Methods and systems for preparing and analyzing cellular samples for morphological characteristics and biomarker expression

Patent metadata

Field	Value
Publication number	US-12590871-B2
Application number	US-202117303092-A
Country	US
Kind code	B2
Filing date	May 19, 2021
Priority date	Nov 20, 2018
Publication date	Mar 31, 2026
Grant date	Mar 31, 2026

Abstract

This disclosure relates generally to the use of automated platforms in the preparation of biomarker-stained cellular samples for microscopic analysis and use of such stained cells in the diagnosis of certain conditions. Disclosed herein is a method of affinity staining a Romanowsky-type stained sample on automated advanced staining systems, wherein the automated advanced stainer destains the sample prior to contact with a biomarker-specific reagent. Also disclosed herein are methods of processing body fluid samples for morphological and biomarker analysis by depositing cells of the sample in a thin layer onto one or more solid supports, staining at least one such solid support with a Romanowsky-type stain and staining at least one such solid support for one or more biomarkers useful for categorizing one or more cells of the sample.

First claim

The invention claimed is:

1 . An automated method of performing a cytological evaluation, the method comprising:
(a) printing a monolayer of cells of a cytology sample onto a surface of a stationary solid support, wherein the monolayer of cells are arranged in parallel rows on the surface of the stationary solid support, wherein the printing comprises flowing a volume of about 0.1 to about 10 μL of the cytology sample through a movable applicator tip to the surface of the stationary solid support, wherein the movable applicator tip is movable in the x-, y-, and z-coordinate directions relative to the surface of the stationary solid support; (b) staining the deposited cells with a Romanowsky-type stain to obtain a Romanowsky-type stained cytology sample; (c) acquiring one or more digital images of the Romanowsky-type stained cytology sample; (d) automatically classifying cells in the acquired one or more digital images of the Romanowsky-type stained cytology sample based on morphology to identify one or more different cell types; (e) counting each of the identified one or more different cell types; (f) incubating the Romanowsky-type stained cytology sample with a cell conditioning solution for at least 5 minutes to obtain a conditioned Romanowsky-type stained cytology sample; and (g) reacting the conditioned Romanowsky-type stained cytology sample with one or more biomarker-specific reagents and a set of detection reagents under conditions sufficient to deposit a brightfield label or a fluorescence label in proximity to a biomarker expressed by the conditioned Romanowsky-type stained cytology sample to provide a biomarker-stained cytology sample.

2 . The method of claim 1 , wherein said solid support is a microscope slide.

3 . The method of claim 1 , wherein the cell conditioning solution comprises a basic or acidic cell conditioning solution.

4 . The method of claim 1 , wherein the brightfield label or fluorescence label is a dye.

5 . The method of claim 4 , wherein the dye is deposited by an affinity enzymatic reaction.

6 . The method of claim 5 , wherein the Romanowsky-type stained cytology sample is a whole blood sample, and said affinity enzymatic reaction is a simplex affinity enzymatic reaction.

7 . The method of claim 5 , wherein the Romanowsky-type stained cytology sample is a whole blood sample, and the affinity enzymatic reaction is a multiplex affinity enzymatic reaction that results in differential staining of a group of biomarkers with fluorescent dyes.

8 . The method of claim 5 , wherein the Romanowsky-type stained cytology sample is a whole blood sample, and the affinity enzymatic reaction is a multiplex affinity enzymatic reaction that results in differential staining of a group of biomarkers with brightfield dyes.

9 . The method of claim 5 , wherein said affinity enzymatic reaction is selected from the group consisting of an immunoenzymatic stain, a genomic in situ hybridization stain, a mRNA in situ hybridization stain, and combinations thereof.

10 . The method of claim 1 , wherein the Romanowsky-type stained cytology sample is fixed in an alcohol-based fixative.

11 . The method of claim 10 , wherein the alcohol is methanol.

12 . The method of claim 1 , wherein said Romanowsky-type cytology stained sample is fixed in an aldehyde-based fixative.

13 . The method of claim 12 , wherein the aldehyde is formaldehyde.

