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Immunophenotyping
Flow cytometry analysis of CD4 PBMC populations. Anti-human CD4 monoclonal antibody was labeled using Buccutite™ Rapid APC Antibody Labeling Kit or Lightning-Link® Rapid APC Antibody Labeling Kit according to manufacturers' instructions. CD4 PBMC populations were then stained and the fluorescence signal was monitored using a flow cytometer in the APC channel.
Immunophenotyping is also used in clinical disease diagnosis to identify specific types of leukemias, lymphomas, or other immunodeficiency disorders, commonly for children. In most cases, it can be used as a secondary, noninvasive, method to verify the presence of a disease subtype. Immunophenotyping may also be used prior to immunotherapy to determine if the therapy target is available, or even to help determine if the identified therapy will ultimately benefit the patient.
In many cases, the identification and quantification of a specific cell type can be related to a certain disorder. For example, is it often necessary to measure CD4-T cell counts when attempting to study specific immune-related disorders. Additionally, the use of CD45R and CD31 in tandem may be helpful in exploring a T cell subpopulation when attempting to narrow down an immunodeficiency subtype. General targets, such as CD56, may also help identify the level of natural killer cells present within a cell population. The table below shows specific markers reported for some disease diagnostic tests.
Examples of Diagnostic Immunophenotyping Markers
| Immunophenotyping Markers Reported For Use in Disease Diagnostic | |
|---|---|
| Chronic Lymphocytic Leukemia | CD20, CD22, CD23+, FMC-7- |
| Mantle Cell Lymphoma | CD20, CD5+/- |
| Prolymphocytic Leukemia | CD20 (+i), sIg (+i), FMC-7, CD5 |
| Follicular Lymphoma | BCL-2, CD43 |
| Diffuse Large B-Cell Lymphoma | BCL-2, CD43 |
| Burkitt Lymphoma | BCL-2,CD10 (+b), CD43 |
| Hairy Cell Leukemia | CD20, CD22, CD11c, CD25, CD103, sIg (I) |
| Adult T-cell leukemia/lymphoma | CD7, CD25, HTLV-1 |
| Natural Killer cells (cell differentiation) | CD57 |
| Activated T cells | CD25 |
| T-cell large granular lymphocyte leukemia | CD5, CD7, CD16, granzyme-B, perforin, CD56 |
| Eosinophilic Otitis Media | eosinophils |
| Multiple Myeloma | plasma cells |
| Common Variable Immunodeficiency | CD21, CD40L |
| Leukocyte adhesion deficiency (LAD) type 1 | CD18, CD11b, CD11c |
| Non-small cell lung cancer | PD-L1 |
Methods
Flow Cytometry
Flow cytometry (or simply, flow) is a technique that allows multiple cell surface markers to be simultaneously characterized by coupling differing antibodies with distinct fluorochromes. Flow cytometry can be used for quantifying immune cell frequencies, and is beneficial when there is a need to identify and sort cells within a complex population, such as a peripheral blood sample.
Flow cytometry analysis of PBMCs using CD45-PE, CD45-APC, CD4-FITC and CD3-APC using the single and double fluorophores. Density plot (Far left) was used to gate out the lymphocytes from other cell populations. Histogram plot (Upper right) and density plot (Lower right) were used for single and multiple fluorophore staining, respectively.
Cluster of differentiation (CD) markers are commonly used in flow for the detection of specific immune cell populations and subpopulations. CD markers are especially useful as immunological cell markers since all human cells naturally exhibit at least one type of CD molecule. CD markers are most commonly helpful in classifying white blood cell malignancies, since every lineage expresses a specific antigen.
In flow, first single cell suspensions are prepared from blood or the lymphoid tissues and stained with cell surface marker-specific antibodies. After appropriate parameters are set into the flow cytometer, the sample is passed through the machine. Scattered light signals pick up the chosen, identified, fluorescent compounds and then report the presence and density of a target cellular protein within the sample.
In analysis, changes in cell subtypes, for example lymphocyte subpopulations, can be expressed as a count within the total cell population or as a ratio relative to other subpopulations (i.e., a CD4 count within a sample, or the ratio of CD4:CD8 within the sample).
Though flow remains the gold standard for diagnostic immunophenotyping due to the ease and economical aspects of the procedure, it does not come without challenges. An appropriate amount of cells must be available as starting material, usually at least one million, for a successful experiment. Experimental reagents are also poorly standardized, and use may vary from protocol to protocol.
A successful flow experiment is also heavily dependent on preparation techniques, and the storage of cell samples, which must be carefully considered. Lastly, though running the sample is relatively easy, collecting and analyzing data requires knowledge of the cellular landscape by experienced personnel.
