June 02, 2023

What is Immunophenotyping?

Immunophenotyping is a technique that utilizes specific antibodies to target and identify unique surface markers or antigens expressed on immune cells, providing a detailed characterization of these cell populations. CD (cluster of differentiation) antibodies, which bind specific cell surface antigens and follow a standardized nomenclature, are commonly used for this purpose. The fluorescence emitted by labeled antibodies can be measured by flow cytometry (FC) and fluorescence-activated cell sorting (FACS™), enabling rapid and simultaneous analysis of multiple cellular parameters.

A newer technique called mass cytometry or CyTOF® (cytometry by time-of-flight) employs metal isotopes as labels and allows robust analysis of up to 60 individual parameters simultaneously (see Cossarizza et al. 2019 for a comprehensive review of methods). Simultaneous measurements with multiple antibodies offer enhanced specificity and sensitivity, facilitating comprehensive and precise characterization of complex cell populations.

Flow cytometry analysis using anti-F4/80 APC antibody

Flow cytometry analysis of C57BL/6 bone marrow cells with isotype control (left) and F4/80 APC Conjugated Monoclonal Antibody (right).


Features and Benefits of Immunophenotyping

  • Accurate Immune Cell Identification

    Immunophenotyping enables precise identification and quantification of immune cells, offering a comprehensive understanding of the immune system. By utilizing antibodies  specific to surface markers on immune cells (e.g. CD markers), flow cytometry analysis allows researchers to discern distinct immune cell populations.
  • Biomarker Discovery

    Immunophenotyping plays a pivotal role in biomarker discovery, revolutionizing diagnostic and prognostic approaches. By evaluating the expression patterns of surface markers on immune cells, researchers can identify novel biomarkers that provide valuable insights into disease progression, treatment response, and personalized medicine.
  • Disease Characterization

    Immunophenotyping serves as a diagnostic tool for disease characterization. By analyzing immune cell subsets and their unique surface markers, researchers gain insights into immune dysregulation, immune-mediated disorders, and the effectiveness of therapeutic interventions. This knowledge paves the way for tailored treatment strategies and improved patient outcomes.
  • Immune Profiling

    Immunophenotyping offers a comprehensive immune profile, facilitating a deeper understanding of immune responses. By evaluating immune cell populations and their phenotypic characteristics, researchers can unravel the complexities of immune regulation, immunodeficiencies, and immune-related disorders.

Immunophenotyping, powered by flow cytometry and targeted antibodies, has revolutionized cellular analysis in immunological studies. With its ability to identify immune cell subsets, discover biomarkers, and characterize diseases, immunophenotyping offers a comprehensive view of the immune system.


Rockland Flow Cytometry Antibodies

CD Markers for Immunophenotyping

Cell Type Human Mouse
B Cells CD19, CD20 CD19, CD20
Cytotoxic T Cells CD8 CD8
Dendritic Cells CD11c CD11c
Endothelial Cells CD146 CD146
Epithelial Cells CD326 CD326
Erythrocyte CD235a CD235a
Granulocytes CD66b Ly6G
Hematopoetic Stem Cells CD34 CD34
Leukocytes CD45 CD45
Monocytes / Macrophages CD14, CD33 CD11b, F4/80
Natural Killer Cells CD56 CD335
Platelets CD41, CD61 CD41, CD61
T Cells CD3 CD3
T Helper Cells CD4 CD4


Flow Cytometry Markers in Disease

Disease Suitable Surface Markers
Acute Myeloid Leukemia (AML) CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD15, CD16, CD19, CD20, CD30, CD33, CD34, CD38, CD45, CD56, CD64, CD71, CD117, CD123, HLA-DR
Adult T-Cell Leukemia/Lymphoma CD7, CD25, HTLV-1
Autoimmune Lymphoproliferative Syndrome (ALPS) CD4, CD8, TCRα, TCRβ
Automosomal Recessive Hyper-IgM (HIGM) CD40
Burkitt Lymphoma BCL-2, CD10 (+b), CD43
Chronic Lymphocytic Leukemia CD20, CD22, CD23, FMC-7
Common Variable Immunodeficiency CD21, CD40L
CTLA4 Haploinsufficiency CTLA4
Diffuse Large B-Cell Lymphoma BCL-2, CD43 
DOCK8 Deficiency DOCK8
Eosinophilic Otitis Media Eosinophils
Familial Hemophagocytic Lymphohistiocytosis Perforin
Follicular Lymphoma BCL-2, CD43
Hairy Cell Leukemia CD11c, CD20, CD22, CD25, CD103, sIg (I)
Immune Dysregulation (IPEX) Foxp3
IRAK4 and UNC93B Deficiency CD62L
Leukocyte Adhesion Deficiency (LAD) Type 1 CD11b, CD11c, CD18
Leukocyte Adhesion Deficiency (LAD) Type 2 CD15s
LRBA Deficiency CTLA4, LRBA
Mantle Cell Lymphoma CD5, CD20
MHC Class II Deficiency HLA-DR
Multiple Myeloma Plasma Cells
Non-Small Cell Lung Cancer (NSCLC) PDL1 (CD274)
Prolymphocytic Leukemia CD5, CD20 (+i), sIg (+i), FMC-7
T-Cell Large Granular Lymphocyte Leukemia CD5, CD7, CD16, CD56, granzyme-B, perforin
X-Linked Hyper-IgM (HIGM) CD40L
X-Linked Agammaglobulinemia (XLA) BTK
X-Linked Lymphoproliferative Syndrome (XLP1/2) SAP, XIAP

Table adapted from Herold & Mitra 2022 and Delmonte & Fleischer 2018