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Clinical Utility of Flow Cytometric Leukocyte Subset Identification

Posted on: October 14, 2020

The common perception is that circulating blood cells are composed of red and white cells.  In reality, the “white cell” population (leukocytes) contains many different cell types.  Most white blood cells, ~60-70%, are in the broad classification of granulocytes.  This population consists primarily of neutrophils, along with lower numbers of eosinophils and basophils.  Lymphocytes comprise ~20-40% of white cells and the remaining leukocytes are part of the monocyte/macrophage lineage.  The lymphocyte population consists of three major populations; T cells, B cells, and large granular lymphocytes, which are sometimes referred to as natural killer (NK) cells.  T cells may be subdivided into T helper and T cytotoxic populations; each of which may be further subdivided into an ever-increasing number of subclasses.(1)  Identifying and enumerating the various leukocyte populations is essential to diagnosing and monitoring many clinical conditions, including HIV infection, leukemias, lymphomas, primary immune deficiencies, paroxysmal nocturnal hemoglobinuria, and transplantation.  

Each leukocyte has a series of proteins on its surface that mediate the cell’s function, thus each cell type may be identified by determining the proteins on its surface. To borrow from Louis Sullivan, form follows function.   Each cell surface molecule plays a role in various aspects of the cell’s function.  Close to 400 of these cell surface proteins have been classified in a system known as “clusters of differentiation”, or CD (2).  For example, mature T cells have a surface protein known as CD3 which plays a role in cell activation.   Flow cytometric detection of CD3 is used to identify T cells.  Subsets of T cells may be identified by detecting both CD3 and CD4 on T helper cells or CD3 and CD8 on T cytotoxic cells.  The common CD proteins used to identify the various leukocyte classes along with the common clinical indications for identifying each cell type are listed in Table 1. Flow cytometry is the principal method used by clinical laboratories to identify CD proteins on the different cell types.

Specific Clinical Conditions in which Flow Cytometric Leukocyte Identification is Applied

HIV Infection -  The human immunodeficiency virus (HIV) has a surface protein known as gp120 which can bind to a CD4 molecule and another protein called a chemokine receptor.  Thus, the primary target of HIV infection is the T helper cell which expresses both CD4 protein and the chemokine receptors (either CXCR4 or CCR5) on the cell surface.  HIV will then destroy the CD4 positive T cells.  HIV infection may be staged by both clinical presentation and the number of CD4 positive T cells in an individual’s blood (3).  Clinical laboratories monitor CD4 counts in HIV infected patients by flow cytometry.

Primary Immune Deficiency -  Over 430 genetic mutations that can affect an individual’s immune capabilities have been identified.  These genetic anomalies cause a broad range of primary immune deficiencies (PID) (4).  Many of these entities are characterized by either an absence of a particular cell type(s) or by reduced cellular function.  PID examples include DiGeorge Syndrome, in which the T cell generating organ (thymus) is absent leading to an absence of mature T cells, Bruton’s Thymidine Kinase deficiency in which B cells are not present or Chronic Granulomatous Disease (CGD), in which neutrophil function is decreased (see below).  Often, a complete lymphocyte analysis (identifying T, B, and NK cells) by flow cytometry is part of the initial diagnostic regimen.  

Leukemia/lymphoma Identification - Leukemias are broadly classified as acute or chronic and lymphoid or myeloid.  Within each classification are many subtypes.  Lymphomas are classified as either Hodgkin’s or non-Hodgkin’s.  As with leukemias, there are many subclassifications within the non-Hodgkin’s lymphoma category.  Treatment and prognosis for these diseases will vary depending upon the type of leukemia/lymphoma a patient has.  Flow cytometry used to identify CD proteins, along with other proteins such as immunoglobulin light chains, is the initial test for classifying these entities and can also be used to detect residual disease following treatment (5). 

