Process for evaluating staining conditions of cells for sorting

• US 7,799,569 B2
• Filed: 03/16/2009
• Issued: 09/21/2010
• Est. Priority Date: 03/28/2003
• Status: Active Grant

• Current Assignee: Inguran LLC
• Original Assignee: Inguran LLC
• Inventors: Durak, Gary, Westfall, Lon A., Vandre, Gary P., Nayak, Niraj V., Wallace, Jeffrey D., Hatcher, Jeremy T.
• Primary Examiner(s): WALLENHORST, MAUREEN
• Application Number:
  US12/404,931
• Publication Number:
  US 20090176271A1
• Time in Patent Office: 554 Days
• Field of Search: 436/63, 436/164, 436/172, 436/174, 435/2, 435/4, 435/29, 435/34, 422/73, 422/82.05, 422/82.08
• US Class Current: 436/63
• CPC Class Codes: A01N 1/0284 Temperature processes, i.e. using a designated change in temperature over time
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12N 5/061 Sperm cells, spermatogonia
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 3/00 Condition responsive control processes apparatus therefor C12M1/36; controlling or regulating in general G05
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1406 Control of droplet point
• CPC Class Codes: G01N 2015/1415 Control of particle position
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/63 optically excited
• 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: G01N 33/48 Biological material, e.g. blood, urine G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence; Haemocytometers counting blood corpuscules distributed over a surface by scanning the surface G06M11/02
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: Y10T 436/10 : Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/115831 : Condition or time responsive
• CPC Class Codes: Y10T 436/12 : Condition responsive control
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2525 : Stabilizing or preserving
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A multi-channel apparatus for classifying particles according to one or more particle characteristics may have a plurality of flow cytometry units, each of which is operable to classify particles in a mixture of particles by interrogating a stream of fluid containing the particles with a beam of electromagnetic radiation. The flow cytometry units may share an integrated platform with: (1) a common supply of particles; (2) a common housing; (3) a common processor for controlling operation of the units; (4) a common processor for receiving and processing information from the units; and (5) a common fluid delivery system. The integrated platform can include a common source of electromagnetic radiation. A method uses a plurality of flow cytometry units sharing the integrated platform to perform a flow cytometric operation, such as analyzing or sorting particles.

Claims

1. A process for evaluating a set of conditions for staining a population of cells for sorting, the population comprising a first type and a second type of cell, the process comprising;

(a) staining a fraction of the population of cells with a fluorescent dye under a set of staining conditions;

(b) exposing the stained cells to electromagnetic radiation as the stained cells are passed through an interrogation location of a flow cytometer at a rate, R;

(c) determining a fluorescence emission characteristic of the exposed cells;

(d) using the determined fluorescence characteristic to classify the exposed cells into two or more sub-populations of cells, one of the sub-populations being an enriched sub-population of the first cell type;

(e) determining a coefficient of variation for the fluorescence emission characteristic of the cells of the enriched sub-population to provide an indication of sorting efficiency for the staining conditions; and

(f) based upon the value of the coefficient of variation for the fluorescence emission characteristic of the cells, determining whether to modify any staining condition under which cells are to be stained or the rate, R, at which the stained cells are to be passed through the interrogation location of the flow cytometer in order to improve sorting efficiency.

2. The process of claim 1, wherein said fraction comprises a first fraction and said set of staining conditions comprises a first set of staining conditions, the process further comprising staining a second fraction of said population of cells under a second set of staining conditions different from the first set and performing steps (b) through (e) with said second fraction of the population of cells.

3. The process of claim 2, wherein the second fraction is stained based on the coefficient of variation for the fluorescence emission characteristic of the cells of the first fraction.

4. The process of claim 2, wherein the second set of staining conditions differs from the first set of staining conditions by at least one of the following:

(1) concentration of the fluorescent dye;

(2) the length of a staining period used to stain the cells; and

(3) a temperature of the cells during a staining period used to stain the cells.

5. The process of claim 2, further comprising staining a third fraction of said population of cells under a third set of staining conditions different from the first and second sets and performing steps (b) through (e) with the third fraction of the population of cells.

6. The process of claim 5, further comprising staining a fourth fraction of said population of cells under a fourth set of staining conditions different from the first, second, and third sets and performing steps (b) through (e) with the fourth fraction of the population of cells.

7. The process of claim 6, further comprising staining a fifth fraction of said population of cells under a fifth set of staining conditions different from the first, second, third and fourth sets and performing steps (b) through (e) with the fifth fraction of the population of cells.

8. The process of claim 2, further comprising selectively varying conditions in the first set of staining conditions based upon said determination of the coefficient of variation for the fluorescence emission characteristic of the cells of the first fraction in order to obtain the second set of staining conditions.

9. The process of claim 2, wherein a modified staining condition as determined by performing step (f) on the second fraction is applied to the staining of the remainder of the population of cells.

