Patrick S. Chang; Caleb Chan; Rajiv Jesudason; Sandra Rost; Cary D. Austin

doi:10.21769/BioProtoc.5797

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Peer-reviewed

An Automated, Ventana Discovery Platform-based Imaging Workflow for Simultaneous Quantification of B Cells, Plasma Cells, and Plasmablasts in FFPE Human Tissues

CA Cary D. Austin email

发布: 2026年09月05日第16卷第17期 DOI: 10.21769/BioProtoc.5797 浏览次数: 44

评审: Alessandro DidonnaSunjay J FernandesAnonymous reviewer(s)

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Cover of Frontiers in Immunology, featuring study using the protocol.

Mar 2026

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Abstract

Accurate, sensitive quantification of B-lineage cells is critical for pharmacodynamic evaluation of B cell–targeted therapies in lupus nephritis (LN) clinical trials. While high-dimensional discovery platforms offer broad profiling, they often lack the sensitivity, quantitative rigor, and throughput needed for precise cell enumeration in renal trial needle biopsies. Traditional immunostaining is hampered by CD20-directed therapeutic masking or downregulation, inadequate sensitivity of CD19 in FFPE tissue, and confounding renal tubular CD138 expression. This protocol details an automated, fit-for-purpose, 5-plex sequential tyramide signal amplification (TSA)-based immunofluorescence assay (CD38, CD79a, CD19, Ki-67, CD138) developed on the Ventana Discovery Ultra platform for deployment on single tissue sections. The workflow anchors B-cell detection on CD79a to ensure sensitivity and utilizes CD38 as an obligate co-marker for CD138+ antibody-secreting cells (ASCs) to definitively exclude the CD138+ epithelial background. Following acquisition via fluorescence whole-slide imaging, a digital analysis pipeline utilizing InstanSeg-based automated segmentation rigorously classifies cell phenotypes to generate precise spatial densities (cells/mm²). This validated protocol maximizes data yield from scarce clinical biopsies while providing high-precision quantitative monitoring of longitudinal therapeutic depletion in the renal microenvironment.

Key features

• Automated trial scalability: Details an automated, TSA-based 5-plex assay optimized for the Ventana Discovery Ultra platform, ensuring high-throughput, reproducible B-lineage quantification in multicenter cohorts.

• Robust lineage identification: Establishes CD79a as an anchor for sensitive B-cell detection in FFPE tissue, avoiding CD20 therapeutic masking/downregulation and CD19 epitope instability.

• High-specificity ASC enumeration: Resolves confounding CD138+ renal tubular background using an obligate CD38/CD138 co-labeling strategy to definitively identify ASCs within the renal microenvironment.

• Single-section phenotypic resolution: Combines multiplexing and InstanSeg segmentation on one 4-μm section to simultaneously quantify B-lineage subsets in tissue-limited renal needle biopsies.

Keywords: Lupus nephritis

B cells

Plasma cells

Plasmablasts

Antibody-secreting cells (ASCs)

Quantitative-multiplex immunofluorescence

FFPE tissue

Spatial density

Graphical overview

Automated multiplex immunofluorescence and digital phenotyping workflow. (A) Formalin-fixed, paraffin-embedded (FFPE) tissue sections (4 μm) undergo automated 5-plex tyramide signal amplification immunofluorescence (CD38, CD79a, CD19, Ki-67, CD138, plus DAPI) on the Ventana DISCOVERY Ultra. (B) Slides are digitized via 7-channel whole-slide imaging (20×) on the EVIDENT SLIDEVIEW VS200 and imported into OMERO. Digital processing includes median blur filtering, background subtraction, InstanSeg cell segmentation, and single-cell MFI calculation. (C) A decision logic tree classifies segmented single cells into B cells (CD79a+/CD138-) and antibody-secreting cells (ASCs; CD138+/CD38+). ASCs are further stratified into plasma cells (Ki-67-) and plasmablasts (Ki-67+) to yield final spatial densities (cells/mm²).

