Vol. 16 •Issue 2 • Page 27
Shining the Light on Clinical Applications of Flow Cytometry
Flow cytometers are complex instruments with several components that must operate correctly to generate accurate and reproducible data.
Sample processing for flow cytometric analysis is relatively straightforward. Virtually any type of cell can be analyzed as long as it is in suspension. Cells present in body fluids such as blood already meet this requirement. Cells from organized tissues such as lymph node, however, must first be dispersed into a single cell suspension. Although straightforward, laboratories must have policies and procedures in place to ensure accurate and precise results.
A critical parameter is cell viability. Nonviable cells are more prone to nonspecific antibody staining. Thus, laboratories must establish criteria for sample age. For samples not meeting these criteria, a viability determination is important to assess sample adequacy.
After the preparation of single-cell suspensions, cells can be stained via a direct or indirect approach. Direct staining employs monoclonal antibodies covalently linked to a fluorochrome. The advantage of this approach is its simplicity. Indirect staining employs an unlabeled monoclonal antibody that’s binding to the target cells and detected by the addition of a second fluorochrome labeled anti-immunoglobulin antibody, specific for the species and isotype of primary antibody. An alternative approach is needed if one is targeting an intracellularly expressed antigen. In this case, cells must first be permeabilized to allow diffusion of the monoclonal antibody into the cell.
Regardless of the approach, the cell suspension is first incubated with the primary antibody, followed by a wash step to remove unbound antibody. Secondary antibody incubation followed by washing is next performed if the indirect approach is utilized. For samples with red blood cell contamination, a lysis step to remove red blood cells is typically included after the antibody staining steps. The cell suspension may then be subjected to an additional wash step.
Finally, cells are fixed in a paraformaldehyde solution to inactivate pathogens that may be present in the sample and stabilize the binding of the antibodies to the cells. Cell suspension is then ready for flow cytometric acquisition and analysis. Following is an overview of several common applications of flow cytometry.
Enumeration of CD4-positive T-lymphocytes
The hallmark of infection with HIV-1 is the loss of CD4 T-lymphocytes, leading to profound immunodeficiency in the final stages of the disease. Serial monitoring of CD4 T-lymphocytes is an important component of the assessment of individuals with HIV infection.1
CD4 T cell enumeration can be performed using two-, three- or four-color immunophenotyping panels. Three and four-color panels are more commonly used because they allow a more rigorous approach to the definition of a lymphocyte gate compared with the two-color approach. Typically, a combination of CD3 and CD4 specific monoclonal antibodies are used to define the proportion of CD3/CD4-dual positive cells in a lymphocyte gate defined by the bright expression of CD45 and low side scatter (Figure 1). Dual expression of CD3 and CD4 is used to exclude other cell types expressing CD4 (e.g., monocytes) from inclusion in the CD4 percent determination. The absolute CD4 count is then calculated as the product of the CD4 proportion and the absolute lymphocyte count (often determined with a hematology analyzer). This dual platform approach for absolute CD4 determination is in common use.
Alternatively, single platform absolute counts may be determined by including a known concentration of beads in the sample tubes that serves as a standard that can be used to calculate the absolute CD4 cell count. Dedicated instruments that determine absolute CD4 T cell counts also are available.
Enumeration of Fetal Hemoglobin Containing Cells
Enumeration of fetal hemoglobin-containing red blood cells is useful for estimating the amount of fetal bleed in women at risk for sensitization to fetal Rhesus D positive red blood cells and to assist in the management of women undergoing intrauterine transfusion. The standard method for enumeration of fetal hemoglobin containing cells has been the Kleinhauer-Betke test. This microscopic-based assay suffers from a lack of precision and objectivity.
