Hematopathology Laboratory

Flow Cytometry

Flow cytometry is performed on cells in liquid suspension (i.e. blood, bone marrow, body fluids or tissue cell suspensions) that have been incubated with fluorescently tagged antibodies directed against specific cell surface proteins. A number of different antibody panels are used, depending on the clinical question to be answered. Our current instruments are capable of examining up to four different antigens simultaneously, although only two or three antigens are currently examined simultaneously for routine diagnostic use. Using the flow cytometer, 5000 cells are routinely counted with each antibody combination, and the percentage of positive cells is calculated. The relative fluorescence intensity of the positive cells indicates the amount of antibody bound to specific binding sites on the cell.

The specific panel of antibodies used is selected by the Hematopathology Laboratory supervisor and/or the director of the laboratory based on the patient's clinical history, referring physician information, and the morphologic appearance of the cells present in the specimen. Antibodies of particular interest may be requested by the referring physician. The laboratory director will issue a written report and interpretation for each case. In addition, printouts of flow histograms will be provided upon request.

Immunophenotyping of Acute Leukemias
The acute leukemia panel is designed to determine whether leukemic blasts are of myeloid or lymphoid origin, and to further classify the cells as B or T cell, monocytic, megakaryocytic, etc. The analysis is performed using three-color flow cytometry. Each antibody combination includes two antibodies directed against lineage-specific cell surface antigens and a third antibody against CD45, which is present on all hematopoietic cells. The inclusion of this third antibody allows the identification of small numbers of blasts in the presence of large numbers of nonmalignant cells, and is based on the blasts' lower expression of CD45. The current panel includes the following antibodies:

If the routine panel is insufficient to adequately characterize the leukemic cells, additional antibodies may be used to determine the expression of other B cell markers, T cell markers, NK cell markers, monocytic markers, erythroid markers, activation antigens, or cell adhesion molecules.

Workup of an acute leukemia generally includes the leukemia panel, and myeloperoxidase and non-specific esterase stains. The laboratory director will issue a written report and interpretation for each case, and flow histograms will be provided upon request.

Specimen requirements: Generally 10-20 ml Heparin-anticoagulated (green top tube) blood or bone marrow. The actual amount of blood needed depends on the number of leukemic cells in the peripheral blood; please contact the hematopathology laboratory at (206) 548-6231 to determine how much blood is necessary from patients with low blast counts. In patients with inaspirable marrow and no circulating blasts, it may be possible to obtain sufficient cells from a bone marrow core biopsy sent in tissue culture medium. Please contact the laboratory for more information.

Immunophenotyping of Lymphomas/Lymphoproliferative Disorders
The lymphoma panel is designed to characterize lymphoproliferative disorders, which usually are comprised of mature B or T cells. For B cell malignancies, demonstration of the presence of a monoclonal population by restricted kappa or lambda immunoglobulin light chain expression is diagnostic. For T cell or NK cell malignancies, there is no convenient clonal marker that can be demonstrated by cell surface marker studies, although in many cases the finding of an abnormal T cell phenotype is strong evidence for a T cell malignancy. To prove clonality for T cell malignancies, gene rearrangement studies need to be performed. The routine lymphoma panel is performed using three-color flow cytometry with the B cell markers CD19, CD20, kappa, and lambda, and the T cell markers CD2 and CD5. If a large granular lymphocyte (LGL) disorder is suspected, the LGL panel is performed, which includes the T cell markers CD3, CD4, and CD8, and the NK cell markers CD16, CD56, and CD57 substituted for the B cell markers. Based on the results of this initial panel, additional B or T cell markers are added to help further classify B cell processes or to try to demonstrate an abnormal phenotype in T cell processes. The laboratory director will issue a written report and interpretation for each case. Flow histograms will be provided upon request.

Specimen requirements: Heparin-anticoagulated blood or bone marrow (green top tube), fresh tissue specimens in tissue culture medium, body fluids

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. 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 three-color T cell subset analysis, which will improve both the quality and efficiency of our testing.

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 of the Laboratory Medicine Department 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) 548-6066.

Specimen requirements: 5 ml heparin-anticoagulated peripheral blood (green top tube) plus 5 ml EDTA-anticoagulated peripheral blood (lavender top tube)

Post-Transplant Monitoring
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

Reticulocyte Count
Flow cytometry is now the method of choice for performing reticulocyte counts. Manual methods are limited in their reproducibility by the low number of cells that can be counted. Flow cytometry allows thousands of cells to be counted rapidly, greatly improving reproducibility. The method is based on the observation that erythrocytes newly released from the bone marrow (reticulocytes) have a higher RNA content than more mature erythrocytes. RNA is detected by staining with a fluorescent dye, and the number of reticulocytes is determined from the number of red cells with fluorescence higher than background. Results are reported by computer printout.

Specimen requirements: 5 ml EDTA-anticoagulated (lavender top tube) peripheral blood

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. The feature common among these proteins is their linkage to the cell membrane via a glycosyl-phosphatidyl-inositol (GPI) linkage. The cell lines most commonly affected are the erythroid, granulocytic and monocytic lineages, and flow cytometry can be used to detect the loss of GPI-linked proteins on each of these lines. For the erythroid cell line, preferential analysis of reticulocytes increases the sensitivity of the assay and recent studies suggest such an assay may be more sensitive than the traditional sucrose hemolysis and Ham's tests used to diagnose PNH. The loss of GPI-linked proteins is also seen in some cases of aplastic anemia having features of PNH and may be a useful test in making this difficult diagnosis. A written interpretive report will be issued by the laboratory directors.

Specimen requirements: Heparin or EDTA-anticoagulated blood or bone marrow (green or lavender top tube)

Custom Flow Cytometry Panels
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.

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Last updated: 6/22/00