PREPARATION OF ANALYSIS TEMPLATE
Details for precise adjustment of regions follows in the next section.
1) Create the following bi-variate dot-plots as shown in Figure 1 and Figure 2:
a. Page 1 upper left: Tube 1, CD19 vs SSC, ungated. Set R1 around the B-cell population (CD19+SSClo). Initially, this region should be as inclusive as possible – set it to the edges of the monocyte and CD19- lymphoid populations.
b. Page 1 upper centre: Tube 1, FSC vs SSC, R1 gated. Set R2 around the B-cell lymphoid population, excluding apoptotic cells, debris, and doublets.
c. Page 1 upper right: Tube 1, CD19 vs SSC, R2 gated.
d. Page 1 middle left: Tube 1, CD3 vs SSC, ungated. Set R3 around the T-cell population (CD3+SSClo).
e. Page 1 middle centre: Tube 1, FSC vs SSC, R1*R2*R3 gated.
f. Page 1 middle right: Tube 1, CD19 vs SSC, R1*R2*R3 gated.
g. Page 1 lower left: Tube 1, CD45 vs SSC, ungated. Set region R4 around leucocytes (CD45+), excluding erythroid cells and debris. Show gate statistics. For this and all following statistics windows, include the following parameters where available: File Name; Sample ID; Patient ID; Patient Name; Acquisition Date; Gated Events; Total Events; X Parameter; Y Parameter; Quad Location; Custom Keyword #1; Custom Keyword #2; Custom Keyword #3; Label; Events; % Gated; X Geo Mean; Y Geo Mean.
h. Page 1 lower centre: Tube 2, CD5 vs kappa, R1*R2 gated. Show quadrant statistics.
i. Page 1 lower right: Tube 2, CD5 vs lambda, R1*R2 gated. Show quadrant statistics.
j. For page 1 layout and approximate region placement, see Figure 1.
k. Page 2 upper left: Tube 3, CD5 vs CD20, R1*R2 gated. Set region R5 (see below). Show regions statistic for this plot.
l. Page 2 upper centre: Tube 3, CD38 vs CD20, R1*R2 gated. Set region R6 (see below). Show regions statistic for this plot.
m. Show gate statistics for the page 2 upper left plot (CD5 vs CD20, R1*R2 gated).
n. Page 2 upper right: Tube 3, CD38 vs CD20, R1*R2*R5*R6 gated.
o. Page 2 middle left: Tube 4, CD5 vs CD22, R1*R2 gated. Set region R7 (see below). Show regions statistic for this plot.
p. Page 2 middle centre: Tube 4, CD22 vs CD81, R1*R2 gated. Set region R8 (see below). Show regions statistic for this plot.
q. Show gate statistics for the page 2 middle left plot (CD5 vs CD22, R1*R2 gated).
r. Page 2 middle right: Tube 4, CD5 vs CD81, R1*R2*R7*R8 gated.
s. Page 2 lower left: Tube 5, CD5 vs CD79b, R1*R2 gated. Set region R7 (see below). Show regions statistic for this plot.
t. Page 2 lower centre: Tube 5, CD5 vs CD43, R1*R2 gated. Set region R8 (see below). Show regions statistic for this plot.
u. Show gate statistics for the page 2 lower left plot (CD5 vs CD79b, R1*R2 gated).
v. Page 2 middle right: Tube 5, CD79b vs CD43, R1*R2*R9*R10 gated.
w. For page 2 layout and approximate region placement, see Figure 2.
ADJUSTING REGIONS
2) If automatic recalculation is on, switch it off now. If automatic recalculation is off, recalculate now.
3) Zoom on the page 1 upper left hand plot (CD19 vs. SSC, ungated) to expand the CD19+SSClo events (see Figure 3).
Figure 3: Zoom in on B-cells in the upper left plot on page 1.
4) Zoom in on page 1 upper middle plot (SSC vs FSC, R1-gated) to expand the mononuclear cells (see Figure 4).
Figure 4: Zoom in on lymphoid cells in upper middle plot on page 1
5) Zoom on the page 1 upper right hand plot (CD19 vs. SSC, R2-gated) to expand the CD19+ SSClo events (see Figure 5).
Figure 5: Zoom in on B-cells in upper right plot on page 1
6) Zoom on the page 1 middle right hand plot (CD19 vs. SSC, R1*R2*R3-gated) to expand the CD19+ SSClo events (see Figure 6).
