Clinical Insights

Flow cytometry is an invaluable tool for multidimensional cellular analysis and continues to expand its applications in clinical research. At MLM Medical Labs, each assay is tailored to the therapeutic area, leveraging advanced markers and techniques.

Below are key markers that can be assessed across multiple disease categories:

ONCOLOGY
Indications: Leukemias, lymphomas, solid tumors.

Assays:

• Leukemia phenotyping
• Minimal residual disease (MRD) detection using multicolor flow cytometry.
• Evaluation of immune checkpoint receptor expression (e.g., PD-1, CTLA-4).
• Absolute count of T cells, B cells and NK cells
• Immunophenotyping of T cells Biology
• FoxP3+ Regulatory T cells (Treg) detection.
• Analysis of CAR-T cell persistence and activation markers (e.g., CD25, CD69, CD71).
• Cytokine production in tumor-infiltrating lymphocytes (TILs) using intracellular staining. Identification of cancer stem cell markers like CD44+, CD133+.
• Drug receptor occupancy assay

Evaluation of tumor-associated fibroblasts (TAFs) and endothelial cells using markers like PDGFRα, FAP, and CD3

IMMUNOLOGY / AUTOIMMUNE DISEASES
Indications: Rheumatoid arthritis, lupus, multiple sclerosis, psoriasis.

Assays:

• • Immune cell profiling to evaluate changes in T cell subsets (e.g., CD4+, CD8+, Th17, Tregs). Activation markers in peripheral blood mononuclear cells (PBMCs) (e.g., CD40L, HLA-DR).
  • Cytokine profiling in response to therapy using intracellular staining (e.g., IL-17, IL-10, TNF-α).
  • B-cell subpopulation analysis (e.g., CD19+, CD27+, IgD+ memory B cells).
  • Analysis of immune checkpoint markers (e.g., PD-1, CTLA-4, Tim-3, LAG-3) on T cells to assess T cell exhaustion in autoimmune diseases (by checking Inhibitory receptors)

INFECTIOUS DISEASES
Indications: HIV/AIDS, hepatitis, COVID-19, bacterial infections.

Assays:

• • CD4/CD8 T-cell ratios in HIV management.
  • Activation and exhaustion markers in viral infections (e.g., PD-1, LAG-3).
  • Intracellular cytokine staining to measure antigen-specific responses (e.g., IFN-γ, IL-2).
  • NK cell activity and granzyme/perforin expression.
  • Assessment of immune reconstitution after antiviral therapy or vaccination.
  • Regulatory T-cells (Tregs) in Chronic Infections: FoxP3+ CD4+ T-cells, which are critical for immune tolerance, can be analyzed to assess immune suppression during chronic infections.
  • Helper (Th) Cell Polarization in Response to Infections – Detection of Th1, Th2, and Th17 subsets by intracellular cytokine staining and surface markers (e.g., IL-4, IFN-γ, IL-17).

HEMATOLOGICAL DISORDERS
Indications: Aplastic anemia, myelodysplastic syndromes (MDS), hemophagocytic lymphohistiocytosis (HLH).

Assays:

• • Evaluation of stem cell phenotypes (e.g., CD34+ hematopoietic stem cells).
  • Apoptosis detection in progenitor cells (e.g., Annexin V staining).
  • Functional assays for immune dysregulation (e.g., perforin/granzyme in HLH).
  • Immunophenotyping of T-cells in Hemophagocytic Lymphohistiocytosis (HLH) T-cell activation and expansion can be monitored by detecting CD3+ T-cells, with further characterization of activation markers like CD69, CD25, and HLA-DR. This is important because it helps us know how HLH involves uncontrolled activation of T-cells and macrophages. CD8+ T-cells may also show elevated expression of CD57 or PD-1, indicating immune exhaustion or dysfunction.
  • In HLH, NK cell dysfunction is a key feature, and CD56+ NK cells can be analyzed for activation and functional defects. Flow cytometry can assess CD16, NKG2D, and NKp46 expression to evaluate NK cell cytotoxic activity. Decreased or dysfunctional NK cells contribute to the excessive immune activation seen in HLH.

Erythropoiesis Monitoring in Aplastic Anemia: In aplastic anemia, the inability to produce mature blood cells is a key feature. Flow cytometry can be used to assess CD71+ (transferrin receptor) and Glycophorin A expression to evaluate the development of erythrocytes from progenitors. Fewer mature erythrocytes or abnormal erythropoiesis may be observed in patients with aplastic anemia.

TRANSPLANTATION
Indications: Solid organ and hematopoietic stem cell transplantation.