14 . The method of claim 13 , wherein the formaldehyde-based fixative is neutral-buffered formalin.

15 . The method of claim 1 , wherein the one or more biomarker-specific reagents bind at least one biomarker selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD 19, CD20, CD23, CD27, CD34, CD38, CD45, CD68, CyclPBERin D1, Myeloperoxidase (MPO), Paired box protein Pax-5 (PAX5), DNA nucleotidylexotransferase (TdT), and Tyrosine-protein kinase ZAP-70 (ZAP-70).

16 . The method of claim 1 , wherein the cell conditioning solution is a citrate-based solution.

17 . The method of claim 1 , wherein the cell conditioning solution has a pH ranging from about 5 to about 6.5.

18 . The method of claim 1 , further comprising capturing one or more digital images of the biomarker-stained cytology sample.

19 . The method of claim 18 , further comprising automatically classifying at least one cell in the captured one or more digital images of the biomarker-stained cytology sample for biomarker-specific staining.

20 . An automated method of performing a cytological evaluation, the method comprising:
(a) printing in concentric circles a monolayer of cells of a cytology sample onto a surface of a stationary solid support, wherein the printing comprises flowing the cytology sample through a movable applicator tip to the surface of the stationary solid support, wherein the movable applicator tip is movable in the x-, y-, and z-coordinate directions relative to the surface of the stationary solid support; (b) staining the deposited cells with a Romanowsky-type stain to obtain a Romanowsky-type stained cytology sample; (c) acquiring one or more digital images of the Romanowsky-type stained cytology sample; (d) automatically classifying cells in the acquired one or more digital images of the Romanowsky-type stained cytology sample based on morphology to identify one or more different cell types; (e) counting each of the identified one or more different cell types; (f) incubating the Romanowsky-type stained cytology sample with a cell conditioning solution for at least 5 minutes to obtain a conditioned Romanowsky-type stained cytology sample; and (g) reacting the conditioned Romanowsky-type stained cytology sample with one or more biomarker-specific reagents and a set of detection reagents under conditions sufficient to deposit a brightfield label or a fluorescence label in proximity to a biomarker expressed by the conditioned Romanowsky-type stained cytology sample to provide a biomarker-stained cytology sample.

21 . An automated method of performing a cytological evaluation, the method comprising:
(a) printing in parallel rows or in concentric circles a monolayer of cells of a cytology sample onto a surface of a stationary solid support, wherein the printing comprises flowing the cytology sample through a movable applicator tip to the surface of the stationary solid support; (b) staining the deposited cells with a Romanowsky-type stain to obtain a Romanowsky-type stained cytology sample; (c) acquiring one or more digital images of the Romanowsky-type stained cytology sample; (d) automatically classifying cells in the acquired one or more digital images of the Romanowsky-type stained cytology sample based on morphology to identify one or more different cell types; (e) counting each of the identified one or more different cell types; (f) incubating the Romanowsky-type stained cytology sample with a cell conditioning solution comprising a proteolytic enzyme for at least 5 minutes to obtain a conditioned Romanowsky-type stained cytology sample; and (g) reacting the conditioned Romanowsky-type stained cytology sample with one or more biomarker-specific reagents and a set of detection reagents under conditions sufficient to deposit a brightfield label or a fluorescence label in proximity to a biomarker expressed by the conditioned Romanowsky-type stained cytology sample to provide a biomarker-stained cytology sample.

Assignees

• Ventana Med Syst Inc
• Roche Diagnostics Operations Inc
• Roche Diagnostics Hematology Inc

Inventors

• Bhaumik Srabani
• Davidson Simon J
• Kosmeder Ii Jerome W
• Mui Katherine K
• Roberts Esteban
• Sebastiao Noemi
• Zahniser David J

Classifications

• G01N2001/305

  Fixative compositions · CPC title

• G01N2001/302

  Stain compositions · CPC title

• G01N33/5094

  for blood cell populations (red blood cells G01N33/80) · CPC title

• G01N1/30 Primary

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