Flow cytometry-fluorescence measurements for characterizing sperm

• US 4,559,309 A
• Filed: 09/01/1982
• Issued: 12/17/1985
• Est. Priority Date: 09/01/1982
• Status: Expired due to Fees

• Current Assignee: Memorial Sloan-Kettering Cancer Center
• Original Assignee: Memorial Sloan-Kettering Cancer Center
• Inventors: Darzynkiewicz, Zbigniew, Evenson, Donald P.
• Primary Examiner(s): Turk, Arnold
• Application Number:
  US06/413,862
• Time in Patent Office: 1,203 Days
• Field of Search: 436/63, 436/94, 436/172, 250/461.2, 424/3
• US Class Current: 436/63
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A process for characterizing sperm motility and viability by staining a sperm sample with Rhodamine 123 and ethidium bromide, and simultaneously measuring the sperm fluorescence emissions at green frequencies 515-575 nm and at red frequencies 600-650 nm, the green counts being correlated with sperm motility and the red counts being correlated with putative dying or dead cells. Additionally, a sample of sperm can be characterized as to type and normality by staining a sample of sperm with acridine orange and simultaneously measuring the sperm fluorescence emissions at green frequencies 515-575 nm and at red frequencies 600-650 nm.

Claims

1. Process for characterizing sperm motility and viability comprising the steps of

staining a sample of the sperm with Rhodamine 123 and ethidium bromide,

applying flow cytographic measuring techniques to count, simultaneously, the sperm fluorescence emissions at green frequencies 515-575 nm and at red frequencies 600-650 nm, the green counts being correlated with sperm motility and the red counts being correlated with putative dying or dead cells.

2. Process of claim 1 further comprising

staining a second sample of the sperm with acridine orange and applying flow cytographic measuring techniques to count, simultaneously, the sperm fluorescence emissions at green frequencies 515-575 nm and at red frequencies 600-650 nm, which counts are correlated with cell type and normality.