Methods and compositions for determining the presence or absence of dna aberrations

Patent metadata

Field	Value
Publication number	US-2019310243-A1
Application number	US-201916379456-A
Country	US
Kind code	A1
Filing date	Apr 9, 2019
Priority date	Apr 9, 2018
Publication date	Oct 10, 2019
Grant date	—

Abstract

The invention consists of methods and compositions for detecting the presence or absence of a DNA aberration by analyzing fluorescence emission characteristics in sperm cells or sperm nuclei, which generally consists of entraining sperm cells or sperm nuclei stained with a DNA selective dye in sheath fluid; exposing the entrained sperm cells or sperm nuclei to electromagnetic radiation; determining a forward fluorescence characteristic and a side fluorescence characteristic of individual events associated with the exposed sperm cells or sperm nuclei; and gating the individual events based on the forward fluorescence characteristic and the side fluorescence characteristic with a criterion.

First claim

What we claim is:

1 . A method of analyzing fluorescence emission characteristics in sperm cells or sperm nuclei, comprising:
entraining sperm cells or sperm nuclei stained with a DNA selective dye in sheath fluid; exposing the entrained sperm cells or sperm nuclei to electromagnetic radiation; determining a forward fluorescence characteristic and a side fluorescence characteristic of individual events associated with the exposed sperm cells or sperm nuclei; gating the individual events based on the forward fluorescence characteristic and the side fluorescence characteristic with a criterion; and determining the presence or absence of a DNA aberration from the gated individual events.

2 . The method of claim 1 , wherein the DNA selective dye is Hoechst 33342.

3 . The method of claim 1 , further comprising the step of orienting the entrained sperm cells or sperm nuclei.

4 . The method of claim 1 , wherein the step of exposing the sperm cells or sperm nuclei to electromagnetic radiation comprises exposing the sperm cells or sperm nuclei to a laser beam with modified beam profile.

5 . The method of claim 1 , wherein the criterion encompasses a subpopulation of oriented sperm cells or sperm nuclei.

6 . The method of claim 1 , wherein the sperm cells or sperm nuclei comprise sperm cells or sperm nuclei from a first and a second mammalian species.

7 . The method of claim 1 , wherein determining the presence of a DNA aberration from the gated individual events comprises detecting more than two peaks or modes, or a peak to valley ratio of 50% or less, 60% or less, 70% or less or 80% or less, on a histogram of fluorescence intensities.

8 . The method of claim 6 , wherein determining the presence of a DNA aberration from the gated individual events comprises detecting a difference between a peak to valley ratio on a histogram of fluorescence intensities of sperm cells or sperm nuclei from the first mammalian species and a peak to valley ratio on a histogram of fluorescence intensities of sperm cells or sperm nuclei from the second mammalian species.

9 . A method of analyzing fluorescence emission characteristics in sperm cells or sperm nuclei, comprising:
entraining sperm cells or sperm nuclei stained with a DNA selective dye in sheath fluid; exposing the entrained sperm cells or sperm nuclei to electromagnetic radiation; determining two or more fluorescence emission characteristics of individual events associated with the exposed sperm cells or sperm nuclei; gating the individual events based on the two or more fluorescence emission characteristics with a first criterion for further processing; gating the further processed individual events with a second criterion; and determining the presence or absence of a DNA aberration from the twice gated individual events.

10 . The method of claim 9 , wherein the first criterion encompasses a first subpopulation of oriented sperm cells or sperm nuclei.

11 . The method of claim 10 , wherein the second criterion encompasses a second subpopulation of sperm cells or sperm nuclei comprising 25% to 75% of the first subpopulation.

12 . The method of claim 11 , wherein the step of determining the presence or absence of DNA aberrations in the sperm cells or sperm nuclei from the twice gated individual events further comprises evaluating a third subpopulation of sperm cells or sperm nuclei within the first subpopulation, wherein the third subpopulation of sperm cells or sperm nuclei excludes the second subpopulation of sperm cells or sperm nuclei.

13 . The method of claim 10 , wherein the second criterion encompasses 25% to 75% of sperm cells or sperm nuclei around a median value of a fluorescence emission characteristic.

14 . The method of claim 9 , wherein the step of determining the presence or absence of a DNA aberration in the sperm cells or sperm nuclei from the twice gated individual events further comprises determining the quantity of peaks or modes in a twice gated fluorescence emission characteristic.

15 . The method of claim 9 , wherein the step of determining the presence or absence of a DNA aberration in the sperm cells or sperm nuclei from the twice gated individual events further comprises analyzing a coefficient of variation of a fluorescence emission characteristic associated with the first subpopulation of sperm cells or sperm nuclei or the second subpopulation of sperm cells or sperm nuclei.

16 . The method of claim 9 , wherein the step of determining the presence or absence of a DNA aberration in the sperm cells or sperm nuclei from the twice gated individual events further comprises generating a first univariate plot based on a fluorescence emission characteristic of the first subpopulation or generating a second univariate plot based on a fluorescence emission characteristic of the second subpopulation.

17 . The method of claim 16 , wherein the step of determining the presence or absence of a DNA aberration in the sperm cells or sperm nuclei from the twice gated individual events further comprises analyzing a peak to valley ratio of the first univariate plot or a peak to valley ratio of the second univariate plot.

18 . The method of claim 9 , further comprising the step of separating tails or midpieces from the sperm nuclei by centrifugation.

19 . A method of analyzing a fluorescence emission characteristic in sperm cells or sperm nuclei comprising:
staining the sperm cells or sperm nuclei with a DNA selective dye; entraining the stained sperm cells or sperm nuclei in sheath fluid; exposing the entrained sperm cells or sperm nuclei to electromagnetic radiation; determining a fluorescence emission characteristic of the exposed sperm cells or sperm nuclei; generating a first multivariate plot based on the fluorescence emission characteristic; providing a first gate on the first multivariate plot; generating a second multivariate plot based on the first gate; providing a first sort region on the second multivariate plot encompassing a first subpopulation of sperm cells or sperm nuclei and a second sort region on the second multivariate plot encompassing a second subpopulation of sperm cells or sperm nuclei; separating the first subpopulation of sperm cells or sperm nuclei and the second subpopulation of sperm cells or sperm nuclei; generating a first univariate plot based on a fluorescence emission characteristic of the separated first subpopulation of sperm cells or sperm nuclei; generating a second univariate plot based on a fluorescence emission characteristic of the separated second subpopulation of sperm cells or sperm nuclei.

20 . The method of claim 19 , further comprising the step of removing tails or midpieces from the sperm nuclei by centrifugation.

21 . The method of claim 20 , wherein the first subpopulation of sperm cells or sperm nuclei comprises 25% to 75% of the sperm cell or sperm nuclei population.

22 . The method of claim 21 , wherein the second subpopulation of sperm cells or sperm nuclei excludes the first subpopulation of sperm cells or sperm nuclei.

23 . The method of claim 22 , wherein the first sort region encompasses the center of the second multivariate plot.

24 . The method of claim 21 , further comprising the step of determining the quantity of peaks or modes of the first univariate plot or the second univariate plot, analyzing a coefficient of variation of the first univariate plot or the second univ

Assignees

• Inguran Llc

Inventors

• Gilligan Thomas B
• Evans Kenneth Michael
• Do Amaral Grossi Daniela

Classifications

• G01N1/30

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title

• G01N15/1459

  the analysis being performed on a sample stream · CPC title

• G01N2015/1006

  for cytology · CPC title

• G01N15/1012

  Calibrating particle analysers; References therefor · CPC title

• G01N15/14

  Optical investigation techniques, e.g. flow cytometry · CPC title