FLOW-0311-HD Flow Cytometry Dry Challenge Scatterplots

Scatterplots

Scatterplot 1
Histogram 1 shows Log SSC vs. FS. Histogram 2 shows Log SSC vs. CD45PC5. This plot is most useful in leukemia diagnosis where intensity and light scatter characteristics are quite variable. In lymphoproliferative disorders the value of this tube is in determining the purity of the acquisition gate and determining the background fluorescence level of the sample based on staining intensity with CD45 and logarithmic light scatter. Note: in this sample that there is only one population identified in this histogram corresponding to lymphocytes. Isotypic controls are not used when assessing surface markers, therefore histogram 3 and 4 show the background fluorescence of each population on FL1 (FITC), FL2 (PE) and FL3 (ECD). All subsequent scatterplots are gated on CD45.

Scatterplot 2
These histograms show a combination of CD8 FITC vs. CD 4 PE and CD3 ECD These markers are associated with cells of T-cell lineage. CD4 and CD8 combined are usually equal to total CD3 as is the case here.

Scatterplot 3
The sample is stained for CD5, CD19 and CD10. Histogram 2 is a combination of CD19 and CD10 showing positive staining in this case. Both markers are consistent with a lymphoid origin of the cell population. CD5 positivity is not expressed on the CD19 positive population as seen in histogram 1. This is an important finding as CD19+ CD5+ cells are predominant in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma. The co-expression of CD10 on the CD19 positive cell and the dim expression of CD19 while not diagnostic, is consistent with a cell of follicular origin. Note: not all CD19+ cells express CD10.

Scatterplot 4
Combination of FMC7, CD23 and CD19. The majority of B cells are positive for FMC7 and CD19, as seen in histogram 2 and negative for CD23 histogram 3. FMC7 positivity is associated with follicular lymphomas if present on a cell that is CD19+CD10+ as this case shows.

Scatterplot 5
This three-colour combination has CD20 and CD2, in addition to CD45. The B cells of interest express normal levels of CD20 and do not co-express CD2.

Scatterplot 6
In the assessment of immunoglobulins, it is often helpful to look at the kappa and lamba light chains on the same fluorochrome. The next three tubes are a control Fab’2 FITC with CD19PE and CD45PECY5, followed by kappa FITC, CD19PE and CD45PC5 and finally lambda FITC with CD19PE and CD45PC5.The control tube, histograms 1 and 2 show CD19PE positive cells with little background staining in FITC. The CD19PE kappa FITC tube shows a small population of CD19+ cells positive for kappa light chains histogram 3.The CD19PE lambda FITC tube shows the majority of B cells positive for lambda light chains histogram 4.

Scatterplot 7
Normally if the kappa and lambda light chains are stained with the same fluorochrome, in this case FITC, they would appear to have the same staining intensity and distribution. This histogram shows an overlay of the kappa and lambda light chains. Note that there are dim lambda events, which are not present in the kappa staining. In addition, kappa to lambda ratio is usually in the range 1-1.5 to 1. In this case the ratio is reversed and is 0.38 to 1.