Determination of neutrophil concentration in semen by measurement of superoxide radical formation.
OBJECTIVE
To develop an assay that measures the concentration of functional neutrophils in human semen.


METHODS
Human blood neutrophils were first isolated to establish linearity and proportionality for the determination of functional neutrophil concentration. Thereafter blood neutrophils in seminal plasma and neutrophils in semen of patients were measured.


METHODS
Blood and human samples were obtained from clinics of the Royal Victoria Hospital.


METHODS
Blood from normal men or from patients presenting with chest pain or trauma were used. Semen samples were also obtained from healthy fertile donors or from unselected patients attending the infertility clinic.


METHODS
Active neutrophil concentration in semen by a colorimetric assay.


RESULTS
The assay developed is based on the reduction of nitroblue tetrazolium of pale yellow color to blue formazan by the superoxide anions produced by stimulated neutrophils. The intensity of the derived blue color is proportional to the concentration of active neutrophils. This assay is simple, requires only 10 minutes of preparation time, and detects neutrophil concentrations greater than 0.5 x 10(6) neutrophils/mL of semen, independent of sperm concentration.
Kovalski
N
N
Urology Research Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada.
de Lamirande
E
E
Gagnon
C
C
D001773:Blood Cells / Q000166:cytology / Q000378:metabolism; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D005609:Free Radicals; D006801:Humans; D008297:Male; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D009580:Nitroblue Tetrazolium; D009195:Peroxidase / Q000032:analysis; D012661:Semen / Q000166:cytology* / Q000201:enzymology; D013094:Spermatozoa / Q000502:physiology; D013194:Staining and Labeling; D013481:Superoxides / Q000378:metabolism*
D005609:Free Radicals; D013481:Superoxides; D009580:Nitroblue Tetrazolium; D009195:Peroxidase