Vol. 12 •Issue 1 • Page 32
Theory, Clinical Applications of Flow Cytometry
Advances in reagents for flow cytometry are certain to expand its application beyond routine immunophenotyping.
Flow cytometry is a technology that facilitates the identification and enumeration of specific cell types in complex biologic samples. The ability to determine the proportion and/or absolute numbers of specific cell types is afforded by the ability of the flow cytometer to rapidly analyze large numbers of cells on a single-cell basis and the use of fluorescently labeled monoclonal antibodies that, by virtue of their ability to bind to specific cell surface or intracellular antigen(s), identify the cell(s) of interest. The definition of the etiology of many diseases at the molecular level and resultant development of reagents to detect these lesions has increased the applications of flow cytometry in the clinical laboratory.
Flow Cytometry Instrumentation
The coordinated functioning of three systems—fluidics, optics and electronics—allow the flow cytometer to identify and enumerate cells.1,2 The fluidics system is the sample handling component of the instrument. Cells in suspension are taken up and injected into a fast flowing stream of sheath fluid (usually a buffered salt solution). The cells enter the center of this fast flowing stream of fluid and are focused, single file, into a stream of cells that pass through a flow cell in which they are “interrogated.”
At the interrogation point of the flow cell, the cells intersect a beam of laser light. When the light hits the cells, photons are scattered in all directions. If the cell has a fluorochrome-labeled monoclonal antibody bound to its surface, the cell will fluoresce. The scattered laser light and fluorescent emissions are directed via a series of mirrors and filters to photodectors that receive the photons of light and convert their energy to electrical signals.
Several photodetectors are present in a standard flow cytometer. The forward and side scatter photodetectors receive laser light that is scattered in the forward and 90o (orthogonal) direction, respectively. Fluorescent emissions are assessed by additional photodetectors, the number of which is dependent upon instrument configuration. Each detector receives fluorescent emissions of wavelengths that are defined by the mirrors and filter systems for each. Clinical flow cytometers typically have four photodetectors, thus allowing the user to label cells with up to four different antibodies (each covalently linked to a different fluorochrome) simultaneously.
The electrical signals generated from the photodetectors provide data characterizing the physical properties of the cells (intrinsic parameters) and the antigenic makeup (extrinsic parameters) as defined by the binding of specific fluorochrome-labeled monoclonal antibodies. The light scattered in the forward direction (forward scatter) is reflective of the cross-sectional area of the cell (i.e., cell size). Larger cells will have a higher forward scatter signal. Light scattered in the 90º direction (orthogonal or side scatter) is reflective of the internal complexity of the cell. Cells with cytoplasmic granules and complex nuclear morphology, such as polymorphonuclear neutrophils, will scatter more light in the 90º direction and generate a higher side scatter signal.
The outcome of signal generation from photodetectors is a digital signal proportional to the number of photons received by a detector. The computer system of the flow cytometric system allocates the signals to channels (using a 1 to 1,024 or 1 to 256 scale). Strong signals are allocated to a higher channel number while weaker signals are allocated to a smaller channel number. The outcome of this analysis is a frequency distribution of light scatter and/or fluorescence intensity. This data presentation allows one to determine the proportion of cells that exhibit specific levels of scattered light or fluorescence.
A dual parameter dot plot allows the user to assess two parameters simultaneously for each cell. Analysis of a whole blood sample by forward and side scatter allows the differentiation of the three major white blood cell populations.
Flow cytometric software allows the user to restrict analysis of cells’ surface characteristics to specific populations of cells via a process termed “gating.” For example, to determine the proportion of CD4 and CD8 T-lymphocytes in the total lymphocyte population in peripheral blood, one gates on the total lymphocyte population based on forward and side scatter or a leukocyte specific monoclonal antibody vs. side scatter. The proportion of CD3/CD8 dual positive and CD3/CD4 dual positive T-lymphocytes is determined based on their frequency in the lymphocyte gated cells.
As stated above, monoclonal antibodies labeled with fluorochromes are used to identify specific cell types based on the presence of cell surface or intracellular antigens. A monoclonal antibody is an immunoglobulin protein derived from an immortalized clone of B-lymphocytes. This clonal population of cells secretes immunoglobulin of a single defined isotype and specificity based on the antigen used to elicit its production. Because of their high degree of specificity for a particular antigen, they are useful tools to detect the presence of their cognate antigen on unknown cells.
Common target antigens used in clinical flow cytometry (Table 1) are associated with cell lineage (e.g., T- vs. B-lymphocyte), cell function (e.g., presence of cytokine or other receptors) or state of maturation (e.g., Pre-B vs. mature B cells). Although monoclonal antibodies are powerful reagents for identifying specific cell types, it is often necessary to employ two or more simultaneously to define cell types of interest. As a result, one must employ several different fluorochromes to be able to distinguish the binding of each monoclonal antibody.
Fluorochromes are molecules that absorb light of one wavelength and emit light of higher wavelength. For flow cytometric2 and other applications, fluorochromes are covalently linked to monoclonal antibody molecules. This provides a mechanism that allows one to determine if the antibody has bound to a cell of interest. A fluorescent signal will be detected by the flow cytometer if a labeled antibody has bound the cell surface.
As stated above, more than one antibody may be required to identify specific cells of interest. As such, multiple antibodies each with a different fluorochrome are often used in flow cytometric analyses.
The use of multiple fluorochrome labeled antibodies is technically straightforward; however, the emission spectra of some fluorochromes commonly used in flow cytometry are similar (Table 2). These overlapping emission spectra can complicate the interpretation of the fluorescent signals, as one detector may receive emissions not only from its intended fluorochrome, but also contaminating emissions from a second fluorochrome with an overlapping emission spectrum.
