Introduction
Immunophenotyping is a laboratory technique employed for the identification and characterization of cells based on the proteins they express on their cell surface or in their cytoplasm. These proteins are often referred to as markers and can be used to evaluate the activation state or other functional characteristics of many cell types, including immune cells. An immunophenotyping experiment consists of cells stained with fluorochrome-conjugated antibodies that are targeted against antigens on the cell surface. Flow cytometry is an indispensable tool used for the analysis of cellular phenotype and immune system function in the biobanking environment. It is also the most advanced technology available for probing human immune phenotypes thereby aiding the characterization of many subsets of cells as well as rare subsets, in a complex mixture of biological samples such as blood. Additionally, due to the wide range of antibody reagents and protocols available, flow cytometry can be used to evaluate both the expression of cell-surface proteins and that of intracellular phosphoproteins and cytokines as well as other functional readouts such as cell viability, proliferation or migration in response to a drug or other treatment. This article focuses on the Immunophenotyping workflow using the flow cytometer, the benefits and the application of immunophenotyping in the biobanking settings as well as the advantages of flow cytometry in immunophenotyping.
Overview of the Flow Cytometer
The flow cytometer is made up of three major parts. The first is the fluidics system, which is in charge of moving the sample from the sample tube to the flow cell. The next component is the optics system, which includes the detecting system that produces the photocurrent and is made up of the lens, filters, and light sources that are used to gather and transfer light around the cytometer. The electronics, which is the third and last part, is where the photocurrent from the detector is converted to digital form.
In flow cytometry, single cells are analysed as they flow across single or multiple lasers while suspended in a buffered salt-based solution. Each particle is evaluated for visible light scatter and one or several fluorescence characteristics. Visible light scatter is measured in two different directions, the forward direction (Forward Scatter or FSC) which can indicate the relative size of the cell and at 90° (Side Scatter or SSC) which indicates the internal complexity or granularity of the cell.
Cell Types and Their Corresponding Markers for Their Immunophenotyping
- T Lymphocytes (T Cells) – are a type of white blood cell vital for the immune system. They play a significant role in both cell-mediated and humoral immunity, protecting the body from infections and potentially fighting cancer. They can be characterized by using markers such as CD3, CD4, CD8, CD45RA, CD45RO, and CD25. CD3 is a pan-T cell marker, while CD4 and CD8 are used to differentiate between helper T cells (CD4+) and cytotoxic T cells (CD8+).
- B Lymphocytes (B Cells) – are a type of white blood cell important for the adaptive immune system. They are primarily responsible for humoral immunity, which involves the production of antibodies. They can be immunophenotyped using markers such as CD19, CD20, CD21, CD22, CD45, and CD79a. CD19 and CD20 are usually used to pinpoint B cell populations, while CD21 and CD22 are involved in B cell receptor signaling.
- Natural Killer (NK) Cells – NK cells play a critical role in innate immunity. They can be immunophenotyped using markers like CD16, CD56, CD3, and CD45. CD16 and CD56 are key NK cell markers, distinguishing them from other lymphocytes.
- Dendritic Cells – Dendritic cells are important for antigen presentation and immune regulation. Dendritic cell subsets can be characterized with markers such as CD11c, CD123, CD86, CD1c, CD141, and CD40.
- Hematopoietic Stem Cells – Hematopoietic stem cells give rise to all blood cell types. Hematopoietic stem and progenitor cells are characterized with markers such as CD34, CD38, CD45, CD90, and CD133.
- Myeloid Cells – Myeloid cells, which include granulocytes and monocytes, can be characterized using markers like CD13, CD14, CD33, CD64, CD117, and CD45. These markers are useful in distinguishing different myeloid cell populations.
- Endothelial Cells – Endothelial cells line blood vessels and they play a significant role in vascular health. Their immunophenotyping is done with markers such as CD31, CD105, CD144, CD146, CD34, and CD45.
Immunophenotyping by Flow Cytometry Workflow
Immunophenotyping by flow cytometry workflow involves sample preparation, antibody selection, antibody staining, flow cytometric analysis, data analysis, quality control, and data storage.
- Sample Preparation: Blood, bone marrow, umbilical cord or other tissue are collected from donors or patients. Cells from these samples are isolated with techniques such as density gradient centrifugation or magnetic activated cell sorting (MACS) based on the sample type and target cells. After the isolation of the target cells, if a viability test is not required, the cells are then fixed in order to preserve the cell structure and permeabilized if required, in order to allow antibody access to intracellular targets.
- Antibody Selection: In antibody selection, fluorophore-conjugated antibodies against markers that define the cell population of interest are chosen. For example: CD3, CD4, and CD8 for T cells; CD19, CD20, CD27, IgM, IgD for B cells; and CD16, CD45, CD56 for Natural Killer (NK) cells.
