ABSTRACT
The analysis of leucocyte population in human semen could be useful in the diagnosis and therapeutic monitoring of male genital infections, but it is difficult due the low percentage of leucocytes, often not easily recognizable from immature cells of spermatogenesis. A method was developed for the isolation and identification of different leucocyte populations in human semen in healthy subjects using anti-CD45-covered magnetic beads. The seminal fluid was incubated with anti-CD45-covered magnetic beads and the samples were placed in contact with a magnet. The CD45-positive cells recovered were analyzed by light microscopy. The leucocyte formula was compared with a leucocyte formula performed on seminal fluid sediment. The method, even if laborious, eliminates all spermatozoa and most of cells of spermatogenetic lineage, thus permitting phenotyping and functional analysis on isolated leucocytes.
Subject(s)
Leukocytes , Semen/cytology , Cell Separation , Centrifugation, Density Gradient , Flow Cytometry , Humans , Leukocyte Common Antigens/immunology , Male , Povidone , Silicon DioxideABSTRACT
DNA extraction is a critical step in PCR analysis and is closely related to its sensitivity. Traditional methods, based on phenol-chloroform extraction, require more time and the use of toxic reagents. GeneReleaser (Bio Ventures Inc.) is a commercial product which releases DNA from whole blood, cell cultures, bacterial colonies and the like. Cells lysis and DNA extraction are accomplished directly in the amplification tube on a thermocycler. We used GeneReleaser in the identification of HIV-1 proviral DNA by PCR on whole blood samples. All samples arrived at our laboratory for HIV-1 detection were treated with two different procedures. The classical one was based on the lysis of separated lymphocytes by proteinase K, while the other consisted in DNA extraction by GeneReleaser from 5 microliters of whole blood in sodium citrate. All samples were amplified for HIV-1 GAG region; to prevent carry-over contamination Uracil N-glycosylase (UNG) sterilization was performed. Amplified sequences were revealed using the DEIA commercial system (Sorin Biomedica, Italy). To verify the suitability both of cell lysates and GeneReleaser DNA-extracted samples for PCR, we amplified a specific sequence of HLA-DQ-alpha gene. Initial data indicate that this new method might reduce the performance time of PCR (DNA extraction time was around 15 minutes) and improve PCR sensitivity.
