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Updated: Aug 8, 2026
Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning
Published on: November 19, 2018
Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using
G H Segal1, M G Edinger, M Owen
1Department of Pathology, Cleveland Clinic Foundation, Ohio 44195.
Insights
Flow cytometry accurately enumerates B-cell subpopulations using multiparameter immunofluorescence. This method precisely identifies B-cell clonality and lineage, correlating well with immunocytology and histopathology for lymphoid proliferations.
Area of Science:
- Immunology
- Hematology
- Flow Cytometry
Background:
- Accurate enumeration of B-cell subpopulations is crucial for diagnosing lymphoid disorders.
- Traditional methods can be labor-intensive and may lack precision in complex lymphoid tissues.
- Multiparameter flow cytometry offers a potential solution for detailed B-cell analysis.
Purpose of the Study:
- To evaluate the efficacy of a three-color immunofluorescence flow cytometric analysis (FCM) for enumerating B-cell subpopulations.
- To assess the correlation of FCM-derived kappa/lambda ratios with immunocytology and histopathology.
- To determine the concordance of FCM-defined immunophenotype, clonality, and lineage with genotypic results.
Main Methods:
- Utilized multiparameter three-color immunofluorescence with phycoerythrin (PE)-anti-CD19, biotinylated anti-HLADr/streptavidin-Duochrome, and fluorescein isothiocyanate (FITC)-conjugated anti-surface immunoglobulin (SIg) antibodies.
- Established fluorescence thresholds using autofluorescence measurements and controlled for non-specific binding with isotype controls.
- Analyzed cell suspensions from 128 samples of lymphoid proliferations.
Main Results:
- FCM achieved accurate and consistent enumeration of B-cell subpopulations.
- Kappa/lambda ratios from FCM correlated well with immunocytology and histopathology in 116 of 128 samples.
- Immunotypic definition of clonality and lineage by FCM was concordant with genotypic results in 64 of 67 cases.
Conclusions:
- Multiparameter three-color immunofluorescence and FCM provide accurate and consistent enumeration of B-cell subpopulations.
- This FCM approach demonstrates strong correlation with established diagnostic methods for lymphoid proliferations.
- FCM is a reliable tool for assessing B-cell clonality, lineage, and phenotypic expression/deletion in lymphoid tissues.
Abstract:
Accurate and consistent enumeration of B-cell subpopulations in lymphoid tissue was achieved through multiparameter three-color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)-anti-CD19 (Leu12) and biotinylated anti-HLADr/streptavidin-Duochrome (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti-surface immunoglobulin (SIg) antibodies, effectively separated non-specific binding and background fluorescence from true B-cell surface FITC immunofluorescence, while concomitantly analyzing for HLADr and CD19 phenotypic expression/deletion. Autofluorescence was measured to establish a fluorescence threshold. A second control measured non-specific binding of isotypic control mouse Ig and non-immune rabbit IgG. Cell suspensions from 128 samples of various lymphoid proliferations were studied. In 116 of the 128 samples, kappa/lambda ratios determined by flow cytometry correlated well with immunocytology results obtained using cytospins from the same cell suspension and with histopathologically established diagnosis. Clonality and lineage as defined immunotypically by flow cytometry was concordant with genotypic results in 64 of the 67 cases evaluated. SIg, HLADr, and CD19 deletions were demonstrated by flow cytometry in 8, 4, and 1 case(s), respectively. Discordance was usually attributable to selective loss of large neoplastic cells in flow cytometry specimens or absent expression of SIg by some cytoplasmic Ig (CIg+) lymphomas.
