Introduction

Immunophenotyping is a technique that couples specific antibodies to fluorescent compounds to measure specific protein expression within a cell population. The protein expression is used to identify and categorize the tagged cells. It is often used to measure CD4-T cell counts when attempting to study specific immunodeficiency disorders and immune-related diseases. Immunophenotyping is a type of flow cytometry testing in which the scattered light signals pick up the chosen fluorescent compounds to report the existence and expression of a target cell protein.[1]

The ability of flow cytometry to determine the presence or absence of cell-surface markers serves as the basis of immunophenotyping. General targets such as CD56 can determine the level of natural killer cells, or more specific targets, such as CD45R combined with CD31, can explore T-cell subpopulations when attempting to narrow down categories of immunodeficiencies.[2] Immunophenotyping is a strong diagnostic tool that can be used in a wide variety of leukemias and immunodeficiency disorders. Immunophenotyping can also be used to further define cancer cell lines by exposing the presence or absence of cancer cell markers that correlate with different degrees of severity.[3]

Specimen Requirements and Procedure

When immunophenotyping for children, the use of a bone marrow sample for the first test is suggested, although subsequent tests can be run from a peripheral blood draw.[4] Peripheral blood draws can be used for the first round of immunophenotyping in adults. Samples collected from bone marrow aspiration and fine-needle aspiration are also options.[5] Blood storage should not be prolonged as it can cause selective loss of cell populations, such as neutrophils and eosinophils, due to their short half-life. Reagents such as Cyto-Chex and formaldehyde can extend the storage time of the blood while waiting to perform cytometry on the sample. When coupling immunophenotyping with flow cytometry, ethylene diamine tetra-acetate (EDTA) is the preferred anticoagulant. If the study is being done on granulocytes, in particular, heparin may be the anticoagulant of choice. Different laboratories may have slightly varied protocols for sample collection and anticoagulant selection.[6]

Following the collection of the sample, the target for the immunofluorescence marker is chosen. The target will vary depending on the reason for testing. Antibodies specific to the desired antigen are chosen to illuminate the antibody-antigen binding complex that is specific to the cell line of choice. The collected blood sample is washed with a lab purchased detergent such as Triton X-100, washed with a buffer, vortexed, and then placed in a centrifuge. The resulting pellet is then washed, resuspended in wash buffer, and then the epitope is stained and immediately analyzed on a flow cytometer.[6]

Diagnostic Tests

Immunophenotyping can be useful as a diagnostic test for a variety of diseases ranging from inherited immunodeficiencies to late-stage leukemia. It can also help support clinical diagnosis if the provider is unsure. Examples of diagnostic ability with target include but are not limited to:

• Acute Myeloid Leukemia [7]
• 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) [8]
• 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 [9]
• Multiple Myeloma: Plasma Cells [10]
• Common Variable Immunodeficiency: CD21, CD40L [11]
• Leukocyte adhesion deficiency (LAD) type 1: CD18, CD11b, CD11c [2]
• Non-small cell lung cancer: PD-L1 [12]

Interfering Factors

Due to the relatively new use of flow cytometry for immunophenotyping, there are several interfering factors:

• Poor standardization regarding the choice of reagents
• How to prepare and store cells to be analyzed
• How to analyze the collected data

Recent attempts have been made to investigate each of the variables that stands as a roadblock for standardization; notably, many are assembled in “Guidelines for the use of flow cytometry and cell sorting in immunological studies,” published in the European Journal of Immunology.[3]

Results, Reporting, and Critical Findings

Results of immunophenotyping and critical findings may vary with age. Reliable reference values are necessary for accurate reporting, and when using immunophenotyping, especially for primary immunodeficiencies, adjusting to lymphocyte count by age is necessary. From birth to 18 years, lymphocyte count trends down, lymphocyte count in a neonate with an immunodeficiency may appear similar to a healthy 18-year-old. Further, the breakdown of lymphocyte subsets fluctuates with age. Both changes must be taken into consideration when determining if the laboratory values for younger individuals are normal or represent critical findings for their age category.[13] 

Age-specific reference values are relatively scarce, which can cause misdiagnosis as immunophenotyping becomes more widespread for the diagnosis of childhood primary immunodeficiencies. Reporting and normal lab values will differ for each cell line and surface protein that is investigated.

Clinical Significance

Immunophenotyping and its ability to identify specific cell lineages allow for the direct diagnosis of many immunodeficiency disorders in children as well as cancers of all ages. In some cases, it can be used as an aid to diseases that are traditionally clinical diagnosis if the clinician is unsure the diagnosis is correct. When used as a clinical test, immunophenotyping is a non-invasive way to investigate the type and cause of specific immunodeficiency subtypes. Immunophenotyping can also be used prior to immunotherapy to determine if the therapy target is available in the cancer cell line. Creating a profile of the cancer cell line with immunophenotyping, is necessary to determine which therapies, if any, will benefit the patient.[12] Clinical use of immunophenotyping is not limited to use in human populations, with studies showing it can help to predict survival time in dogs afflicted with chronic lymphocytic leukemia (CLL). Its use in predicting the severity of CLL in humans has been established, and it appears the results are similar in dogs.[14] 

Although not as common as immunophenotyping with blood and bone marrow, samples can also be obtained from cerebrospinal fluid. Cerebrospinal fluid immunophenotyping via flow cytometry can compare T-cell populations as well as the CD4: CD8 ratio within the cerebrospinal fluid, expanding the scope of immunophenotyping as a diagnostic tool.[15] Flow cytometry immunophenotyping also has a role to play in the detection of minimal residual disease after the treatment of adult and pediatric lymphoblastic leukemia. It allows clinicians to detect aberrant leukemia-specific antigens that coincide with possible disease recurrence.[16]

Quality Control and Lab Safety

The increasing use of mass cytometry-based immunophenotyping requires increased reproducibility of experiments and normalization of protocols between various labs. Current trends work to increase reproducibility and decrease technical variations. This goal is achievable with the use of a consistent reference sample from a single blood donation to mimic stable population frequencies. The use of mass cytometry for phenotyping creates more separation between negative and positive populations when compared to using it to determine the functional characteristics of cells. To increase quality control when investigating functional characteristics, the use of semi-automated “tethered” gating approaches (FlowJo) when analyzing data provides superior quality control compared to the fully automated methods (SPADE, viSNE) algorithms which are superior for phenotyping.[17]

The mapping of the human immune system termed the “Human Immunology Project,” pushes for “measurement of variations in the human immune system.” To meet this goal, the project is pushing for standardizing assays to distinguish true variation from the artifact. The project pushes for quality control through the reduction of diversity of reagents, “definitions of standard antibody panels for immunophenotyping, point-of collection automation of sample processing, setting the fluorescence of standard beads to defined target channels for reproducible setup across instruments, central analysis by one of few coordinated experts and use of automated gating algorithms.”[18] Currently, even well-studied cell lines are defined differently in studies making standardization difficult.