- Antibody Staining: To perform antibody staining, it is required to add antibody to the cell suspension and incubate at 4°C in the dark for 20–30 minutes to protect the fluorophore from photodegradation because the fluorophores are photosensitive. For intracellular markers such as cytokines, additional fixation with fixatives such as paraformaldehyde (PFA) and permeabilization with reagents like Triton X-100, saponin, Tween-20, or digitonin is required before antibody staining. The choice of permeabilization reagent depends on the specific experiment and target antigen: mild detergents such as Tween-20 or saponin are suitable for cytoplasmic or soluble nuclear antigens, while harsher detergents such as Triton X-100 or NP-40 are suitable for nuclear antigens. This is followed by washing of the cells with phosphate-buffered saline solution containing a protein such as fetal bovine serum or bovine serum albumin (BSA) to remove unbound antibodies from the cells in order to reduce background noise.
- Flow Cytometry Analysis: The stained cell suspension is introduced into the flow cytometer where cells pass one after the other through a laser beam. As each cell passes through the laser beam the detectors measure the scattered light and fluorescence emitted by each cell. By evaluating the intensity and pattern of fluorescence, the expression of specific surface markers on each cell can be determined. If the immunophenotyping analysis requires sorting of the cells, a flow cytometer with a cell sorter is required for this purpose because not all flow cytometers are incorporated with cell sorters.
- Data Analysis: Software such as FlowJo, FCS Express, Cytobank, BD FACSDiva, OMIQ, Floreada.io, Cyflogic and MACSQuantify are used to analyse the data collected by the flow cytometer.
- Quality Control: Quality controls (unstained, single stain, Fluorescence Minus One (FMO)) should be used and reproducibility checks should also be performed.
- Data Storage: Results from the immunophenotyping analysis are typically stored in databases associated with the biobank. This data can be used for research, diagnostic, or quality control purposes.
Benefits of Immunophenotyping in Biobanking
- Cell Characterization: Enables the identification and characterization of specific cell populations within samples.
- Quality Control: Helps maintain the quality of stored samples by verifying cell viability and surface marker expression.
- Research Purposes: Provides valuable data for research studies investigating diseases, treatments, and cell behavior.
- Diagnostic Potential: Can be used to identify specific cell types relevant to diagnostics or monitoring disease progression.
- Long-Term Preservation: Ensures that detailed information about cell populations is archived alongside the samples for future use. The combination of immunophenotyping and biobanking with flow cytometry enhances the value of stored samples by providing detailed information about the cellular composition, which can be crucial for various research and clinical applications.
Applications of Immunophenotyping in the Biobanking Environment
- Sample Characterization: Immunophenotyping is the most used application in flow cytometry. It utilizes the unique ability of flow cytometry to simultaneously analyse mixed populations of cells for multiple parameters. Prior to long-term storage of biological samples, immunophenotyping can be used to characterize the cell composition of samples (blood, bone marrow, tissue). Cells isolated from solid tissues can be analysed with flow cytometry thereby enabling the study of tissue-specific cell populations and their functions.
- Human Leukocyte Antigen (HLA) Typing and Matching: In umbilical cord biobanks, flow cytometry is used to determine the HLA type of both the donor and the recipient. This involves using fluorescently labelled antibodies that bind to the specific HLA molecules on the surface of the cells. By analysing the fluorescence intensity, the HLA types can be identified and matched. This is important for successful transplantation.
- Quality Control: Immunophenotyping can be used to assess the impact of freezing and thawing on cell populations. This helps to ensure that stored samples retain their desired characteristics and viability.
- Diagnosis and Monitoring of Disease Progression: Immunophenotyping can be used to identify abnormal cell populations based on the surface markers on these cells, thereby aiding the diagnosis and classification of disease conditions such as hematological malignancies like leukemia and lymphoma. It is also used to monitor changes in cell populations over time in disease conditions such as leukemia, lymphoma, HIV/AIDS and autoimmune disorders, which can be useful in tracking disease progression or treatment response.
- Standardization and Consistency: By using standardized flow cytometry protocols and antibody panels, researchers can ensure consistency in sample characterization across different time points and different biobanks.
Advantages of Flow Cytometry in Immunophenotyping
- High Throughput: Flow cytometry allows for the rapid analysis of large numbers of cells, enabling high-throughput screening.
- Multi-Parameter Analysis: Flow cytometers can detect multiple markers simultaneously, providing a comprehensive view of cellular phenotype.
- Single-Cell Resolution: Flow cytometry provides information about individual cells, allowing for the detection of rare cell populations.
- Quantitative Data: Flow cytometry provides quantitative data on marker expression, allowing for accurate comparisons between samples.
Conclusion
Flow cytometry is an indispensable tool for characterizing, preserving, and analyzing cell populations in biobanking, supporting both basic research, diagnosis, and clinical applications.
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About the Author
Bsc. Zoology, BMLS, Msc. Biobanks & Complex data management (In view)
Tijani Abdulrahman is a medical laboratory scientist and a biobanking professional with 10+ years of demonstrated history in working in the medical science practice industry. Skilled in laboratory medicine, biobanking, quality management system and biomedical research. Currently a master’s student at the University Cote d’Azur, France with focus on Biobanking and complex data management. With a multidisciplinary background Tijani Abdulrahman strives to champion biobank and biomedical enabled research in order to strengthen biobank and biomedical innovation.