10. The process of claim 1, wherein multiple fractions of cells are stained in step (a), each fraction being separately stained under a unique set of staining conditions, wherein steps (b) through (e) are performed for each separately stained fraction, and wherein step (f) comprises using the respective coefficients of variation to determine the set of staining conditions to be used to separately stain additional cells in said population.

11. The process of claim 1, wherein the cells are sperm cells.

12. The process of claim 11, wherein the first cell type comprises X chromosome bearing sperm cells.

13. The process of claim 1, wherein step (a) comprises staining the cells with Hoechst 33342.

14. The process of claim 1, wherein said fluorescence emission characteristic comprises a feature of a fluorescence pulse waveform corresponding to movement of a cell through the interrogation location, and wherein said feature is indicative of at least one of the following:

(1) total fluorescence intensity; and

(2) peak fluorescence intensity.

15. The process of claim 1, further comprising determining before step (f) whether the coefficient of variation from step (e) is equal to or less than a predetermined coefficient of variation and repeating steps (a) through (e) using a different set of staining conditions to stain a different fraction of the cells each time until the coefficient of variation determined in step (e) is equal to or less than the predetermined coefficient of variation, and wherein step (f) comprises determining to use the staining conditions to stain additional cells if the coefficient of variation from step (e) for the respective staining conditions is equal to or less than the predetermined coefficient of variation.

16. A process for evaluating a set of conditions for staining a population of cells for sorting, the population comprising a first type and a second type of cell, the process comprising.

(a) staining a fraction of the population of cells with a fluorescent dye under a set of staining conditions;

(b) exposing the stained cells to electromagnetic radiation as the stained cells are passed through an interrogation location of a flow cytometer at a rate, R;

(c) determining a fluorescence emission characteristic of the exposed cells;

(d) using the determined fluorescence emission characteristic to classify the exposed cells into two or more sub-populations of cells, one of the subpopulations being an enriched sub-population of the first cell type;

(e) determining a coefficient of variation for the fluorescence emission characteristic of the cells of the enriched sub-population to provide an indication of sorting efficiency for the staining conditions;

(f) based upon the value of the coefficient of variation for the fluorescence emission characteristic of the cells, determining whether to modify any staining condition under which the fraction of cells are to be stained or the rate, R, at which the stained cells are passed through the interrogation location of the flow cytometer in order to improve sorting efficiency; and

(g) applying the modified staining condition to the remainder of the population of cells.

17. The process of claim 16, further comprising sorting the remainder of the population of cells in a flow cytometer to obtain an enriched sample population of the first cell type.

18. The process of claim 16, wherein said fraction comprises a first fraction and said set of staining conditions comprises a first set of staining conditions, the process further comprising staining a second fraction of said population of cells under a second set of staining conditions different from the first set and performing steps (b) through (e) with said second fraction of the population of cells.

19. The process of claim 18, wherein the second fraction is stained based on the coefficient of variation for the fluorescence emission characteristic of the cells of the first fraction.

20. The process of claim 18, wherein the second set of staining conditions differs from the first set by at least one of the following:

(1) concentration of the fluorescent dye;

(2) the length of a staining period used to stain the cells; and

(3) the temperature of the cells during a staining period used to stain the cells.

21. The process of claim 18, further comprising staining a third fraction of said population of cells under a third set of staining conditions different from the first and second sets and performing steps (b) through (f) with the third fraction of the population of cells.

22. The process of claim 21, further comprising staining a fourth fraction of said population of cells under a fourth set of staining conditions different from the first, second, and third sets and performing steps (b) through (f) with the fourth fraction of the population of cells.

23. The process of claim 22, further comprising staining a fifth fraction of said population of cells under a fifth set of staining conditions different from the first, second, third and fourth sets and performing steps (b) through (f) with the fifth fraction of the population of cells.

24. The process of claim 18, further comprising prior to step (g), selecting a set of staining conditions for use in step (g) that results in a coefficient of variation level for the fluorescence emission characteristic that is equal to or less than a predetermined coefficient of variation.

25. The process of claim 24, wherein the predetermined coefficient of variation is about 1.3% or less.

26. The process of claim 16, further comprising determining before step (f) whether the coefficient of variation from step (e) is equal to or less than a predetermined coefficient of variation and repeating steps (a) through (e) using a different set of staining conditions to stain a different fraction of the cells each time until the coefficient of variation determined in step (e) is equal to or less than the predetermined coefficient of variation, and wherein step (f) comprises determining to use the staining conditions to stain additional cells if the coefficient of variation from step (e) for the respective staining conditions is equal to or less than the predetermined coefficient of variation.

27. The process of claim 16, wherein the cells are sperm cells.

28. The process of claim 27 wherein the first cell type comprises X chromosome bearing sperm cells.

29. The process of claim 16, wherein step (a) comprises staining the cells with Hoechst 33342.

30. The process of claim 16, wherein said fluorescence emission characteristic comprises a feature of a fluorescence pulse waveform corresponding to movement of a cell through the interrogation location, and wherein said feature is indicative of at least one of the following:

(1) total fluorescence intensity; and

(2) peak fluorescence intensity.