Flow Cytometry

Flow Cytometry: Principles and Clinical Applications in Hematology

General Principles

Flow cytometry measures optical and fluorescence characteristics of single cells (or any other particle, including nuclei, microorganisms, chromosome preparations, and latex beads). Physical properties, such as size (represented by forward angle light scatter) and internal complexity (represented by right-angle scatter) can resolve certain cell populations. Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA. Additionally, antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state. Upon returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The use of multiple fluorochromes, each with similar excitation wavelengths and different emission wavelengths (or “colors”), allows several cell properties to be measured simultaneously. Commonly used dyes include propidium iodide, phycoerythrin, and fluorescein, although many other dyes are available. Tandem dyes with internal fluorescence resonance energy transfer can create even longer wavelengths and more colors. The  list of clinical applications and cellular characteristics that are commonly measured. Several excellent texts and reviews are available.

 

 

 

Common clinical uses of flow cytometry.

Field Clinical application Common characteristic measured
Immunology Histocompatibility cross-matching IgG, IgM
Transplantation rejection CD3, circulating OKT3
HLA-B27 detection HLA-B27
Immunodeficiency studies CD4, CD8
Oncology DNA content and S phase of tumors DNA
Measurement of proliferation markers Ki-67, PCNA1
Hematology Leukemia and lymphoma phenotyping Leukocyte surface antigens
Identification of prognostically important subgroups TdT, MPO
Hematopoietic progenitor cell enumeration CD34
Diagnosis of systemic mastocytosis CD25, CD69
Reticulocyte enumeration RNA
Autoimmune and alloimmune disorders
Anti-platelet antibodies IgG, IgM
Anti-neutrophil antibodies IgG
Immune complexes Complement, IgG
Feto-maternal hemorrhage quantification Hemoglobin F, rhesus D
Blood banking Immunohematology Erythrocyte surface antigens
Assessment of leukocyte contamination of blood products Forward and side scatter, leukocyte surface antigens
Genetic disorders PNH CD55, CD59
Leukocyte adhesion deficiency CD11/CD18 complex

 

Common clinical uses of flow cytometry.

Inside a flow cytometer, cells in suspension are drawn into a stream created by a surrounding sheath of isotonic fluid that creates laminar flow, allowing the cells to pass individually through an interrogation point. At the interrogation point, a beam of monochromatic light, usually from a laser, intersects the cells. Emitted light is given off in all directions and is collected via optics that direct the light to a series of filters and dichroic mirrors that isolate particular wavelength bands. The light signals are detected by photomultiplier tubes and digitized for computer analysis. Fig. 1 is a schematic diagram of the fluidic and optical components of a flow cytometer. The resulting information usually is displayed in histogram or two-dimensional dot-plot formats.

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Figure 1.

Schematic of a flow cytometer.

A single cell suspension is hydrodynamically focused with sheath fluid to intersect an argon-ion laser. Signals are collected by a forward angle light scatter detector, a side-scatter detector (1), and multiple fluorescence emission detectors (2–4). The signals are amplified and converted to digital form for analysis and display on a computer screen.

DNA Content Analysis

The measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as propidium iodide, that intercalate into the DNA helical structure. The fluorescent signal is directly proportional to the amount of DNA in the nucleus and can identify gross gains or losses in DNA. Abnormal DNA content, also known as “DNA content aneuploidy”, can be determined in a tumor cell population. DNA aneuploidy generally is associated with malignancy; however, certain benign conditions may appear aneuploid. DNA aneuploidy correlates with a worse prognosis in many types of cancer but is associated with improved survival in rhabdomyosarcoma, neuroblastoma, multiple myeloma, and childhood acute lymphoblastic leukemia (ALL). In multiple myeloma, ALL, and myelodysplastic syndromes, hypodiploid tumors cells portend a poor prognosis. In contrast, hyperdiploid cells in ALL have a better prognosis. For many hematologic malignancies, there are conflicting reports regarding the independent prognostic value of DNA content analysis. Although conventional cytogenetics can detect smaller DNA content differences, flow cytometry allows more rapid analysis of a larger number of cells.

Immunophenotyping Applications in Hematology

The distributed nature of the hematopoietic system makes it amenable to flow cytometric analysis. Many surface proteins and glycoproteins on erythrocytes, leukocytes, and platelets have been studied in great detail. The availability of monoclonal antibodies directed against these surface proteins permits flow cytometric analysis of erythrocytes, leukocytes, and platelets. Antibodies against intracellular proteins such as myeloperoxidase and terminal deoxynucleotidyl transferase are also commercially available and permit analysis of an increasing number of intracellular markers.

erythrocyte analysis

The use of flow cytometry for the detection and quantification of fetal red cells in maternal blood has increased in recent years. Currently in the United States, rhesus D-negative women receive prophylactic Rh-immune globulin at 28 weeks and also within 72 h of delivery. The standard single dose is enough to prevent alloimmunization from ∼15 mL of fetal rhesus D+ red cells. If feto-maternal hemorrhage is suspected, the mother’s blood is tested for the presence and quantity of fetal red cells, and an appropriate amount of Rh-immune globulin is administered. The quantitative test most frequently used in clinical laboratories is the Kleihauer-Betke acid-elution test. This test is fraught with interobserver and interlaboratory variability, and is tedious and time-consuming. The use of flow cytometry for the detection of fetal cells is much more objective, reproducible, and sensitive than the Kleihauer-Betke test. Fluorescently labeled antibodies to the rhesus (D) antigen can be used, or more recently, antibodies directed against hemoglobin F. This intracellular approach, which uses permeabilization of the red cell membrane and an antibody to the γ chain of human hemoglobin, is precise and sensitive. This method has the ability to distinguish fetal cells from F-cells (adult red cells with small amounts of hemoglobin F). Fig. 2 is a histogram of a positive test for feto-maternal hemorrhage. Although the flow cytometry method is technically superior to the Kleihauer-Betke test, cost, instrument availability, and stat access may limit its practical utility.

