Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2001-02-22
2002-10-29
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S039000, C436S063000, C436S172000, C250S461200
Reexamination Certificate
active
06472168
ABSTRACT:
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is related to Japanese Patent Application No. 2000-52953, filed on Feb. 29, 2000 whose priority is claimed under 35 USC § 119, the disclosures of which are incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for counting sperms (sperm cells) by the flow cytometry.
2. Related Art
For treating male infertility in the urology department, properties concerning fertility such as concentration, motility, teratosis of sperm in semen have been examined as a means for specifying the cause. It was recently reported that there was an inclination of decreasing a concentration and amount of sperm in male adults under influence of endocrine-disrupting chemicals. A number of sperm-related reports due to epidemiological investigations were made, but various results including positive and negative ones were present on lowering the sperm properties. Since there is no standardization of an assay of sperm concentration, credibility or statistical significance of those data is questionable.
Known methods of counting sperms in conventional semen examination include a method of using a hemocytometer as shown in the guideline issued by WHO, a method of observation with microscope by using a sperm counter by Markler Chember and so on. However, these methods have a problem that, since they mean a subjective method of counting sperms with eyes, the resultant data differ even between samples of the same origin, depending upon investigating institutes or inspectors. Moreover, such a method of counting sperms need so much time that it is not suitable for treating a large number of samples. In addition, there are some problems in accuracy and reproducibility, because semen shows so high viscosity that there occurs certain unevenness on the distribution of sperm cells, and because there are a very few number of sperm cells to be counted.
In order to overcome those problems, methods of counting sperms with an automatic homocytometer [Journal of Studying Artificial Insemination, Vol. 7, No. 2 May, 1985], an electric resistance type particle counter [Fukuoka Medical Journal, 88(8): 294-297] and a flow cytometer [U.S. Pat. No. 4,559,309] had already been tried.
Contaminants such as leukocyte, erythrocyte, granulocyte, bacteria, oil drops, etc. are often found in semen. It is pointed out that, also in trials of the automation, some errors have occurred in the counting data under the influence of these contaminants. In particular, such contaminants give so remarkable influence in assaying semen having a low concentration of sperms due to hypospermia that the number of sperms may hardly be determined accurately. Though some trials to avoid the influence of those contaminants with a trypsin and lipase solution were made, the effects were so insufficient that the number of sperms could hardly be determined accurately.
SUMMARY OF THE INVENTION
The present invention has been established in view of the circumstances as described above, and it is an object of the present invention to provide a process for accurately counting the number of sperms.
The present invention provides the process for counting sperms comprising the step of:
(1) mixing a specimen containing sperms with an aqueous solution containing a cationic surfactant in an amount effective in removing contaminants contained in the specimen to give a surfactant-treated specimen,
(2) staining the sperms contained in the surfactant-treated specimen with a staining liquid containing a dye for staining nucleic acid to give a measuring specimen,
(3) introducing the measuring specimen into a flow cell in a flow cytometer and irradiating the stained sperms in the measuring specimen with excitation light,
(4) detecting a scattered light signal and a fluorescent light signal emitted from the sperms irradiated with the excitation light,
(5) preparing a two-dimensional distribution involving two axes of a scattered light intensity and a fluorescent light intensity based on the scattered light signal and the fluorescent light signal, and
(6) specifying a region of a sperm mass on the two-dimensional distribution and counting the number of sperm in the region of the sperm mass.
REFERENCES:
patent: 4559309 (1985-12-01), Evenson et al.
patent: 5985572 (1999-11-01), Macfarlane
Takacs et al. 1987. ACTA Biochim. Biophys. Hung. vol. 22, No. 1, pp. 45-58, BIOSIS Abstract enclosed.*
Janos et al. 1986. Magy. Allatorv. Lapja. vol. 41, No. 8, pp. 459-463, BIOSIS Abstract enclosed.*
Omura et al., Usefulness of Particle Size Distribution Analyzer for Counting the Number of Sperm, Fukuoka Medical Journal, 88(8): 297-297.
Sasaki, et al., Simplified Method for Rapid Calculation of Sperm Numbers in Bull Semen Diluted with Extender Containing Egg Yolk by Automatic Blood Cell Counter, Journal of Studying Artificial Insemination, vol. 7, No. 2, May, 1985.
Hamaguchi Yukio
Matsumoto Teruya
Okada Hiroshi
Cohan, Pontani, Lieberman & Pavane
Sysmex Corporation
Tate Christopher R.
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