Flow cytometry allows rapid identification and quantification of subpopulations of cells in suspension through assessment of physical properties and antigen expression. Suitable specimens include fresh blood, bone marrow, body fluids, or tissue samples. (See specimen handling requirements.) Samples are incubated with flourescently-tagged antibodies directed against specific cell surface proteins. The antibodies used are generally tailored to specimen type, clinical history and suspected diagnosis. Our current clinical instruments are capable of simultaneously examining up to 10 different antigens, providing a very high level of sensitivity and allowing identification of abnormal populations at diagnosis as well as the identification of small abnormal populations in the post-therapy setting (minimal residual disease testing). The following section describes specific panels offered.
The standard acute leukemia panel is designed to determine whether leukemic blasts are of myeloid or lymphoid origin, and to then further classify the neoplastic cells (e.g., B lymphoblasts, T lymphoblasts, myeloid blasts, abnormal promyelocytes, monoblasts, etc.). The analysis is performed using a series of panels (typically 4) utilizing 10-color flow cytometry to identify and then characterize the immunophenotype of the blasts. The blasts are identified initially on the basis of CD45 versus side scatter characteristics (see figure 1a) and are then evaluated for the expression of B lymphoid, T lymphoid, myeloid, and monocytic markers (see figures 1b-1d).
Initial antibody panels include testing for the following surface markers.
|Myeloid||CD13, CD15, CD33, CD117|
|B cell||CD19, CD20, immunoglobulin kappa and lambda light chains|
|T Cell||CD2, CD3, CD4, CD5, CD7, CD8|
|Other||HLA-DR, CD38, CD10, CD71|
Figure 1: Acute myeloid leukemia
If the routine panel is insufficient to adequately characterize the leukemic cells, evaluation of additional antibodies including cytoplasmic markers (CD3, CD79a, myeloperoxidase, TdT), erythroid markers (glycophorin), or megakaryocytic markers (CD41 and CD61) may be required.
The standard lymphoma panel is designed to identify abnormal populations of B cells, T cells or NK cells.
The B cell panel involves evaluation of 8 antibodies in a single assay allowing a differential (CD45 versus side scatter), identification of B cells (CD19, CD20), evaluation of B cells for clonality (immunoglobulin kappa and lambda light chains), and evaluation for aberrant antigen expression (CD5, CD10, CD38) on B cells. If indicated by clinical history or initial immunophenotypic screening, additional antibodies (for example, CD23 for chronic lymphocytic leukemia; and CD11c, CD25 and CD103 for hairy cell leukemia) may be evaluated. Figure 2 shows an example of a CD5 positive, CD10 negative B cell lymphoma that lacked CD23, suspicious for mantle cell lymphoma.
Figure 2: Clonal CD5 positive B cell lymphoma
The standard T/NK cell panel involves evaluation of 9 antibodies in a single tube using CD45 versus side scatter to perform an initial differential and employing evaluation of CD2, CD3, CD4, CD5, CD7, CD8, CD34, and CD56 to further characterize the lymphoid cells. In some cases, additional markers (including CD16, CD25, CD30, CD52, alpha beta T cell receptor, or gamma delta T cell receptor) may be evaluated to allow further characterization of T or NK cells populations or to provide information regarding antigen expression prior to antibody driven therapies (e.g., Campath or Ontak). Furthermore T cell clonality can be investigated by evaluating expression of T cell receptor beta chain expression and expression of KIR receptors can be evaluated on NK cell. Figure 3 demonstrates an example of an abnormal T cell population (shown in yellow) consistent with a T cell lymphoproliferative disorder.
Figure 3: T cell lymphoproliferative disorder
The laboratory has recently validated a flow cytometric method for identification of the neoplastic cells in Classical Hodgkin lymphoma and if this entity is suspected on the basis of clinical findings or the morphology of a cytospin preparation in an appropriate specimen (tissue less than 48 hours since removal; peripheral blood and bone marrow are not appropriate specimens for this assay), antibodies to evaluate for Classical Hodgkin lymphoma (including CD15 and CD30) may be added.
Plasma Cell Neoplasms
Plasma cell neoplasms are evaluated using a 7 color panel in which CD45 versus CD38 expression is used to screen for an abnormal plasma cell population. Plasma cell populations are then evaluated for light chain expression (cytoplasmic kappa and lambda immunoglobulin light chain expression) and for aberrant expression of CD19, CD45 and CD56. In patients with no clinical history, specimens are generally screened for an abnormal B cell population as well to exclude the possibility of a B cell lymphoma with plasmacytoid differentiation. Figure 4 demonstrates an example of a clonal plasma cell population from a patient with a clinical history of multiple myeloma.
Figure 4: Abnormal plasma cell population
Immunophenotyping for evaluating myeloid stem cell disorders
(myeloproliferative disorders and myelodysplastic syndromes)
Recent studies have demonstrated and validated the presence of immunophenotypic abnormalities in the setting of myeloid stem cell disorders. All hematopoietic cells, including myeloid blasts, have scheduled and tightly regulated patterns of antigen expression with maturation and differentiation that commonly become abnormal with neoplastic transformation. Abnormalities seen include the expression of antigens at an abnormally increased or decreased level of intensity in comparison to that normally seen at a distinct stage of maturation for a particular lineage; aberrant co-expression of markers associated with maturity with markers of immaturity; uniform expression of an antigen that typically shows variable expression; and, expression of significant amounts of non-lineage specific antigens such as CD5, CD7 or CD56 on myeloid blasts. In the laboratory, we evaluate a series of antibodies known to demonstrate aberrant patterns of expression in myeloid stem cell disorders. Of note, although helpful in many cases, flow cytometry is often not abnormal in low grade myelodysplastic syndromes and chronic phase myeloproliferative disorders, thus correlation with clinical history, morphology, and genetic testing (including cytogenetics, JAK2 mutational status or PCR for a BCL-ABL fusion) may be required for diagnosis.
