Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Summary

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Abstract

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Introduction

Lymphoid neoplasms are caused by the uncontrolled malignant proliferation of lymphocytes. These cells are vital components of the immune system and localize to the primary and secondary immune organs, such as the bone marrow, lymph nodes, spleen, and other mucosa-associated lymphoid system. Lymphoid neoplasms are a heterogeneous group of disorders who are classified based on a constellation of features, including morphology, immunophenotype, genetic features, and clinical presentation. While each parameter plays a part, lineage remains a defining feature and forms the basis for the WHO classification system which recognizes neoplasms derived from B cells, T cells, and....

Protocol

All tissues used in this protocol were obtained under the Singapore NHG Domain Specific Review Board B study 2014/00693.

1. Selection and Validation of Antibodies

NOTE: Before proceeding with the establishment of any multiplexed panel, ensure that all antibodies stain robustly, identifying only the target antigen of interest. The aim is to select antibodies that specifically recognize the antigen of interest in tissue sections.

1. For an antibody with a well-established research use, or routine clinical use in IHC, confirm conditions such as epitope retrieval and antibody dilution by performing conventio....

Results

mf-IHC images for a DLBCL sample with C-MYC and BCL2 gene rearrangement (double-hit lymphoma) are shown in Figure 1. Figure 2 illustrates the simulated bright-field immunohistochemical images. Figure 3 indicates the generation of percentage data. Figure 4 displays the details of a median formula for the generation of numeric data. Figure 5 s.......

Discussion

mf-IHC has the potential to enable pathologists to refine diagnosticcriteria in lymphoid pathology and to analyze the role of biomarkers in specific cell types toward a prediction of clinical outcome. As a new research method, mf-IHC is increasingly applied to the quantitative and spatial identification of multiple immune parameters of tumor cells¹⁷. The detection of mf-IHC for the co-expression of tumor biomarkers has been shown to be reproducible and reliable⁵. However, t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibody diluent	DAKO	REF S3022	Blocking
Peroxidase Blocking Solution	DAKO	S2023	For peroxide blocking
Vectra multispectral automated microscope	Perkin Elmer	Vectra2.0.8	Spectral imaging
absolute Ethanol	EMSURE	1.00983.2500	Ethyl alcohol for rehydration
Amplification Diluent	PERKIN ELMER	FP1135	Fluorophore diluent buffer
Anti-Mouse IgG [Goat] HRP-Labeled	PERKIN ELMER	NEF822001EA	Secondary antibody
Anti-Rabbit IgG [Goat] HRP-Labeled	PERKIN ELMER	NEF812001EA	Secondary antibody
BCL2	DAKO	clone 124 ( Lot No. 20031561)(RRID-AB578693)	primary antibody
BCL6	LEICA	NCL-L-Bcl6-564(Lot No.6050438)(RRID-AB563429)	primary antibody
CD20	DAKO	Clone L26 (Lot No.20028627) (RRID-AB442055)	primary antibody
c-MYC	ABCAM	Y 69 clone ab32072 (Lot NO.GR29511133)(RRID-AB731658)	primary antibody
Cy 5	PERKIN ELMER	FP1171	Appropriate tyramide based fluorescent reagent
Graphpad Prism 7	Graph pad		Statisitcal software
HistoClear Clearing Agent	SIGMA	H2779-1L	Histoclear for dewaxing and clearing
inForm Advanced Image Analysis Software	Perkin Elmer	Inform Software 2.2.1	Data Analysis software
KI67	DAKO	Clone MIB-1 (Lot No.20040401) (RRID-AB2314699)	primary antibody
KOS MILESTONE multifunctional tissue processor	Milestone		Microwave for Heat induced epitope retrieval
Microwave	PANASONIC	NN-ST651M	Microwave stripping
Mowiol	SIGMA ALDRICH	81381 Aldrich Mowiol® 4-88	mounting media
Opal 570	PERKIN ELMER	FP1488	Appropriate tyramide based fluorescent reagent
Opal520	PERKIN ELMER	FP1487B21	Appropriate tyramide based fluorescent reagent
Opal540	PERKIN ELMER	FP1494	Appropriate tyramide based fluorescent reagent
Opal620	PERKIN ELMER	FP1495	Appropriate tyramide based fluorescent reagent
Poly-L-lysine coated slide	FISHER SCIENTIFIC	120-550-15	Slide for tissue section adhesion in routine histological use
Spectral DAPI	PERKIN ELMER	FP1490	nucleic acid stain
Target Retrieval Solution, pH9.0(10x)	DAKO	S2367	For HIER
Tris Buffer saline (TBS)	1st BASE	BUF3030 20X4L	for buffer wash
Tween 20	SIGMA ALDRICH	P1379-1L	Tween