis, decision-point bias.
4. AMR/linearity: Verified range with any limitations documented.
5. Reference range: Verified or established, with population description.
6. Additional studies: Carryover, interference, LoD/LoQ, stability.
7. Limitations: Any identified limitations affecting clinical use.
8. Conclusion: Statement of acceptance or rejection with rationale.
9. Laboratory director signature: Required for go-live approval per CLIA.

Checkpoint B: Post-Draft Alignment (Mandatory)

1. Does the precision study meet the minimum 20-day/2-run/2-level protocol per CLSI EP05?
2. Does the method comparison include at least 40 specimens spanning the clinically relevant range?
3. Is the AMR verified at a minimum of 5 concentration levels including boundary values?
4. Is the reference range verified using 20 subjects (or established using 120+ subjects for LDTs)?
5. Has the laboratory director reviewed and signed the validation report?

Quality Audit

• Validation type correctly classified (verification vs. full validation vs. modification)
• Precision study follows CLSI EP05-A3 protocol (20 days, 2 runs, 2 levels)
• Total CV% meets acceptance criteria (manufacturer claim or clinical requirement)
• Method comparison includes >= 40 specimens across the concentration range
• Regression statistics and Bland-Altman analysis documented
• Bias at medical decision points compared to TEa
• AMR verified at >= 5 concentration levels per CLSI EP06
• Dilution protocol validated if reportable range exceeds AMR
• Reference range verified (20 subjects) or established (120+ subjects)
• Interference studies performed for hemolysis, lipemia, icterus
• LoD/LoQ established for analytically sensitive assays
• Validation report signed by laboratory director before go-live
• CAP checklist requirements met for the specific test discipline

Guidelines

• Distinguish verification (FDA-cleared, confirming manufacturer claims) from validation (LDT or modified method, establishing specifications from scratch); the regulatory burden differs significantly
• Follow CLSI EP05-A3 exactly for precision studies: 20 days minimum, 2 runs per day, 2 replicates per run — shortcuts undermine statistical power and invite CAP citations
• Include specimens at clinical decision points in the method comparison (e.g., glucose at 126 mg/dL, HbA1c at 6.5%); demonstrating acceptable performance at decision points is more clinically meaningful than overall correlation
• When the manufacturer's reference range cannot be verified, perform a full establishment study with 120+ subjects per partition rather than adopting reference ranges from literature
• Document all deviations from the planned validation protocol and justify why the deviation does not invalidate results
• Do not go live until the laboratory director has reviewed all data and signed the validation report — this is a CLIA regulatory requirement
• Retain all raw validation data, worksheets, and statistical analyses for the life of the test system or as required by institutional policy (minimum 2 years per CLIA)
• Plan for ongoing performance monitoring after go-live: compare the first month of patient results to the comparator method if running in parallel