Rejection treshholds and seasonal variability of epifluorescent sperm tunel assay

Abstract

OBJECTIVE: To determine: seasonal and batch effects on positive and negative controls results of TUNEL assay. To analyze cut-off points for rejection of assay. DESIGN: Retrospective review. MATERIALS AND METHODS: TUNEL assay was performed using 10 ul sperm spread on each of 5 microscope slides. Two slides for negative and 2 for positive controls were analyzed. 200 sperm per slides were counted. All positive controls were incubated with 500 ul of DNAse type I for 10 min at 37C incubator prior to adding enzyme solution and labeling solution. For negative controls only labeling solution was added. All slides were then incubated for 1 h at 37C, counter stained with DAPI and analyzed by the same observer. Results were reported as percentage of TUNEL positive (FITC) sperm per total number of sperm (DAPI). RESULTS: 125 positive and 126 negative assays were scored between April of 2009 and 2012 by the same observer. Mean % TUNEL and 95% CI for negative control was 1.02 (0.94-1.13) and 98.6% (98.5-98.8) for positive controls. Distribution curves fitting showed Weilbull as most appropriate model. Normal Q-Q plot and weighted percentiles analysis revealed that assay should be rejected if negative TUNEL is> 2% or positive TUNEL1⁄4 2% or positive control is <1⁄497%. TUNEL assays is extremely reproducible as evident by very close clustering of values. Seasonal variability although surprising may be explained by variability in temperatures in the culture rooms or other factors like DNAse stability. Seasonal variability does not increase assay rejection rate. Supported by: Robert Dow.