Solid Organ Transplantation - Lymphocyte phenotyping by flow cytometry is commonly used for both solid organ and peripheral blood stem cell transplantation.  Individuals receiving a solid organ transplant are placed on immunosuppressive medication.  These drugs may target a CD protein resulting in reduced numbers or functions of cells. For example, OKT3 is a monoclonal antibody that results in CD3 being modulated off the T cell surface.  Rituximab is an antibody targeting CD20 present on B cells.  Its use results in B cell destruction.  Campath, an anti-CD52 antibody, will eliminate all lymphocytes.  Flow cytometry for TBNK identification may be used to monitor individuals being treated with any of these and other medications (6).  Another use of flow cytometry for solid organ transplantation is performing cross-matching for identifying a potential solid organ donor. In this procedure, donor lymphocytes are incubated in recipient serum.  Any antibodies the recipient has to the donor cells are identified with fluorescently labeled antibodies against IgG or IgM.  Co-labeling the cells with anti CD3 to identify T cells, and anti CD19, to identify B cells, allows one to identify either IgG or IgM antibodies targeting HLA Class 1 (present on both T and B cells) or HLA class 2 (present only on B cells) proteins (7).  The presence of these antibodies may be a contraindication to that donor-recipient combination.  

Peripheral Blood Stem Cell Transplantation - Bone marrow stem cells express CD34, a protein that is lost as lymphocytes mature.  CD34 positive stem cells are normally not found in peripheral blood.  One potential treatment modality for some acute leukemias is an autologous peripheral blood stem cell transplant.  Patients are treated with a colony-stimulating factor to mobilized bone marrow stem cells into the peripheral blood.  Flow cytometry is used to identify the CD34 stem cells that have been so mobilized.   When an appropriate number of stem cells is present in the blood, they are harvested and stored until the patient has been successfully treated to remove leukemic cells from the bone marrow.  The stem cells are then transplanted back into the host to repopulate the bone marrow with normal cells (8).  

Neutrophil Deficiencies -  Many of the genetic mutations mentioned above may affect neutrophils, leading to a variety of deficiencies.  Two of the more common ones are identified using flow cytometry.  Chronic Granulomatous Disease (CGD)is characterized by a reduced oxidative burst capability of neutrophils.  The DiHydro Rhodamine reduction assay, performed by flow cytometry, measures the neutrophil oxidative burst capability which will below or absent in a patient with CGD (9).  Leukocyte Adhesion Deficiency, Type 1, (LAD-1) is a defect in the gene encoding the CD18 protein resulting in no CD18 being produced.  CD18, coupled with CD11a forms a complex called LFA-1.  LFA-1 is essential for neutrophils to exit the circulation and enter damaged tissue (10).  Recurrent soft tissue infections are a common symptom of this entity and CD18 may be identified by flow cytometric methods.

Paroxysmal Nocturnal Hemoglobinuria (PNH)-  PNH is a disease in which cell surface proteins anchored to glycol phosphatidyl inositol (GPI)  are absent from the cell surface.  This defect affects subsets of both neutrophils and red blood cells.  These proteins include CD55 and CD59.  For many years, these two proteins were used to identify PNH cells.  More recently, FLAER (fluorescein‐labeled proaerolysin), a protein that binds directly to the GPI anchor, has been used in a flow cytometric assay to detect PNH (11).  A combination of FLAER and antibodies to CD24 or CD157 are currently used in most PNH assays. 

FlowMetric Diagnostics Clinical Flow Cytometry Assays-  FlowMetric, a CLIA High Complexity Laboratory, offers lymphocyte phenotyping by Flow Cytometry.  The standard TBNK assay identifies and enumerates total T cells, total B cells, total NK cells, T helper, and T cytotoxic cells.  This assay is useful as part of a general screen for immune deficiencies or for monitoring patients receiving immunosuppressive medications targeting one or more of the lymphocyte subclasses.  We also offer a CD4 T cell assay that may be used for monitoring HIV patients during treatment. This assay reports quantitative levels for both total T cells and T helper cells.   If a CD4:CD8 ratio is also desired, we do offer a T cell subset assay that adds a CD8 cytotoxic T cell quantitation.  Additional assays for the other entities described in this blog may be available for research or clinical trial studies.  Please address specific requests to either Dr. Julie Bick, chief scientific officer, (jbick@flowmetric.com) or Dr. Tom Alexander, clinical laboratory director (talexander@flowmetric.com).  