Background

Accurate spatial enumeration of B-lineage cells within the renal microenvironment is essential for understanding the pathogenesis of lupus nephritis (LN), where B cells and antibody-secreting cells (ASCs), which include plasma cells (PCs) and plasmablasts (PBs), mediate kidney injury [1]. Monitoring these populations in formalin-fixed, paraffin-embedded (FFPE) kidney tissue is critical for evaluating the pharmacodynamic efficacy of B cell–targeted therapies in clinical trials. Traditional immunohistochemical (IHC) approaches often rely on single markers such as CD20 for B cells and CD138 for plasma cells. However, in the context of CD20-directed depletion therapies, CD20 is unreliable for quantifying total B-lineage burden because it can be masked or internalized following therapeutic depletion [2,3]. Additionally, while CD19 is a canonical B-cell marker often used in flow cytometry studies [4], its detection in FFPE tissue lacks the sensitivity required for rigorous quantitative analysis due to poor epitope preservation. CD138 (Syndecan-1), often used as a standalone marker for ASC identification, is unreliable in renal tissue due to substantial endogenous CD138 expression on renal tubular epithelium, creating a significant risk of false-positive ASC identification [5,6]. While high-dimensional spatial platforms offer broad inflammatory cell profiling, they often lack the throughput and quantitative rigor required for precise cell enumeration in large multicenter clinical cohorts. Furthermore, reliance on serial sections for multiple markers is often unfeasible for scarce LN needle biopsy material.

This multiplex protocol (see Graphical overview) provides an automated, sequential tyramide signal amplification (TSA) workflow [7] (CD38, CD79a, CD19, Ki-67, CD138) developed on the Ventana Discovery Ultra platform. Key advantages include establishing CD79a as a requisite anchor, which remains detectable, even when CD20 is lost or masked [8,9], providing far greater sensitivity than CD19 in FFPE sections [10,11]. To ensure specific renal ASC identification, the protocol employs an obligate CD38/CD138 co-labeling strategy to definitively distinguish them from the confounding renal tubular epithelium. Finally, the workflow allows for the efficient use of tissue by combining all markers and InstanSeg-based digital segmentation [12] on a single 4-μm section, maximizing data yield from material-constrained clinical trial samples.

A limitation of this methodology is the requirement for specific automated staining hardware and digital analysis expertise. While validated specifically for LN, this robust, fit-for-purpose workflow is adaptable for any FFPE tissue where high-precision B-lineage enumeration is required. Furthermore, the method is agile and amenable to swapping in or out these markers with other markers of interest.

Materials and reagents

Biological materials

1. FFPE human tissue (validation samples were de-identified clinical remnants acquired from Avaden Biosciences; however, the protocol is broadly applicable to clinical trials, clinical remnants, or other commercial specimens)

a. Target tissue: Designed for lupus nephritis (LN) needle biopsies, but adaptable to other tissue types; if feasible, standardization of fixation (e.g., 16–24 h in 10% neutral buffered formalin at ambient temperature), automated processing, and embedding in histology-grade paraffin is recommended

b. Control tissue: Secondary lymphoid tissue with germinal centers (e.g., palatine tonsil) to validate B-cell maturation spectrum, including B cell (CD19, CD79a), ASC (CD38, CD138), and proliferation Ki-67 markers; tonsillar epithelium provides a reference for the epithelial CD138 expression

Reagents

Note: All reagents should be stored at 4 °C unless otherwise noted.

1. Discovery CC1 (predilute) (Roche Diagnostics, catalog number: 950-500); store at room temperature (RT)

2. DISCOVERY inhibitor (Roche Diagnostics, catalog number: 760-4840)

3. CD38 (clone: SPC32) antibody (Leica Biosystems, catalog number: NCL-L-CD38-290)

4. Bovine serum albumin (BSA) (Fisher Scientific, SKU #501657297)

5. Phosphate-buffered saline (PBS) 1×, sterile-filtered (Life Technologies, SKU #J61196.AP); store at RT

6. Goat Ig block (Roche Diagnostics, catalog number: 760-6008)

7. OmniMap anti-mouse HRP (Roche Diagnostics, catalog number: 760-4310)

8. Discovery Rhodamine 6G (Roche Diagnostics, catalog number: 760-244)

9. Ultra CC2 (Roche Diagnostics, catalog number: 950-223); store at RT

10. CD79a (clone: SP18) antibody (Roche Diagnostics, catalog number: 790-4432)

11. OmniMap anti-rabbit HRP (Roche Diagnostics, catalog number: 760-4311)

12. Discovery FAM (Roche Diagnostics, catalog number: 760-243)

13. CD19 (clone: LE-CD19) antibody (Agilent, catalog number: M7296)

14. Discovery Cy5 (Roche Diagnostics, catalog number: 760-238)

15. Ki-67 (clone: D3B5) antibody (Cell Signaling, catalog number: 12202S); store at -20 °C

16. Discovery Red610 (Roche Diagnostics, catalog number: 760-245)

17. CD138 (clone: B-A38) antibody (Roche Diagnostics, catalog number: 760-4248)

18. Opal 780 Reagent Pack (Akoya Biosciences, catalog number: FP1501001KT); store at -20 °C in powder form

19. DAPI nucleic acid stains (ThermoFisher, catalog number: D1306); store at -20 °C

20. 1× Plus automation amplification diluent (Perkin Elmer, catalog number: FP1609)

