Method Validation Edition

Reference interval software for method validation Establish, partition, verify, and transfer reference intervals per EP28-A3C — parametric, non-parametric, robust, bootstrap, and Harrell-Davis quantile methods with a full range of transformations.

Establish reference ranges that reflect real patient populations

Clinicians interpret every result against the reference interval. If the interval doesn’t represent the population being tested — wrong quantile method for the sample size, no partitioning for biologically distinct subgroups, non-normal data forced through a parametric calculation — the clinical interpretation is unreliable. Patients get flagged as abnormal when they’re not, or genuinely abnormal results slip through. IVD manufacturers need reference intervals for product labelling; clinical laboratories need to establish, verify, or transfer them for every analyte they report.

Analyse-it covers the full EP28-A3C workflow — five quantile methods, seven transformations, partitioning by any combination of factors, and both transfer and verification workflows. Choose the right method for the data in front of you, not the one the software happens to support.

We use Analyse-it for the analysis of data necessary to file 510k. We chose Analyse-it because it works in Excel, includes CLSI protocols, and, unlike EP-Evaluator, lets us analyze data directly from equipment without typing.

Thomas D Harrigan, Ph.D.
Technical Product Manager
Alfa Wassermann Diagnostic Technologies

What's included

Five quantile methods for any distribution and sample size

Parametric, non-parametric with three computation approaches — (N+1)p, Np+½, and (N+⅓)p+⅓ — robust bi-weight, bootstrap, and Harrell-Davis. Confidence intervals on all reference limits. Compare methods on the same dataset to assess how sensitive the limits are to the estimation approach.

Partition by sex, age, ethnicity, or any combination

Partition by any combination of factors within a single analysis — such as sex and age group for an analyte like alkaline phosphatase where both matter. Each subgroup maintains its own sample, its own outlier screening, and its own reference limits.

Seven transformations for non-normal distributions

Log, square root, cube root, reciprocal, Box-Cox, Manly exponential, and two-stage exponential/modulus. Shapiro-Wilk and Anderson-Darling normality tests plus normal Q-Q plots before and after transformation.

Tukey outlier screening

Tukey outlier box plot to identify and flag extreme values before they distort the reference limits.

Transfer and verify existing reference intervals

Transfer an existing interval to a new measurement procedure using the regression function from a method comparison study. Verify transferred limits using a binomial test for the proportion falling inside the reference interval — typically 20 samples per partition per EP28-A3C.

Example analyses

See reference interval results in detail — quantile methods, partitioning, transformations, and transfer/verification — using CLSI example datasets you can download and follow along with.

EP28-A3C — Table 4
Calcium reference intervals. Partitioned by sex. Non-parametric (N+1)p quantile method, 120 observations per group. Histogram with reference limits and 90% CIs.

EP28-A3C — Table 5
ALT reference intervals. Partitioned by sex. Log transformation for non-normal data. Non-parametric quantile reference limits on transformed data. Histograms, Q-Q plot, and Tukey box plot.

EP28-A3C
Mercury reference interval transfer. Partitioned by sex. Transferred reference limits verified against 20 observations per group using binomial proportion test.

EP28-A3C — Appendix B
Calcium reference interval. Simple reference interval using robust bi-weight quantile method with bootstrapped confidence intervals.

Part of the Method Validation Edition

Reference intervals are one part of the Method Validation Edition, alongside measurement system analysis, method comparison, and diagnostic performance.

Software you can trust

Validated calculations you can defend at inspection Every calculation is performed by Analyse-it — no Excel formulas, no third-party functions. Results are validated against CLSI reference datasets, published datasets, and thousands of internal test cases before every release. See how we develop and validate Analyse-it →

Data stays in your facility Analyse-it runs entirely within Microsoft Excel on your PC. No cloud processing, no data transmission. Pre-submission data, patient-adjacent data, and in-process results stay within your facility under your own data governance controls.

Standard Excel workbooks anyone can open Every analysis is an ordinary .xlsx workbook. Share with colleagues, submit to regulatory affairs, archive for audit, open on any PC with Excel. No proprietary format, no licence required to view results. Colleagues and auditors see exactly what you see.

Results that can’t be accidentally broken Analysis output contains computed values, not formulas. Nothing to accidentally overwrite, no cell references to break, no formula errors to introduce. The results you reported are exactly what you’ll find when you reopen the workbook months or years later for an audit.

Technical details

CLSI protocols

EP28-A3C: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory

Establish reference limits

Normal (parametric) quantile
Non-parametric quantile: (N+1)p, Np+½, (N+1/3)p+1/3
Harrell-Davis quantile
Bootstrap quantile
Robust bi-weight quantile
Confidence intervals on all reference limits

Transfer / verify

Transfer using method comparison regression function
Binomial test for proportion inside reference interval

Partitioning & transformations

Partition by factor(s) new in v4.00
Reciprocal transformation
Log transformation
Square and cube root
Box-Cox new in v3.52
Manly exponential new in v4.00
2-stage exponential / modulus new in v4.00

Normality testing

Shapiro-Wilk test
Anderson-Darling test
Normal Q-Q plot

Plots

Frequency distribution histogram with normal overlay and reference limits
Tukey outlier box plot
Normal Q-Q plot with Lilliefors confidence band

Method Validation Edition Reference interval software for method validation Establish, partition, verify, and transfer reference intervals per EP28-A3C — parametric, non-parametric, robust, bootstrap, and Harrell-Davis quantile methods with a full range of transformations.