Flow Cytometric Evaluation of Canine Bone Marrow Differential Cell Counts

Abstract

Three flow cytometric techniques were evaluated for determination of differential cell counts on canine clinical bone marrow specimens. Techniques included staining bone marrow specimens with 2'7'-dichlorofluorescein (DCF) or 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluation of forward-angle light scatter vs. side-angle light scatter plots. Flow cytometric evaluation of bone marrow cells stained with DCF failed to separate bone marrow cells into distinct cell populations. Staining with DiOC6 resulted in separation of bone marrow cells into populations of mature and immature erythroid cells, mature and immature myeloid cells, and lymphocytes. The scatter plot method resulted in identification of mature and immature erythroid cells, immature myeloid cells, metamyelocytes, and bands and segmenters. Lymphocytes could not be differentiated from mature erythroid cells by the scatter plot method. When the results of the DiOC6 method and the scatter plot method were compared with manual bone marrow differential cell counts, the scatter plot method had more similar mean values and higher correlation coefficients. The scatter plot method has the potential of providing rapid semiquantitative assessment of bone marrow differential cell counts in dogs for specimens that contain low numbers of lymphocytes.

Flow Cytometric and Immunophenotypic Evaluation of Acute Lymphocytic Leukemia in Dog Bone Marrow

“…Because erythroid cells have low granularity and may overlap in size with lymphoid cells, erythremic myelosis may be difficult to distinguish from lymphocytic leukemia. 13,14 Scatter plot analysis has been used for analysis of leukemias in the peripheral blood of dogs. 6,7,21 Scatter plot analysis correctly predicted the lineage of leukemic cells in 7 of 8 dogs.…”

“…Previous studies have shown that rubriblasts and rubricytes are not lysed by this procedure. 13 After 10 minutes of incubation at room temperature, tubes were centrifuged, and the cell pellet was washed and resuspended in 1.0 mL of Dulbecco's phosphate-buffered saline solution containing 1% sheep serum and 2 mM sodium azide. The cell count was adjusted to 1 ϫ 10 7 cells/mL.…”

“…i For all samples, cell populations were displayed by forward-angle versus side-angle light scatter properties (scatter plot technique) as described. 13 A total of 30,000 cells were analyzed for each blood or bone marrow specimen. Scatter plots were evaluated by use of templates previously developed for differential cell counting ( Fig 1A).…”

“…Scatter plots were evaluated by use of templates previously developed for differential cell counting ( Fig 1A). 13,14,19 The bone marrow template enabled identification of im-mature and mature nucleated erythroid cells, immature myeloid cells, metamyelocytes, and mature myeloid cells. Blood and bone marrow samples were also analyzed by a 2nd scatter plot with forward-angle light scatter on the x-axis and fluorescence intensity on the y-axis.…”

“…In previous studies, we used a flow cytometric forwardangle versus side-angle light scatter plot technique (scatter plot technique) to identify subpopulations of myeloid and erythroid cells in nonmalignant canine bone marrow specimens. 13 A template was developed to identify immature and mature nucleated erythroid cells, immature myeloid cells, metamyelocytes, and mature myeloid cells ( Fig 1A). Use of the scatter plot technique did not permit quantification of lymphocytes because the lymphoid cells overlapped with erythroid cells.…”