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Updated: Apr 26, 2026
Flow Cytometric Characterization of Murine B Cell Development
Published on: January 22, 2021
Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell
Lisa M Rimsza1, William A Day, Sarah McGinn
1Department of Pathology, University of Arizona, Tucson, AZ, USA. lrimsza@email.arizona.edu.
Insights
Colorimetric in situ hybridization (CISH) accurately detects B cell clonality in mature B cell neoplasms using formalin-fixed paraffin-embedded biopsies. This sensitive method improves upon existing techniques for diagnosing B cell lymphomas.
Area of Science:
- Hematopathology
- Molecular Pathology
- Diagnostic Cytogenetics
Background:
- B cell clonality detection aids in diagnosing B cell lymphomas.
- KAPPA and LAMBDA light chain expression analysis is key for clonality assessment.
- Current slide-based methods have limitations in detecting light chain expression in lymphomas.
Purpose of the Study:
- To evaluate the utility of a novel colorimetric in situ hybridization (CISH) method for B cell clonality detection.
- To assess the sensitivity and multiplexing capacity of CISH for mature B cell malignancies.
- To compare CISH performance against established methods like flow cytometry and immunohistochemistry (IHC).
Main Methods:
- KAPPA and LAMBDA mRNA detection using two-color chromogenetic in situ hybridization (ISH) on a Ventana Benchmark XT.
- Utilized haptenated riboprobes targeting conserved mRNA regions with silver (black) for KAPPA and a novel chromogen (pink) for LAMBDA.
- Tested on 79 formalin-fixed paraffin-embedded (FFPE) B cell lymphoma biopsies, including follicular, mantle cell, marginal zone, lymphoplasmacytic, small lymphocytic, and diffuse large B cell types.
Main Results:
- CISH classified 49.4% of cases as KAPPA-restricted and 36.7% as LAMBDA-restricted, with 11.3% indeterminate.
- Of the 70 cases with restricted light chain expression by CISH, 98.6% were concordant with reference methods (flow cytometry or IHC).
- Optimized CISH demonstrated visualization of mRNA in benign B cells and detected lower mRNA levels than current slide-based methods.
Conclusions:
- Optimized CISH enables clonality assessment in FFPE biopsies for mature B cell neoplasms by detecting lower mRNA levels.
- CISH demonstrated high accuracy in this preliminary study, comparable to flow cytometry and IHC.
- CISH offers potential for broader application in light chain ISH and is poised to become a valuable diagnostic tool.
Background:
Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies.
Methods:
The KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator.
Results:
39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted; while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant.
Conclusions:
Optimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool.
Virtual Slides:
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856.
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