IHC is another method of immunophenotyping used for visualizing immune cell localization and may be more common for the analysis of other types of cell samples, including tissue sections, bone marrow, or even cerebrospinal fluid. Commonly tumor tissues are chosen as they can be isolated and snap-frozen for IHC investigation; histology can be combined as well to further explore the sample in question. A tumor tissue microarray can additionally be assembled for streamlined processing which allows the staining of potentially several dozen tumor samples simultaneously.
IHC is particularly handy in allowing for the visualization of not only the tissue architecture of expressed proteins, but also in analyzing the patterns of involvement and cytological features of cells, which may not be possible by flow.
When performing IHC for immunophenotyping, some things must be considered. IHC stains are not standardized, which in part contributes to the difficulty and/or impossibility of quantifying results. Additionally, while the cost of IHC is relatively inexpensive, the equipment needed to perform this technique may be costly. Moreover, IHC is subject to error, simply because of the numerous hands-on steps required.
In flow, first single cell suspensions are prepared from blood or the lymphoid tissues and stained with cell surface marker-specific antibodies. After appropriate parameters are set into the flow cytometer, the sample is passed through the machine. Scattered light signals pick up the chosen, identified, fluorescent compounds and then report the presence and density of a target cellular protein within the sample.
In analysis, changes in cell subtypes, for example lymphocyte subpopulations, can be expressed as a count within the total cell population or as a ratio relative to other subpopulations (i.e., a CD4 count within a sample, or the ratio of CD4:CD8 within the sample).
Though flow remains the gold standard for diagnostic immunophenotyping due to the ease and economical aspects of the procedure, it does not come without challenges. An appropriate amount of cells must be available as starting material, usually at least one million, for a successful experiment. Experimental reagents are also poorly standardized, and use may vary from protocol to protocol.
A successful flow experiment is also heavily dependent on preparation techniques, and the storage of cell samples, which must be carefully considered. Lastly, though running the sample is relatively easy, collecting and analyzing data requires knowledge of the cellular landscape by experienced personnel.
| Digital Catalogs on CD Antibodies: | Application Note: | |
| Assaywise Letter: |
Immunohistochemistry (IHC)
Human lung adenocarcinoma tissue samples were fixed and stained for EpCAM. The tissue sections were incubated with an anti-EpCAM rabbit monoclonal antibody (Left) or isotype IgG as a negative control (Right). The sample were then incubated with poly-HRP conjugated Goat anti-Rabbit IgG and the signal was developed with Stayright™ Purple solution. Stayright™ Purple generates a clean purple color stain without background.
IHC is particularly handy in allowing for the visualization of not only the tissue architecture of expressed proteins, but also in analyzing the patterns of involvement and cytological features of cells, which may not be possible by flow.
When performing IHC for immunophenotyping, some things must be considered. IHC stains are not standardized, which in part contributes to the difficulty and/or impossibility of quantifying results. Additionally, while the cost of IHC is relatively inexpensive, the equipment needed to perform this technique may be costly. Moreover, IHC is subject to error, simply because of the numerous hands-on steps required.
Product Ordering Information
Table 1. Fluorochrome Brightness Reference for Flow Cytometry
| Fluorophore ▲ ▼ | Spectrum Viewer ▲ ▼ | Ex ▲ ▼ | Em ▲ ▼ | Filter(nm) ▲ ▼ | Laser Line ▲ ▼ | Brightness ▲ ▼ |
| Alexa Fluor 350 | Link | 346 | 442 | 450/40 | 355 | 2 |
| iFluor 350 | Link | 345 | 450 | 450/40 | 355 | 6 |
| mFluor UV375 | Link | 351 | 387 | 450/40 | 355 | 2 |
| mFluor UV455 | Link | 357 | 461 | 450/40 | 355 | 2 |
| mFluor UV460 | Link | 358 | 456 | 450/40 | 355 | 2 |
| mFluor Violet 505 | Link | 393 | 504 | 450/40 | 405 | 6 |
| mFluor Violet 545 | Link | 393 | 543 | 450/40 | 405 | 2 |
| Pacific Orange | Link | 400 | 551 | 525/50 | 405 | 2 |
| mFluor Violet 540 | Link | 402 | 535 | 450/40 | 405 | 2 |
| mFluor Violet 450 | Link | 406 | 445 | 450/50 | 405 | 5 |
Table 2. Ordering Info for Readilink Antibody Labeling Kits Products
Table 3. IHC Chromogens For Product Ordering Information
| Cat# ▲ ▼ | Product Name ▲ ▼ | Size ▲ ▼ |
| 45900 | ReadiUse™ Stayright™ Purple *HRP Chromogen Premixed with Hydrogen Peroxide* | 5 mL |
| 45905 | Stayright™ Purple | 5 mL |