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Regression analysis is the method of choice for the production of covariate-dependent reference limits. There are currently no recommendations on what sample size should be used when regression-based reference limits and confidence intervals are calculated. In this study we used Monte Carlo simulation to study a reference sample group of 374 age-dependent hemoglobin values. From this sample, 5000 random subsamples, with replacement, were constructed with 10-220 observations per sample. Regression analysis was used to estimate age-dependent 95% reference intervals for hemoglobin concentrations and erythrocyte counts. The maximum difference between mean values of the root mean square error and original values for hemoglobin was 0.05 g/L when the sample size was > or = 60. The parameter estimators and width of reference intervals changed negligibly from the values calculated from the original sample regardless of what sample size was used. SDs and CVs for these factors changed rapidly up to a sample size of 30; after that changes were smaller. The largest and smallest absolute differences in root mean square error and width of reference interval between sample values and values calculated from the original sample were also evaluated. As expected, differences were largest in small sample sizes, and as sample size increased differences decreased. To obtain appropriate reference limits and confidence intervals, we propose the following scheme: (a) check whether the assumptions of regression analysis can be fulfilled with/without transformation of data; (b) check that the value of v, which describes how the covariate value is situated in relation to both the mean value and the spread of the covariate values, does not exceed 0.1 at minimum and maximum covariate positions; and (c) if steps 1 and 2 can be accepted, the reference limits with confidence intervals can be produced by regression analysis, and the minimum acceptable sample size will be approximately 70.

Regression-based reference limits: determination of sufficient sample size