Assays:

• • Mixed lymphocyte reaction (MLR) to assess donor-specific immune responses and predict graft rejection. MLR can be analyzed using flow cytometry to track lymphocyte proliferation and identify T-cell activation markers such as CD69, CD25, and CD4/CD8 subsets.
  • To evaluate the regulatory T-cell population, that plays a key role in maintaining transplant tolerance and preventing rejection T-regulatory cell monitoring (e.g., CD4+CD25+FoxP3+ this can help in monitoring graft tolerance and immune suppression efficacy).
  • Assessment of donor chimerism using lineage-specific markers to determine engraftment status and identify graft rejection or relapse. This can be achieved by using lineage specific markers CD3 for T cells, CD19 for B cell and CD33 for myeloid cells that can help to distinguish donor and recipient cells
  • Detection of anti-HLA antibodies and B-cell activation helps to understand donor-specific antibodies (DSA), which are a cause of antibody-mediated rejection (AMR) in solid organ transplantation. Flow cytometry can be used to assess B-cell activation markers like CD19, CD20, CD27
  • T-Cell Subset Analysis: To assess the activation and function of different T-cell populations (e.g., CD4+, CD8+ T-cells), which are critical in acute rejection and graft survival. Using Flow cytometry we can assess T-cell subsets CD4+, CD8+, their activation markers like CD25, CD69, CD38, and cytokine production  IFN-γ, TNF-α, IL-2. These assays can help identify immune responses specific to graft rejection.
  • B-Cell Monitoring and Germinal Center Activity: To identify the potential for post-transplant B-cell activation and antibody production, which can contribute to chronic rejection. Using flow cytometry B-cell activation markers CD20, CD19, CD38, CD27, germinal center markers CXCR5, and plasma cell differentiation markers CD138, to detect and identify antibody-mediated rejection or a predisposition.

RESPIRATORY DISEASES
Indications: Asthma, chronic obstructive pulmonary disease (COPD), interstitial lung disease, Cystic fibrosis

Assays:

• • Phenotyping of airway immune cells (e.g., eosinophils, neutrophils) in bronchoalveolar lavage (BAL) fluid.
  • To assess the cellular composition of the inflammatory response in the lungs, particularly in conditions like asthma, COPD, and ILD.
  • Flow Cytometry Application: Flow cytometry is used to quantify immune cells (e.g., eosinophils, neutrophils, macrophages, dendritic cells, T-cells) in BAL fluid. Eosinophilic inflammation is commonly seen in asthma, while neutrophil infiltration is prevalent in COPD and ILD. Markers like CD45, CD11b, CD16, and CD14 are commonly used to differentiate these cell types.
  • Assessment of T-helper cell polarization (e.g., Th1/Th2/Th17): this can help in determining the underlying immune response in these diseases.
  • Intracellular cytokine staining to measure IL-13, IL-5 in response to treatment.
  • Fibrocyte Profiling in ILD (e.g., IPF):  Fibrocytes contribute to the fibrotic response in ILD and idiopathic pulmonary fibrosis (IPF), and their presence can correlate with disease progression. Flow cytometry can be used to identify fibrocytes in lung tissue and peripheral blood based on markers such as CD45, collagen I, and CD34. Monitoring these cells could help assess the degree of fibrosis and response to antifibrotic treatments.

NEUROLOGICAL DISORDERS
Indications: Multiple sclerosis, Alzheimer’s disease, Parkinson’s disease.

Assays:

• • Evaluation of blood-brain barrier integrity via immune cell trafficking markers.
  • Analysis of microglia activation markers in CSF (e.g., CD11b, CD45).
  • T-cell and B-cell subset profiling in neuroinflammatory diseases.

• Multiple Sclerosis (MS):
  • Chronic neuroinflammatory disease where the immune system attacks the myelin sheath, leading to neuronal damage. In MS, there is often an imbalance between Th1 and Th17 cells. Disease can be identified using T-cell Subset Profiling. Here, T-cell subsets CD4+ T-helper cells, CD8+ cytotoxic T-cells can be analyzed, and their specific activation markers, such as CD25 and CD69, in response to autoantigens
  • Regulatory T-cell (Treg) Analysis: Quantification of CD4+CD25+FoxP3+ Tregs in peripheral blood or cerebrospinal fluid (CSF) can reveal immune dysregulation in MS, as patients may exhibit impaired Treg function, leading to insufficient suppression of autoimmunity.
  • For B-cell Subsets and Plasma Cells: Flow cytometry can analyze B-cell subsets CD19+, CD27+ memory B-cells and plasma cells CD38+ as there is emerging evidence of the involvement of B-cells in the pathogenesis of MS, particularly in the secondary progressive stage.
    
    
• Alzheimer’s Disease (AD)
  • Detection of cytokines such as TNF-α, IL-6, IL-1β, and IFN-γ in immune cells like T-cells,   B-cells, monocytes from blood or CSF to assess the neuroinflammatory environment in AD.

