Size-based separation of dissociated fixed tissues

Patent metadata

Field	Value
Publication number	US-11768137-B2
Application number	US-201916571785-A
Country	US
Kind code	B2
Filing date	Sep 16, 2019
Priority date	Apr 14, 2017
Publication date	Sep 26, 2023
Grant date	Sep 26, 2023

Abstract

The present disclosure provides a method of separating cellular particles from a tissue sample and then sorting the cellular particles into two or more cellular particle populations.

First claim

The invention claimed is:

1. A method of segregating cellular particles from a representative sample comprising: (i) obtaining the representative sample, wherein the representative sample is derived from at least one fixed tissue specimen that has been homogenized, wherein any heterogeneity of cells within the at least one fixed tissue specimen is substantially uniformly distributed within the representative sample; and (ii) sorting cellular particles in at least a first aliquot of the representative sample by size into at least a first cellular particle population and a second cellular particle population, wherein the cellular particles are not tagged or labeled prior to or during the step of sorting.

2. The method of claim 1 , wherein the cellular particles include cells.

3. The method of claim 1 , wherein the cellular particles include cell nuclei.

4. The method of claim 1 , wherein the second cellular particle population comprises cellular particles derived from tumor cells.

5. The method of claim 4 , wherein the cellular particles derived from tumor cells have an average diameter ranging from between about 12 μm to about 50 μm or from between about 8.5 μm to about 30 μm.

6. The method of claim 5 , wherein the tumor cells are derived from at least one of a whole tumor, a partial tumor, a metastatic tumor, a partial metastatic tumor, or lymph nodes.

7. The method of claim 1 , wherein the representative sample is derived from at least one of fixed residual surgical material or a fixed biopsy sample.

8. The method of claim 1 , wherein the representative sample is derived from a fixed tissue specimen embedded in paraffin.

9. The method of claim 1 , wherein the cellular particles are sorted with a microfluidic device.

10. A method of segregating cells from a homogenized sample comprising:
(i) obtaining a homogenized sample, wherein the homogenized sample is derived from one or more fixed tissue specimens, and where any aliquot of the homogenized sample substantially uniformly expresses the heterogeneity of the one or more fixed tissue specimens; and
(ii) sorting untagged or unlabeled cells in the homogenized tissue sample by size, wherein the cells are sorted into at least a first cellular population enriched in cells having an average diameter of less than 12 μm and a second cellular population enriched in cells having an average diameter of greater than 12 μm, and wherein the cells within the homogenized sample are sorted using a microfluidic device.

11. The method of claim 10 , wherein the microfluidic device is selected from the group consisting of a deterministic lateral displacement device, a hydrophoretic filtration device, a hydrodynamic filtration device, a microfluidic device utilizing inertial focusing in curved channels, and a microfluidic device utilizing inertial focusing in straight channels.

12. The method of claim 10 , wherein the microfluidic device does not require staining of the cells prior to sorting.

13. The method of claim 10 , further comprising analyzing at least one of the first or second cellular populations for one of an RNA biomarker or a protein biomarker.

14. The method of claim 10 , further comprising sequencing at least one of the first or second cellular populations.

15. The method of claim 14 , wherein an amount of genomic material available for sequencing in the first or second cellular populations is at least 0.05 micrograms.

16. The method of claim 15 , wherein the amount of genomic material is at least 0.1 micrograms.

17. The method of claim 14 , wherein at most four amplification cycles are conducted prior to sequencing.

18. The method of claim 14 , wherein the sequencing employs next-generation sequencing.

19. The method of claim 10 , wherein the first and second cellular populations provide matched tumor and normal samples for a patient.

20. The method of claim 10 , wherein the first cellular population is enriched in normal cells and the second cellular population is enriched in tumor cells.

21. A method of segregating cellular particles from a sample comprising sorting cellular particles in the tissue sample by size into at least a first cellular particle population and a second cellular particle population, wherein the cellular particles are not tagged or labeled prior to or during the step of sorting; wherein the sample is derived from at least one fixed tissue specimen, and where any aliquot of the sample substantially homogeneously expresses the heterogeneity of the at least one fixed tissue specimen.

Assignees

• Ventana Med Syst Inc

Inventors

• Alexander Nelson
• Barhoumi Aoune
• Gallegos Lisa
• Stanislaw Stacey

Classifications

• G01N33/57595

  involving intracellular compounds · CPC title

• G01N33/575 Primary

  for cancer · CPC title

• G01N2001/4088

  filtration · CPC title

• G01N2001/2866

  Grinding or homogeneising · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title