A Comparison of IHC and Flow Cytometry in Detecting Hematologic Malignancies

Karen Gaur^*
*Correspondence: Karen Gaur, Department of Hematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India, Email:

Introduction

The accurate and timely detection of hematologic malignancies is critical for guiding effective therapy and improving patient prognosis. Immunophenotyping, the process of identifying cells based on the antigens they express, is central to the diagnosis and classification of blood cancers. Two widely employed methods in this domain are Immunohistochemistry (IHC) and Flow Cytometry (FCM). While both techniques share the objective of identifying specific cellular markers, they differ in methodology, applications, strengths and limitations. A nuanced understanding of these differences is essential for pathologists, hematologists and oncologists involved in the diagnosis and monitoring of hematologic neoplasms. Immunohistochemistry is a histological technique that utilizes antibodies to detect specific antigens within fixed tissue sections. IHC provides morphological context by allowing visualization of antigen expression in relation to cellular architecture, thereby enabling assessment of the pattern, intensity and distribution of marker expression [1,2].

Description

This is particularly valuable in cases involving bone marrow biopsies or lymph node excisions, where architectural features such as follicular patterns, sinusoidal involvement, or interstitial infiltrates are diagnostically relevant. IHC is indispensable in the diagnosis of lymphomas, especially when evaluating paraffin-embedded tissue and is widely used for classifying subtypes according to the WHO criteria. Antibodies against markers such as CD3, CD20, CD30, BCL2 and Ki-67 help characterize T-cell and B-cell populations, detect proliferative indices and determine clonality. Flow cytometry, in contrast, is a quantitative technique that analyzes the physical and antigenic properties of individual cells in suspension. Using fluorescent-labeled antibodies, FCM can rapidly analyze thousands of cells per second, producing high-dimensional data that offers detailed immunophenotypic profiles. This approach is highly sensitive and capable of detecting Minimal Residual Disease (MRD), making it invaluable in the diagnosis and follow-up of leukemias and lymphoproliferative disorders. FCM excels in detecting abnormal populations based on aberrant expression of lineage-specific markers, maturation antigens, or combinations not normally seen in healthy hematopoiesis. Commonly analyzed markers include CD19, CD45, CD10, CD34, HLA-DR and lineage-specific antigens such as MPO and TdT [3].

While IHC offers the advantage of contextual histological information, it is generally more limited in terms of multiplexing capacity compared to flow cytometry. Typically, only a small number of markers can be evaluated per tissue section and analysis is often semi-quantitative. In contrast, modern multicolor flow cytometry panels can simultaneously assess 8 to 20 or more antigens on a single cell, allowing for more refined classification and the detection of subtle immunophenotypic changes indicative of malignancy. Moreover, FCM is generally faster, with results often available within hours, whereas IHC may require longer processing times, especially in formalin-fixed paraffin-embedded specimens. Despite these differences, the two methods are complementary rather than mutually exclusive. In the diagnosis of acute leukemia, for example, flow cytometry is frequently used for initial classification and to distinguish between myeloid and lymphoid lineages. IHC then plays a confirmatory role, especially when assessing the tissue context or evaluating myeloid sarcomas and lymphoblastic infiltrates in solid organs. In the workup of lymphomas, IHC is essential for identifying histological subtypes and detecting markers such as BCL6, CD23, or cyclin D1, while flow cytometry is often employed for peripheral blood or bone marrow evaluation to detect disseminated disease or clonality [4].

Both techniques also present challenges. Flow cytometry requires viable cells and thus cannot be performed on fixed tissue, limiting its use in archival samples. Cell viability, sample handling and cellular yield are crucial for obtaining reliable FCM data. IHC, meanwhile, may suffer from antigen degradation due to fixation and variability in staining protocols or antibody specificity can affect interpretation. Furthermore, subjective interpretation of staining patterns in IHC may lead to interobserver variability, whereas FCM offers more objective numerical data, albeit requiring expertise in gating strategies and interpretation. Advances in technology are increasingly blurring the boundaries between these two approaches. Multiplex IHC and immunofluorescence techniques are expanding the capabilities of tissue-based analysis, allowing multiple antigens to be assessed in the same section. Similarly, mass cytometry and spectral flow cytometry offer even higher dimensionality in cell analysis, potentially revealing novel diagnostic and prognostic biomarkers. Integration of these modalities with molecular techniques such as Next-Generation Sequencing (NGS), Fluorescence In Situ Hybridization (FISH) and digital pathology platforms is paving the way for more precise and comprehensive hematologic cancer diagnostics [5].

Conclusion

In conclusion, both immunohistochemistry and flow cytometry are indispensable tools in the detection and characterization of hematologic malignancies. Their respective advantages in spatial resolution and quantitative multi-parameter analysis underscore their complementary roles in diagnostic workflows. Optimal utilization depends on the clinical context, type of specimen and the specific diagnostic questions at hand. The synergy between IHC and FCM, supported by emerging technologies and integrated diagnostics, is poised to enhance the accuracy, speed and depth of hematologic cancer evaluation, ultimately contributing to better patient outcomes and more individualized therapeutic strategies.

Acknowledgement

None.

Conflict of Interest

None.

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