Table 1- Cell surface Proteins Commonly Used to Identify Leukocyte Subclasses by Flow Cytometry

Cell Type

Surface Molecules

Clinical Indications for Monitoring

Total T cells

CD2, CD3, CD7

PID, leukemias, Immunosuppressive medications

T helper cells

CD3, CD4

PID, HIV infection, leukemias

T cytotoxic cells

CD3, CD8

PID, Leukemias

B cells

CD19, CD20, immunoglobulin light chain

PID, Leukemias/Lymphomas, Rituximab treatment or other immunosuppressive medications

LGL (NK) cells

CD16, CD56, CD57


Stem Cells


Leukemias/lymphomas, stem cell transplantation


CD16, CD18, CD24, CD55, CD59, GPI anchor



    1.  Abbas, A.K., A. H.Lichtman, and S. Pillai, Cellular and Molecular Immunology. 9th Edition, 2018 pp. 225-250.
    2. https://docs.abcam.com/pdf/immunology/Guide-to-human-CD-antigens.pdf. Accessed 09/22/2020.
    3. Selik, R.M., et al., “Revised Surveillance Case Definition for HIV Infection — United States, 2014”, CDC Recommendations and Reports, April 11, 2014 / 63(RR03);1-10
    4. Tangye, S.G., “Human Inborn Errors of Immunity: 2019 Update on the Classification from the International Union of Immunological Societies Expert Committee”, J Clin Immunol.,  2020 Jan;40(1):24-64.
    5. DiGiuseppe JA and BL Wood, “Applications of Flow Cytometric Immunophenotyping in the Diagnosis and Posttreatment Monitoring of B and T Lymphoblastic Leukemia/Lymphoma”, Cytometry B Clin Cytometry, 96(4), 2019, pp. 256-265)
    6. Stadler, M., et al., “Quantification of Immunosuppression by Flow Cytometry in Stable Renal Transplant Recipients”, Therapeutic Drug Monitoring: 25(1), 2003, pp 22-27. 
    7. Liwski, R.S., et sl., “Rapid optimized flow cytometric crossmatch (FCXM) assays: The Halifax and Halifaster protocols”, Human Immunology, 79(1),  2018, Pages 28-38.
    8. Korbling, M. and E.J Freireich, “Twenty-five years of peripheral blood stem cell transplantation”, Blood , 117 (24): 2011, pp 6411–6416.
    9. Mauch, L., et al., “Chronic Granulomatous Disease (CGD) and Complete Myeloperoxidase Deficiency Both Yield Strongly Reduced Dihydrorhodamine 123 Test Signals but Can Be Easily Discerned in Routine Testing for CGD”, Clin Chem., 53(5), 2007, pp 890-896.
    10. Abbas, A.K., A. H.Lichtman, and S. Pillai, Cellular and Molecular Immunology. 9th Edition, 2018 pp. 461-462.
    11. Zhao, J.J., et al., “Reduced cell surface levels of GPI‐linked markers in a new case with PIGG loss of function”, Human Mutations”, 38(10): 2017, pp. 1394–1401.



thomas-alexander Authored by: Dr. Thomas Alexander, Ph.D., D(ABMLI)



Dr. Thomas Alexander, Ph.D., D(ABMLI) is a highly respected clinical immunologist with more than 30 years of clinical flow cytometry experience, holding a Director Level Board Certification from the American Board of Medical Laboratory Immunology. Dr. Alexander joined the FlowMetric team in 2019 as Clinical Lab Director for our CLIA certified laboratory, after retiring from a 37-year position as Scientific Director of Immunology with Summa Health in Akron OH. Dr. Alexander’s has an academic appointment as a Professor of Pathology at Northeastern Ohio Medical University, and previously served as Chief of Clinical Pathology with Western Reserve Hospital in Cuyahoga Falls, OH.

During Dr. Alexander’s career his expertise has been recognized with several awards including: Gelfand Lecturer, Ohio University Phi Delta Epsilon Medical Fraternity, 2016; American Society for Microbiology Distinguished Lecturer, 2014-2016; Akron Medical Technology Program Commencement Speaker selected by the students, 2002, 2005, 2009, 2012, 2013, 2019 and the AMLI Distinguished Service Award, 2008. Most recently in 2020, Dr. Alexander was awarded the AMLI Erwin Neter Award and Lectureship.

With an extensive background in immunology research and clinical practice, Dr. Alexander has been instrumental in supporting FlowMetric’s continued excellence in clinical flow cytometry.

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