The application of flow cytometry with a fetal hemoglobin specific monoclonal antibody provides a more rapid, objective and precise assay for enumeration of fetal hemoglobin-containing cells.2 Because hemoglobin is an intracellular antigen, cells must first be permeabilized prior to incubation with the monoclonal antibody to allow entry into the cell. Otherwise, this test is similar to other standard immunophenotyping assays. Using a single parameter histogram of antifetal hemoglobin fluorescence of gated red blood cells, one can determine the proportion of fetal hemoglobin-containing cells in the red blood cell population.
Flow Cytometry in Transplantation
Flow cytometry has several applications in the field of transplantation. The determination of CD34 expressing cells is the standard method for evaluating the repopulating capacity of stem cell products to be used in hematopoeitic stem cell (HSC) transplantation.3 The number of CD34 positive cells in stem cell products is determined in whole blood samples of HSC donors or in pheresis products collected for transplantation. The CD34 number guides the number of collections that need to be performed to obtain an adequate CD34 cell dose for infusion. This assay is frequently performed as a two-color panel, including CD45 as a gating reagent and a CD34 specific monoclonal antibody. Multiple gates are applied to identify and enumerate the typically infrequent CD34 positive cells (Figure 2).
In solid organ transplantation, flow cytometry is commonly used for the pretransplant screening of recipients for HLA alloantibody and for the detection of donor-specific HLA antibodies.4 Individuals awaiting a renal transplant are routinely monitored for the presence of human leukocyte antigen (HLA) alloantibody to identify those individuals presensitized to HLA antigens and identify the target specificity of these antibodies. In this fashion, potential donors who have HLA antigens to which a recipient is sensitized can be excluded from consideration for donation. Panels of purified HLA antigens can be coupled to beads that are incubated with patient serum. If HLA antibodies are present, they will bind to the bead(s) that are coated with their cognate antigen. An FITC-labeled anti-human IgG secondary antibody is added to detect bound HLA antibody. In this way, beads to which HLA antibodies have bound will exhibit green fluorescence.
Flow cytometric crossmatching is the most sensitive method for detecting donor-specific HLA alloantibody. Serum from the potential recipient is incubated with donor lymphocytes, obtained from blood, lymph node or spleen. After washing unbound antibody, an FITC-labeled anti-human IgG secondary antibody is added to detect bound HLA antibody on the donor lymphocytes. The presence of antibody bound to donor T (defined by costaining with a CD3 antibody) and/or B (defined by costaining with a CD19 antibody) lymphocytes are indicated by green cell fluorescence.
Paroxysmal Nocturnal Hemoglobinuria (PNH)
Red blood cells from individuals with PNH are exquisitely susceptible to complement mediated lysis. This susceptibility is the result of a genetic defect resulting in the lack of a glycophosphatidylinositol (GPI) molecule involved in anchoring molecules to the cell surface. In the case of PNH, the lack of (or reduced) expression of cell surface complements regulatory proteins (CD55 and CD59) as a result of defective GPI anchors that results in increased susceptibility to complement mediated lysis. Flow cytometric evaluation of red blood cells or polymorphonuclear neutrophils using fluorochrome-labeled monoclonal antibodies to CD55 and CD59 is a useful method for the diagnosis of PNH.5 Their reduced or absent expression supports the diagnosis of PNH.
Immunophenotyping Hematologic Malignancies
Flow cytometry also is an important adjunct for the diagnosis, classification and monitoring of patients with hematologic malignancies. Using panels of fluorochrome-labeled monoclonal antibodies, flow cytometric analysis facilitates the determination of cell lineage (e.g., T or B cell), state of maturation (e.g., pre B cells in acute lymphoblastic leukemia) and aberrant antigen expression (e.g., expression of myeloid antigens on lymphoid neoplasms).6 Flow cytometry also can be used to monitor patients for the presence of minimal residual disease after therapy.
Find a complete list of references beginning the Wednesday after publication at www.laboratorian.advanceweb.com by clicking on “References” on the left-hand navigation bar, then the title of the article.
Dr. Schmitz is an assistant professor and Flow Cytometry Laboratory director, Department of Pathology and Lab Medicine, University of North Carolina, Chapel Hill.