Figure 6: Zoom in on B-cell area showing contamination in middle right plot on page 1
7) Adjust R3 to include CD3+SSClo events (see Figure 7)
Figure 7: Adjust R3 to enclose CD3+SSClo events in middle left plot on page 1
8) Adjust R4 to include leucocytes and exclude erythrocytes and debris (see Figure 8).
Figure 8: Adjust R4 to enclose leucocyte events and exclude erythrocytes/debris in lower left plot on page 1
9) Adjust gates R1 & R2:
- R2 – adjust to exclude apoptotic events and debris (events to the bottom of the plot) and cellular aggregates (events to the top of the plot). See Figure 9.
Figure 9: Adjust R2 to exclude erythrocytes/debris and cellular aggregates in upper middle plot on page 1
b. R1 – assess the characteristics of contaminating events in the R1*R2*R3-gated CD19 vs. SSC plot and reduce the size of R1 to exclude as many CD3+ events whilst excluding as few CD19+SSClo events as possible (see Figure 10).
Figure 10: Exclude contaminating CD3+ events by raising the lower edge of R1 but do not raise it so high as to exclude any B-cell events. This should be done using both upper right and middle right plots on page 1.
c. Repeat this process for R2 if necessary (see Figure 11).
Figure 11: Exclude contaminating CD3+ events by adjusting R2 but do not exclude any B-cell events. This should be done using both upper middle and centre plots on page 1.
10) Recalculate now and repeat step (9) if necessary.
11) Adjust the quadrants on the page 1 lower middle plot (CD5 vs. lambda, R1*R2 gated). Use the CD5-negative events to set the vertical delineater between lambda+ and lambda- events. If there is no clear delineation between the CD5+ and CD5- events, change the date file to show file tag .001 (i.e. the CD45/CD14/CD19/CD3 file for the same patient). Use the CD3 and CD14 expression as negative controls to set the horizontal and vertical quadrants respectively. See Figure 12.
Figure 12: If it is not clear where to set the horizontal quadrants in the lower middle and right plots, change these files to show tube 1 and adjust the horizontal quadrant to the upper edge of CD3 expression. Set the vertical quadrants using the positive and negative populations present in the CD5- fraction.
12) Copy the quadrants to the page 1 lower right plot (CD5 vs kappa, R1*R2 gated). If necessary, adjust the quadrants using the same approach as in step (12).
13) Adjust R5 – R10 to include CLL cells and exclude normal B-cells. The distribution patterns for typical CLL cells with a polyclonal background are shown in Figure 2. Contaminating events may include T-cells, NK-cells, and Monocytes; the profiles for these potential contaminants are shown in Figure 13, Figure 14, and Figure 15 respectively. Note that only T-cells should interfere with analysis of typical CLL, and will identify the same “space” as CLL cells in tubes 3 and 5. Other contaminants should be excluded by the complete gating strategy, but care needs to be taken with atypical CLL cases.
Figure 13: T-cell antigen expression profile for the CLL MRD reagents. The vast majority of T-cell events would normally be excluded using regions R1 and R2. Contaminating T-cell events may satisfy both CLL regions in tubes 3 and 5 but not in tube 4.
Figure 14: NK-cell antigen expression profile for the CLL MRD reagents. The vast majority of NK-cell events would normally be excluded using regions R1 and R2. Contaminating NK-cell events should be excluded from CLL regions in all tubes.
Figure 15: Monocyte antigen expression profile for the CLL MRD reagents. The vast majority of monocyte events would normally be excluded using regions R1 and R2. Contaminating monocyte events should be excluded from CLL regions in all tubes.
More precise instruction for setting individual regions are as follows. Regions R5-R10 should only contain events that the operator is confident represent CLL cells. The events included should form a homogeneous cluster in the relevant plots (except the CD38 vs. CD20 plot as CLL events may have heterogeneous CD38 expression). The cluster should be discrete from other gated B-cell events. If there is not a discrete cluster of events, then the results for that file should not be included in the analysis (if using automated analysis, set the relevant regions to include zero events).