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Figure 2.

Hemoglobin F test for feto-maternal hemorrhage.

Most adult RBCs do not have any hemoglobin F and are included in the large peak on the left. A few adult red cells have a small amount of hemoglobin F and are called F cells. Higher quantities of hemoglobin F in fetal cells yield a higher fluorescence signal and allow discrimination between fetal cells and adult F cells.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder that leads to intravascular hemolysis with associated thrombotic and infectious complications. PNH can arise in the setting of aplastic anemia and may be followed by acute leukemia. The disease is caused by deficient biosynthesis of a glycosylphosphatidylinositol linker that anchors several complement and immunoregulatory surface proteins on erythrocytes, monocytes, neutrophils, lymphocytes, and platelets. On erythrocytes, deficiencies of decay-accelerating factor and membrane-inhibitor of reactive lysis render red cells susceptible to complement-mediated lysis. Conventional laboratory tests for the diagnosis of PNH include the sugar water test and the Ham’s acid hemolysis test. Problems associated with these tests include stringent specimen requirements and limited specificity. Antibodies to CD55 and CD59 are specific for decay-accelerating factor and membrane-inhibitor of reactive lysis, respectively, and can be analyzed by flow cytometry to make a definitive diagnosis of PNH. In affected patients, two or more populations of erythrocytes can be readily identified, with different degrees of expression of CD55 and CD59 (Fig. 3 )

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Figure 3.

Diagnosis of PNH.

Control individuals (A) show high expression of CD55 and CD59 on all red cells. In PNH (B), some stem cell clones produce RBCs with decreased expression of CD55 and CD59. In the PNH patient (B), two distinct populations are present: normal red cells with high CD55 and CD59 expression and a second population with low CD55 and CD59 expression.

leukocyte analysis

Immunologic monitoring of HIV-infected patients is a mainstay of the clinical flow cytometry laboratory. HIV infects helper/inducer T lymphocytes via the CD4 antigen. Infected lymphocytes may be lysed when new virions are released or may be removed by the cellular immune system. As HIV disease progresses, CD4-positive T lymphocytes decrease in total number. The absolute CD4 count provides a powerful laboratory measurement for predicting, staging, and monitoring disease progression and response to treatment in HIV-infected individuals. Quantitative viral load testing is a complementary test for clinical monitoring of disease and is correlated inversely to CD4 counts. However, CD4 counts directly assess the patient’s immune status and not just the amount of virus. It is likely that both CD4 T-cell enumeration and HIV viral load will continue to be used for diagnosis, prognosis, and therapeutic management of HIV-infected persons.

Perhaps the best example of simultaneous analysis of multiple characteristics by flow cytometry involves the immunophenotyping of leukemias and lymphomas. Immunophenotyping as part of the diagnostic work-up of hematologic malignancies offers a rapid and effective means of providing a diagnosis. The ability to analyze multiple cellular characteristics, along with new antibodies and gating strategies, has substantially enhanced the utility of flow cytometry in the diagnosis of leukemias and lymphomas. Different leukemias and lymphomas often have subtle differences in their antigen profiles that make them ideal for analysis by flow cytometry. Diagnostic interpretations depend on a combination of antigen patterns and fluorescence intensity. Several recent review articles are available. Flow cytometry is very effective in distinguishing myeloid and lymphoid lineages in acute leukemias and minimally differentiated leukemias. Additionally, CD45/side scatter gating often can better isolate the blast population for more definitive phenotyping than is possible with forward scatter/side scatter gating.

Quantification of Soluble Molecules

Soluble antigens or antibodies can be quantified by flow cytometry if standard cells or beads are used. For example, OKT3 is a mouse anti-human antibody useful in treating transplant rejection. Circulating concentrations of OKT3 can be quantified by incubating with normal CD3-positive lymphocytes, followed by a fluorescently labeled anti-mouse antibody. Fluorescence values are compared to a calibration curve generated with known amounts of OKT3. Recently, multiplex assays for several antigens have become possible by the use of beads indexed by incorporating two different dyes.

Summary

Flow cytometry is a powerful technique for correlating multiple characteristics on single cells. This qualitative and quantitative technique has made the transition from a research tool to standard clinical testing. Applications in hematology include DNA content analysis, leukemia and lymphoma phenotyping, immunologic monitoring of HIV-infected individuals, and assessment of structural and functional properties of erythrocytes, leukocytes, and platelets. Smaller, less expensive instruments and an increasing number of clinically useful antibodies are creating more opportunities for routine clinical laboratories to use flow cytometry in the diagnosis and management of disease.

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