• • Amyloid-Beta and Tau-Specific Immune Responses: Flow cytometry can identify amyloid-beta (Aβ) and tau protein-specific immune responses in peripheral immune cells, providing insights into how the immune system responds to amyloid plaques and tau tangles.
  • 3. Parkinson’s Disease (PD), A neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra, leading to motor dysfunction. Flow cytometry can assess the involvement of natural killer (NK) cells in PD, as they may play a role in the degeneration of dopaminergic neurons. CD56+CD3- NK-cells can be analyzed for cytotoxic activity and activation markers CD69, CD25.

CARDIOVASCULAR DISEASES
Indications: Atherosclerosis, myocardial infarction, heart failure.

Assays:

• • Monocyte and macrophage phenotyping (e.g., CD14, CD16, CD11c).
  • Measurement of endothelial progenitor cells (EPCs) using CD34, VEGFR-2 markers.
  • Detection of circulating inflammatory cytokines via intracellular flow cytometry.
  • T-cell Subset Profiling CD4+, CD8+, especially Th17 cells, contribute to heart failure. Flow cytometry can be used to measure these subsets and their activation markers CD69, CD25. Tregs are involved in controlling inflammation and may help improve outcomes in chronic heart failure by limiting tissue damage.

DERMATOLOGY
Indications: Psoriasis, atopic dermatitis, cutaneous T-cell lymphoma.

Assays:

• • Skin-specific immune profiling (e.g., CCR4+ T cells, Th17).
  • Cytokine production assays in response to allergens (e.g., IL-4, IL-22).

VACCINE DEVELOPMENT
Indications: Influenza, malaria, COVID-19, cancer vaccines.

Assays:

• • Antigen-specific T-cell response measurement (e.g., tetramer staining). It enables researchers to track immune responses to specific pathogens, vaccines, or even cancer antigens. Tetramer staining is a technique where tetramers (which are complexes made of four MHC molecules loaded with a specific peptide) are used to directly stain and identify antigen-specific T-cells. These tetramers bind to T-cell receptors (TCRs) on CD8+ T-cells (for MHC class I peptides) or CD4+ T-cells (for MHC class II peptides) that are specific for the peptide-MHC complex
  • B-cell memory and plasma cell quantification (e.g., CD27+CD38+).
  • Functional assays for antibody-dependent cellular cytotoxicity (ADCC).
  • Vaccine-Induced Inflammation and Immune Activation: Monocyte and Dendritic Cell Activation CD14+, CD86+, HLA-DR+ Monocytes and dendritic cells are essential for initiating and amplifying immune responses. Flow cytometry can measure their activation using markers like CD14, CD86, and HLA-DR, which are critical for antigen presentation and cytokine production following vaccination.
  • Cytokine Production in Immune Cells (IL-1β, IL-6, TNF-α): Vaccination often leads to an initial inflammatory response that can be assessed by IL-1β, IL-6, and TNF-α levels using flow cytometer.

RARE GENETIC DISORDERS
Indications: Primary immunodeficiency diseases (e.g., SCID, CGD). Wiskott-Aldrich Syndrome (WAS),

Assays:

• • Functional assays for NADPH oxidase activity (e.g., DHR test for CGD). Oxidative Burst (DHR Test): The dihydrorhodamine (DHR) assay measures the oxidative burst activity in neutrophils. In CGD, neutrophils fail to generate reactive oxygen species (ROS) due to defective NADPH oxidase. Phagocyte Function Assessment: Flow cytometry can assess neutrophil and monocyte activation and function in response to pathogens, which is impaired in CGD.
  • Evaluation of lymphocyte populations in SCID (e.g., absence of T/B/NK cells) Flow cytometry is used to identify the absence or reduction in the number of T-cells (CD3+), B-cells (CD19+), and NK-cells (CD56+), which is a hallmark of SCID.
  • Genetic correction efficacy using flow cytometry in gene therapy trials.

    Wiskott-Aldrich Syndrome (WAS): Using flow T-cell and Platelet can assess T-cell activation (CD4+ and CD8+ subsets) and identify defective platelet populations (CD41+), which are a hallmark of WAS. CD43 Expression: WAS cells show reduced expression of CD43 on T-cells and platelets.

• • For T-cell Subset Analysis: The functionality of Th1, Th2, and Th17 subsets, as well as the presence of regulatory T-cells (Tregs), can be analyzed to gauge immune dysfunction.
  • Ataxia-Telangiectasia (AT) : T-cell and B-cell Profiling: Flow cytometry can detect defects in both T-cells (CD3+) and B-cells (CD19+), as well as abnormalities in NK-cells (CD56+). AT disease is caused due to the ATM gene mutation that leads to defective DNA repair, which can be analyzed by assessing cell cycle arrest or apoptotic markers  using  Annexin V,  and 7-AAD

Conclusion:

Flow cytometry provides a versatile platform for the detailed analysis of immune and cellular responses in a variety of diseases. While specific markers vary across different disease types, some common markers can be used across many of the disease indications mentioned. These markers are often associated with general immune cell populations, activation states, or specific functional characteristics.