Risks and Challenges in Interpreting Simultaneous Analyses of Multiple Cytokines

PURPOSE: To determine the inherent risks of handling results below the lowest detectable value in the analysis of multiple cytokines in the aqueous humor of patients with retinal diseases by comparing possible statistical strategies to lower the risk of mis interpretation or over interpretation of results. Furthermore, in analyzing multiple cytokines simultaneously, the challenge of multiple comparison arises. METHODS: The analyses were based on parallel testing of 43 cytokines in 58 aqueous humor samples from patients with macular hole or epiretinal membrane. Substitution of values below the detection limit with 0.1 ×, 0.5 ×, or 1.0× of the lowest level of quantitation was compared with handling as missing value. The impact of correction for multiple comparisons was assessed using the Holm correction. RESULTS: When comparing macular hole with epiretinal membrane, not substituting the missing data revealed a difference (P < 0.05) for five compared with wight cytokines after their substitution, indicating an increased risk for under-estimating group differences (type II error). Correcting for multiple comparisons revealed a relevant risk of over estimating group differences (type I error). CONCLUSIONS: Physiologic cytokine concentrations in ocular fluids typically range at or below the lowest level of quantitation. Handling of results below this cutoff as missing leads to increased type II errors. Not correcting for multiple comparisons increases the risk of a type I error. Taken together, both harbor a systematic inherent risk of misinterpretation of the results. TRANSLATIONAL RELEVANCE: Ignoring the inherent risks of data misinterpretation in analyses of ocular fluid samples may result in mis leading conclusions regarding their biological relevance.