Spectral overlap is accounted for by special circuitry or software that corrects for this phenomenon. This process of subtracting signals from one detector that are due to overlapping emission spectra from another fluorochrome is termed fluorescence compensation.
Sample processing for flow cytometric analysis is 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 in solid tissue, for example lymph node cells, must first be dispersed via mechanical or enzymatic means into a single cell suspension.
A second critical parameter is the viability of the cell suspension. Non-viable cells are more prone to non-specific antibody staining. Thus, for samples in which cell viability is questionable, 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. Indirect staining employs an unlabeled monoclonal antibody that’s binding to the target cells and is detected by the addition of a second fluorochrome labeled antibody, specific for the species and isotype of the primary antibody.
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 solution of paraformaldehyde to inactivate pathogens that may be present in the sample and stabilize the binding of the antibodies to the cells. At this point, the cell suspension is ready for flow cytometric acquisition and analysis.
Flow cytometry has evolved into an important technology in the clinical laboratory with applications in several specialties in laboratory medicine. The following is a description of several typical applications of flow cytometry in the clinical laboratory.
•Enumeration of CD4-positive T-lymphocytes
The hallmark of infection with the Human Immunodeficiency Virus Type 1 (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.3
The absolute number of CD4 lymphocytes is reflective of the degree of immune deficiency in HIV infected individuals and may be used as a guide for timing the institution of antiretroviral therapy as well as monitoring the level of immune reconstitution following after initiation of therapy. Typically, a combination of CD3 and CD4 specific monoclonal antibodies are used to define the frequency of CD3/CD4-dual positive lymphocytes. 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). Alternatively, the absolute count may be determined with the use of a standard counting bead preparation.
•Enumeration of fetal hemoglobin containing cells
Enumeration of fetal hemoglobin containing red blood cells is useful for quantitating the level 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.4
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 anti-fetal hemoglobin fluorescence, 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 evaluation of the repopulating capacity of stem cell products to be used in hematopoeitic stem cell transplantation.5 Specifically, the number of CD34 positive cells in stem cell products is determined. In solid organ transplantation, flow cytometry is commonly used for the pre-transplant screening of recipients for HLA alloantibody and for the detection of donor-specific HLA antibodies.6
Individuals awaiting a renal transplant are routinely monitored for the presence of HLA alloantibody to identify those individuals pre-sensitized 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. When a donor is identified for an individual awaiting a renal transplant, serum from the patient is tested for the presence of donor-specific HLA alloantibody. This is termed the pretransplant crossmatch and if positive (indicating that the recipient has donor specific antibody), it may be a contraindication to transplantation. Flow cytometric crossmatching is the most sensitive method for detection of donor-specific HLA alloantibody.
•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 complement regulatory proteins (CD55 and CD59) as a result of defective GPI anchoring 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.7
•Immunophenotyping of hematologic malignancies
Hematologic malignancies represent a diverse group of white blood cell neoplasms. These clonal proliferations of various white blood cells are diagnosed by microscopic examination of blood, bone marrow, lymph nodes or other tissues. Flow cytometry 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, state of maturation and aberrant antigen expression.8 Flow cytometry can also be used to monitor patients for the presence of minimal residual disease after therapy.
Advances in reagents for flow cytometry are certain to expand its application beyond routine immunophenotyping. Assessment of immune function is one area that is likely to see increased clinical application of flow cytometry. Reagents such as tetrameric Class I MHC/peptide complex allow the enumeration of antigen-specific CD8 T cells. In addition, the flow cytometry based detection of intracellular cytokine production also allows the determination of antigen specific CD4 and CD8 responses. As vaccines to infectious agents and malignancies are further developed, these techniques will play an important role in assessing the effectiveness of these therapeutic approaches.
Dr. Schmitz is with the Department of Pathology & Laboratory Medicine, University of North Carolina — Chapel Hill.
1. Longbardi-Givan A. Flow Cytometry: First Principles, 2nd Edition. Wiley, 2001.
2. McCoy JP. Basic principles in clinical flow cytometry, p. 31-64. In: Keren DF, McCoy JP, Jr. and Carey JL (ed.), Flow Cytometry in Clinical Diagnosis, 3rd Ed. ASCP Press, Chicago, IL, 2001.
3. Fahey JL, Taylor JM, Detels R, Hofmann B, Melmed R, Nishanian P, Giorgi JV. The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus type 1. New Eng J Med 1990;322:166-172.
4. Davis BH, Olsen S, Bigelow NC, Chen JC . Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry. Transfusion 1998;38:749-756.
5. Sutherland DR, Keeney M, Chin-Yee I. Standardized flow cytometry assays for enumerating hematopoietic stem progenitor cells, p. 185-196. In: NR Rose, RG Hamilton and B Detrick (ed.), Manual of Clinical Laboratory Immunology, ASM Press, Washington, DC, 2002.
6. Bray RA, Gebel HM. Clinical utility of flow cytometry in allogeneic transplantation, p. 507-541. In: Keren DF, McCoy JP, Jr, and Carey JL (ed.), Flow Cytometry in Clinical Diagnosis. ASCP Press, Chicago, IL, 2001.
7. Richards SJ, Rawstron AC, Hillmen P. Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry 2000;42:223-233
8. Wood BL, Kidd PG. Immunophenotyping of leukemia and lymphoma by flow cytometry, p. 148-159. In: NR Rose, Hamilton RG and Detrick B (ed.), Manual of Clinical Laboratory Immunology, ASM Press, Washington, DC, 2002.