FLOW-0211-HD Flow Cytometry Dry Challenge Scatterplots

Scatterplots

Scatterplot 1
Log SSC versus CD45PC5. This plot is an essential part of leukemia diagnosis. From this histogram, a 'fingerprint' of each specific case can be determined, based on staining intensity with CD45 and logarithmic light scatter. There appears to be a large population of cells overlapping the mature granulocyte population. Isotypic controls were not used when assessing surface markers, therefore histogram 2 shows the background fluorescence of each population on FL1 (FITC) and FL2 (PE), as noted by the different intensity for each coloured population. Note the high autofluorescence of the unstained cells in this sample. While not diagnostic, this high autofluorescence is common in promyelocytic leukemias.

Scatterplot 2
This slide shows a combination of CD14 (clone MO2) versus CD36, the thrombospondin receptor and CD14 (clone MY4 ) versus CD64 (FcR1). The use of two separate CD14 antibodies can be useful in identifying a clonal monocytic population. In this case, the cells are negative for both markers. Cells of interest are also negative with CD36, usually associated with cells monocytic or platelet origin. CD64 is weakly expressed and is associated with cells of myelomonocytic origin and may present on mature granulocytic cells after activation.

Scatterplot 3
CD4 is weakly positive on the cells of interest. Monocytes express CD4, however usually at levels below those of T helper cells. The combination of CD15 and CD13 show positive staining in this case. Both markers are consistent with a myeloid origin of the cell population.

Scatterplot 4
CD10 when positive with CD19 is important in the diagnosis of acute lymphoblastic leukemia or in mature follicular lymphomas. In this case, the cells are negative for CD10, CD19 and CD20. The majority of cells in this case are also negative for CD2 a T-cell associated antigen.

Scatterplot 5
Note the high expression of CD33. This is common in acute monocytic leukemia's and is also seen in promyelocytes. CD33 is usually weakly expressed on mature granulocytes. The cells are negative for CD34, HLA-DR and CD7. HLA-DR negative, strong CD33 positive myeloid cells should alert the flow cytometrist to the possibility of a promyelocytic leukemia.

Scatterplot 6
When assessing intracellular markers, isotypic controls may be of assistance in determining the level of non specific staining within the permeabilized population. Note the extremely high background on the FITC control.

Scatterplot 7
Assessment of cytoplasmic Myeloperoxidase, CD3 and CD79a is an essential part of leukemia immunophenotyping. This 4-colour analysis allows assessment of T (cCD3) B (cCD79a) and myeloid populations (cMPO) in a single tube. This case is negative for cCD3 and cCD79a and strongly positive with MPO.