One minor criterion for a diagnosis of systemic mastocytosis using WHO criteria is the demonstration of abnormal antigen expression on mast cells. The laboratory uses an 8 color panel to isolate mast cells and evaluate mast cell populations for the presence of aberrant expression of CD2 and CD25. Figure 5 demonstrates an abnormal mast cell population from a patient with systemic mastocytosis.
Figure 5: Abnormal mast cell population
T Cell Subset Test
The UW Hematopathology Laboratory has been performing T cell subset testing almost 10 years in cooperation with the AIDS clinical trial group, and has consistently been one of the country's top laboratories for T cell subset testing based on periodic proficiency testing administered by the National Institute of Allergy and Infectious Diseases. Most recently we have received approval to perform 3 color T cell subset analysis, which will improve both the quality and efficiency of our testing.
T Cell Subsets
The absolute number of CD4 cells in the peripheral blood is used to follow the immunologic status of patients with HIV infection.
Our panel includes the T cell markers CD3, CD4, and CD8 and the pan-hematopoietic marker CD45, which is used for gating and quality control. The percentage of cells bearing each marker is determined by this method. In addition, the absolute number of CD4+ and CD8+ T cells is determined by including a calibrated number of fluorescent beads with the patient specimen, and comparing the number of lymphocytes to the number of beads that pass through the flow cytometer. This method eliminates the need for a separate lymphocyte count and allows determination of CD4 and CD8 counts on specimens that are up to 72 hours old. Results are reported by computer printout and include the absolute CD4 count, absolute CD8 count, and CD4/CD8 ratio. The Virology Division performs testing for the presence of antibodies to the human immunodeficiency virus (HIV) and the presence of HIV proteins and RNA in patient specimens.
For further information regarding HIV testing, please contact Community Services at 206.520.4600.
Specimen requirements: 5 mL heparin-anticoagulated peripheral blood (green top tube) plus 5 mL EDTA-anticoagulated peripheral blood ( lavender top tube).
The degree of immunosuppression in post-transplant (renal, cardiac, liver, pancreas, heart-lung) patients receiving antithymocyte globulin (ATG) or OKT3 is routinely followed by lymphocyte counts. The transplant antibody panel includes CD2, CD3, CD4, CD8, and CD19. The rejection antibody panel determines only the total number of T cells based on CD3 staining and is generally used to follow the therapeutic efficacy of ATG or OKT3 therapy. Results are reported by computer printout.
Specimen requirements: 5 mL heparin-anticoagulated peripheral blood (green top tube) plus 5 mL EDTA-anticoagulated peripheral blood (lavender top tube).
Bronchoalveolar Lavage Analysis (BAL)
The clinical role of the bronchoalveolar lavage (BAL) in the management of patients with interstitial lung diseases (ILD) continues to be explored. The cellular analysis of BAL, although not diagnostic, does offer clues to the etiology and/or the "activity" of the underlying parenchymal abnormality in ILD. For instance, a predominance of lymphocytes and an excess of T helper cells (increased CD4:CD8 ratio) may be useful in supporting the diagnosis of sarcoidosis.
A cell count and differential is performed on each BAL specimen. If greater than 10% of the nucleated cells are lymphocytes, the percentage of CD4+ and CD8+ cells is determined by flow cytometry, and a CD4:CD8 ratio is calculated. The Hematopathology Laboratory director and Dr. Ganesh Raghu, Associate Professor in Pulmonary and Critical Care Medicine, will issue a written report and interpretation for each case.
Specimen requirements: Bronchoalveolar lavage fluid.
Paroxysmal Nocturnal Hemoglobinuria (PNH) Assay
In Paroxysmal Nocturnal Hemoglobinuria (PNH), a clonal marrow stem cell population gives rise to circulating mature hematopoietic cells lacking the expression of a variety of different cell surface proteins whose common feature is their linkage to the cell membrane via a glycosyl-phosphatidyl-inositol (GPI) linkage, a linkage that is deficient in the PNH clone. Similar findings may also be seen in aplastic anemia and myelodysplasia, although the frequency of GPI-deficient cells is lower. GPI-deficiency is most easily assessed in the erythroid, granulocytic and monocytic lineages, and flow cytometry is the method of choice for their detection. The flow cytometric assay evaluates for a loss of expression of the following GPI-linked antigens: CD59 on red cells, CD14 and FLAER on monocytes, and CD24 and FLAER on granulocytes. The assay can detect as little as 0.01% GPI-deficient cells in each cell lineage.
Specimen requirements: 5 mL heparin-anticoagulated peripheral blood (green top tube) or 5 mL EDTA-anticoagulated peripheral blood (lavender top tube).
Custom Flow Cytometry
Customized antibody panels for research or clinical use can be designed in consultation with the laboratory directors and supervisors. Issues such as cell activation, expression of adhesion molecules, or expression of specific cell surface proteins can be addressed with special panels. Please inquire as to pricing and availability.
Last updated: 07/29/2020