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Enumeration of semen leucocytes by fluorescence in situ hybridisation technique

Aim—To determine whether the fluorescent in situ hybridisation technique (FISH) using a total human DNA genomic probe can be used to enumerate semen leucocytes. Methods—Semen samples from five donors were subjected to a mild KC1 solution. These samples were then biotin labelled under FISH conditions using a total human DNA genomic probe and the leucocyte counts were determined. To check the accuracy of the technique a monoclonal antibody against the common leucocyte antigen CD45 [KC56(T-200)] served as a control. An isotypic control for [KC56(T-200)], the immunoglobulin [MsIgG1], served as a secondary control. Results—Semen leucocytes stained by the FISH technique were easily detected because of their distinct bright yellow colour, while the sperm cells were red. The leucocyte count ranged from 0·5 to 4·9 × 106 per ml of semen. KC56(T-200) and its isotypic control MsIgG1, which served as control for the FISH technique, accurately identified 94% and 97% of the semen leucocytes of a control donor, respectively. Conclusions—The FISH technique using a total human DNA probe can accurately and effectively enumerate the overall leucocyte population in semen.