21. Antibody diluent/block (Perkin Elmer, catalog number: ARD1001EA)

22. Dawn ultra liquid dish detergent (Procter & Gamble, catalog number: 24386798)

23. ProLong Gold antifade reagent (Invitrogen, catalog number: P36930)

Solutions

1. 3% BSA (see Recipes)

2. CD38 antibody solution (see Recipes)

3. CD19 antibody solution (see Recipes)

4. Ki-67 antibody solution (see Recipes)

5. TSA-DIG stock solution (see Recipes)

6. TSA-DIG working solution (see Recipes)

7. Anti-DIG Opal 780 stock solution (see Recipes)

8. Anti-DIG Opal 780 working solution (see Recipes)

9. DAPI solution (see Recipes)

10. Slide cleaning solution (see Recipes)

Recipes

Notes:

1. Recipes for working antibody solutions and DAPI are calculated for a 10-slide batch. This volume specifically accounts for the 1-mL dead volume required by Ventana reagent dispensers to prevent bubble introduction.

2. All solutions should be stored at 4 °C, except the slide cleaning solution.

1. 3% BSA

Reagent	Final concentration	Quantity or volume
BSA	3%	30 g
PBS	1×	1 L
Total	n/a	1 L

Filter-sterilize (0.2 μm). Stable at 4 °C for up to 1 year.

2. CD38 antibody solution

Reagent	Final concentration	Quantity or volume
CD38 stock solution	46 μg/mL	20 μL
3% BSA (Recipe 1)	n/a	1,980 μL
Total	0.46 μg/mL	2 mL

Stable at 4 °C for up to 2 weeks.

3. CD19 antibody solution

Reagent	Final concentration	Quantity or volume
CD19 stock solution	83 μg/mL	20 μL
3% BSA (Recipe 1)	n/a	1,980 μL
Total	0.83 μg/mL	2 mL

Stable at 4 °C for up to 2 weeks.

4. Ki-67 antibody solution

Reagent	Final concentration	Quantity or volume
Ki-67 stock solution	270 μg/mL	2.22 μL
3% BSA (Recipe 1)	n/a	1997.78 μL
Total	0.3 μg/mL	2 mL

Stable at 4 °C for up to 2 weeks.

5. TSA-DIG stock solution

Reagent	Final concentration	Quantity or volume
Opal TSA-DIG	n/a	1 vial
Dimethyl sulfoxide (DMSO)	n/a	75 μL
Total	n/a	75 μL

Opal TSA-DIG and DMSO are part of the Opal 780 Reagent Pack. Stable at 4 °C for up to 1 month.

6. TSA-DIG working solution

Reagent	Final concentration	Quantity or volume
Opal TSA-DIG stock solution (Recipe 5)	n/a	20 μL
1× Plus automation amplification diluent	n/a	1,980 μL
Total	1:100	2 mL

Store at 4 °C; it should be used within 3 weeks of preparation for the best outcome.

7. Anti-DIG Opal 780 stock solution

Reagent	Final concentration	Quantity or volume
Anti-DIG Opal 780	n/a	1 vial
Deionized H₂O	n/a	300 μL
Total	n/a	300 μL

Anti-DIG Opal 780 is part of the Opal 780 Reagent Pack. Stable at 4 °C for up to 1 month.

8. Anti-DIG Opal 780 working solution

Reagent	Final concentration	Quantity or volume
Anti-DIG Opal 780 stock solution (Recipe 7)	n/a	80 μL
Antibody Diluent/Block	n/a	1920 μL
Total	1:25	2 mL

Prepare in an ASSY, LP PREP KIT dispenser. Store at 4 °C; it should be used within 3 weeks of preparation for the best outcome.

9. DAPI solution

Reagent	Final concentration	Quantity or volume
DAPI stock solution	5 mg/mL	8 μL
Deionized H₂O	n/a	1 mL
PBS	1×	199 mL
Total	0.2 μg/mL	200 mL

Prepare in an ASSY, LP PREP KIT dispenser. Stable at 4 °C for up to 1 month.

10. Slide cleaning solution

Reagent	Final concentration	Quantity or volume
Dawn Ultra liquid dish soap	n/a	50 mL
Deionized H₂O	n/a	150 mL
Total	1:4	200 mL

Store at RT.

