Multi-color flow cytometric analysis of samples with low cell numbers
Aspects of the present disclosure include methods for processing a biological sample. Methods according to certain embodiments include contacting a biological sample having cells with an assay reagent that includes one or more analyte-specific binding members to produce a biological sample assay composition and introducing the biological assay composition into an inlet of a flow cytometer having an integrated acoustic separator. Systems, including a flow cytometer with integrated acoustic separator having one or more acoustic concentrator devices suitable for practicing the subject methods are also described.
1. A method comprising:
contacting a biological sample derived from a tumor comprising cells, cellular debris, tissue matter, cell aggregates and non-cellular components comprising connective tissue, free lipids, proteins and nucleic acid fragments with an amount of assay reagent comprising one or more analyte-specific binding members to produce a biological sample assay composition comprising cells labelled with an analyte-specific binding member and excess assay reagent;
introducing the biological sample assay composition into an inlet of a flow channel of an acoustic separator that is integrated into a flow cytometer, wherein the biological sample assay composition is introduced directly following preparation into the flow cytometer without intervening steps and wherein the integrated acoustic separator comprises:
a flow channel comprising an inlet and an outlet and walls that extend from the inlet to the outlet; and
an acoustic field generator positioned adjacent to the flow channel and configured to produce an acoustic field in the flow channel;
conveying the biological sample assay composition along the walls of the flow channel of the acoustic separator with a laminating wash buffer flowing therebetween; and
applying an acoustic standing wave in the flow channel in a manner sufficient to move one or more of the cellular debris, tissue matter, cell aggregates, non-cellular components and excess assay reagent from the biological sample assay composition flowing along the walls of the flow channel into the laminating wash buffer flowing therebetween, thereby producing a waste fluid.
2. The method according to claim 1 , wherein the method further comprises monitoring the waste fluid with a detector positioned at the outlet of the flow channel.
3. The method according to claim 1 , wherein the biological sample has a volume of 50 μl or less.
4. The method according to claim 1 , wherein the biological sample is selected from the group consisting of a fine needle aspirate, tissue biopsy, pediatric sample, nasal lavage, bronchial lavage, pleural lavage and a lysed blood sample.
5. The method according to claim 4 , wherein the biological sample is a fine needle aspirate.
6. The method according to claim 1 , wherein the method further comprises detecting cells of the biological sample composition at the outlet of the acoustic separator.
7. The method according to claim 6 , wherein detecting comprises detecting light scatter from the cells of the biological sample composition.
8. The method according to claim 6 , wherein detecting comprises detecting fluorescence from the cells of the biological sample composition at the outlet of the acoustic separator.
9. The method according to claim 1 , further comprising determining the makeup of the biological sample composition.
10. The method according to claim 9 , wherein determining the makeup of the biological sample composition comprises counting the number of each type of cell in the biological sample composition.
11. The method according to claim 9 , further comprising adjusting one or more parameters of the acoustic separator in response to the determined makeup of the biological sample composition.
12. The method according to claim 11 , wherein the method comprises adjusting the flow rate of the biological sample composition through the acoustic separator in response to the determined makeup of the biological sample composition.
13. The method according to claim 11 , wherein the method comprises adjusting the acoustic field strength in the acoustic separator in response to the determined makeup of the biological sample composition.
14. The method according to claim 1 , wherein the analyte-specific binding member is an antibody or antibody fragment.
15. The method according to claim 14 , wherein the antibody or antibody fragment specifically binds to a target selected from CD14, CD4, CD45RA, CD3 or a combination thereof.
16. The method according to claim 1 , wherein contacting the biological sample with the assay reagent comprises labeling one or more cells in the biological sample composition with a detectable label.
17. The method according to claim 16 , wherein the detectable label is an optically detectable label.
18. The method according to claim 17 , wherein the optically detectable label comprises a fluorescent dye.
19. The method according to claim 1 , wherein laminating wash buffer has a greater density than the biological sample assay composition.
20. The method according to claim 19 , wherein the laminating wash buffer has a density that is 5% or greater than the biological assay composition.
21. The method according to claim 1 , wherein the acoustic standing wave is applied in a manner sufficient to move one or more non-cellular components from the biological sample assay composition into the laminating wash buffer based on the size of the non-cellular components.
22. The method according to claim 1 , wherein the assay reagent is dry.
23. The method according to claim 1 , wherein the method further comprises re-directing the biological sample assay composition through the acoustic separator for one or more additional washing intervals.