Laboratory supplies

1. Superfrost Plus glass slides (Thermo Fisher Scientific, Waltham, MA, USA, catalog number 4951PLUS-001 or equivalent)

Critical: Use of high-adhesion slides is critical to prevent tissue detachment during sequential heat-induced epitope retrieval cycles.

2. DISCOVERY, REAGENT DISP. CARD, Detection 1 (Roche Diagnostics, catalog number: 05280192001)

3. DISCOVERY, REAGENT DISP. CARD, Detection 2 (Roche Diagnostics, catalog number: 05280206001)

4. NEXES REAGENT DISP. CARD, CNTRSTAIN 1 (Roche Diagnostics, catalog number: 05271720001)

5. ASSY, LP PREP KIT (Roche Diagnostics, catalog number: 7475144001); brown dispenser to prevent light damage to the solution

6. Nexes Primary Antibody 1 Dispenser (Roche Diagnostics, catalog number: 05269857001)

7. Nexes Primary Antibody 2 Dispenser (Roche Diagnostics, catalog number: 05269865001)

8. Nexes Primary Antibody 3 Dispenser (Roche Diagnostics, catalog number: 05269873001)

9. 24 × 50 mm No. 1 SuperSlip coverslip (VWR, catalog number: 48404-453)

10. Acrodisc PF syringe filter with 0.8/0.2 μm Supor membrane non-pyrogenic, sterile (Pall Life Sciences, catalog number: 4658)

Equipment

1. Desert Chamber Oven (Biocare Medical, model: DRY2008US)

2. Ventana DISCOVERY ULTRA (Roche Diagnostics, catalog number: 05987750001)

3. SLIDEVIEW VS200 Universal Whole Slide Imaging Scanner (EVIDENT, model: VS200), including the following filter cubes:

a. DAPI LED Filter Set 378/52X/BS409/447/60M zeropixel w/ BX3 cube (EVIDENT, model: OSF-DAPIZLEDBX3)

b. CFP Zero LED Filter (EVIDENT, model: OSF-CFPZLEDBX3)

c. FITC LED Filter Set 474/27X/BS495/525/45M zeropixel w/ BX3 cube (EVIDENT, model: OSF-FITCZLEDBX3)

d. ET-Gold/Yellow Filter Set 546/10×, BS556, 572/23M w/ BX3 cube (EVIDENT, model: OCT-49304BX3)

e. ET-Red#1 FISH Filter Set 580/25×, BS600, 625/30M w/ BX3 cube (EVIDENT, model: OCT-49306BX3)

f. ET-CY5 Filter Set 640/30×, BS660, 690/50M w/ BX3 cube (EVIDENT, model: OCT-49009BX3)

g. Cy7 LED Filter Set 735/28×/BS757/809/81M zeropixel w/ BX3 cube (EVIDENT, model: OSF-CY7ZLEDBX3)

Software and datasets

Staining and scanning

1. Ventana NexES, VSS (Roche Diagnostics, version 12.5.4; included with the instrument)

2. VS200 ASW (EVIDENT, version 4.3; included with the instrument)

Data analysis

The digital analysis pipeline described herein (including image processing, segmenting whole-slide images, and single-cell feature extraction) was successfully performed on a high-performance computing node (HPC) with 128 GB RAM, an Intel Xeon Gold 6248 CPU, and an NVIDIA V100 GPU with 32 GB VRAM to efficiently run InstanSeg and handle large OME-TIFF files locally.

1. NGFF-Converter (Glencoe Software, version 2.0.2; freely available at https://www.glencoesoftware.com/products/ngff-converter/)

2. OMERO (Glencoe Software, version OMERO.web 5.29.2; freely available at https://pypi.org/project/omero-web/)

3. InstanSeg (v0.1.0; freely available at https://github.com/instanseg/instanseg)

4. Custom Python analysis scripts (Threshold Explorer and tissue area calculations). Scripts were executed in Python 3.12. The specific repository version 0.1.0, source code, and full environment dependencies (listed in pyproject.toml) are freely available at https://github.com/Genentech/sp_pipeline_public. Please refer to the repository’s README file for the installation of dependencies.

Procedure

文章信息

稿件历史记录

提交日期: May 29, 2026

接收日期: Jul 20, 2026

在线发布日期: Aug 12, 2026

出版日期: Sep 5, 2026

版权信息

如何引用

Chang, P. S., Chan, C., Jesudason, R., Rost, S. and Austin, C. D. (2026). An Automated, Ventana Discovery Platform-based Imaging Workflow for Simultaneous Quantification of B Cells, Plasma Cells, and Plasmablasts in FFPE Human Tissues. Bio-protocol 16(17): e5797. DOI: 10.21769/BioProtoc.5797.