14) Adjust R5: If polyclonal B-cells are present, set the right edge of R5 to the left of the CD5+CD20bright mature B-cell population. If B-progenitors are present (i.e. CD5-CD20dim) set the lower edge of R5 to delineate CLL cells and B-progenitors. If no B-progenitors are present, set the lower edge of R5 to the same level as the horizontal quadrant in the page 1 lower middle plot (CD5 vs kappa, R1*R2 gated). Alternatively, set the plot to show the data from file tag.001 using CD3 expression to define the lower limit of positivity for CD5 APC. If there is no clear distinct CLL population by CD5 vs CD20 expression, it is unlikely that this tube will be informative.
15) Adjust R6: If polyclonal B-cells are present, set the right edge of R6 to the left of the CD20bright mature B-cell population. If B-progenitors are present (i.e. CD38brightCD20dim) set the upper edge of R6 to delineate CLL cells and B-progenitors. If no B-progenitors are present, the position of the upper edge of R6 is not relevant.
16) If there is a population of cells that are suspicious of having a CLL-phenotype with respect to CD20/CD38 expression but are not clearly separated from the normal B-cells, adjust the gate in the CD38 vs CD20 plot from R1*R2 to R5*R2*R1. This may improve the separation between the remaining CLL cells and normal B-cells – if so, adjust R6 accordingly. If not, adjust R6 so as not to exclude any CLL cells. Return the gate to R1*R2. See Figure 16.
Figure 16: If a discrete population is not apparent in the CD38 vs CD20 plot (upper middle on page 2), adjust the gate to show events in R5*R2*R1 (a). Optimise R6 so that CLL cells are not excluded whilst excluding as many normal B-cells as possible (b). Return the gate to R2*R1 (c).
17) Assess the upper right plot, which displays events defined as CLL cells. The cells should appear as a single population with homogeneous antigen expression. See Figure 17.
Figure 17: The events in the upper right plot should appear as a single homogeneous population. There may be broad distribution of CD38 expression but discrete event clusters, as seen in the (a), should not occur. Re-adjust R5 and R6 if necessary
18) Adjust R7: If polyclonal B-cells are present, set the right edge of R7 to the left of the CD22bright mature B-cell population. If B-progenitors are present (i.e. CD5-CD22dim) set the lower edge of R5 to delineate CLL cells and B-progenitors. If no B-progenitors are present, set the lower edge of R5 to the same level as the horizontal quadrant in the page 1 lower middle plot (CD5 vs kappa, R1*R2 gated). Alternatively, set the plot to show the data from file tag.001 using CD3 expression to define the lower limit of positivity for CD5 APC.
19) Adjust R8: R8 should encapsulate a population of cells with dim/moderate CD22 expression and negative/dim CD81 expression. If polyclonal mature CD22brightCD81mod B-cells are present, the right edge of R8 should be to the midline or left of the mature B-cell CD81 expression level and the upper edge of R8 should be to the midline or below the mature B-cell CD22 expression level. Contaminating T-cells will have little or no CD22 expression and similar CD81 expression to the mature B-cells.
20) If there is a population of cells that are suspicious of having a CLL-phenotype with respect to CD5/CD22 expression but are not clearly separated from the normal B-cells, adjust the gate in the CD5 vs CD22 plot from R1*R2 to R8*R2*R1. This may improve the separation between the remaining CLL cells and normal B-cells – if so, adjust R7 accordingly. Return the gate to R1*R2. See Figure 18.
Figure 18: If a discrete population is not apparent in the CD5 vs CD22 plot (middle left on page 2), adjust the gate to show events in R8*R2*R1 – i.e. events with weak CD22 and CD81 (a). This may provide a clear delineation between CLL cells and polyclonal B-cells. Optimise R7 so that CLL cells are not excluded whilst excluding as many normal B-cells as possible (b). Return the gate to R2*R1 (c).
21) If there is a population of cells that are suspicious of having a CLL-phenotype with respect to CD22/CD81 expression but are not clearly separated from the normal B-cells, adjust the gate in the CD22 vs CD81 plot from R1*R2 to R7*R2*R1. This may improve the separation between the remaining CLL cells and normal B-cells – if so, adjust R8 accordingly. Return the gate to R1*R2. See Figure 19.
Figure 19: If a discrete population is not apparent in the CD22 vs CD81 plot (middle left on page 2), adjust the gate to show events in R7*R2*R1 – i.e. events with weak CD22 and strong CD5 (a). This may provide a clear delineation between CLL cells and polyclonal B-cells. Optimise R8 so that CLL cells are not excluded whilst excluding as many normal B-cells as possible (b). Return the gate to R2*R1 (c).
22) Assess the middle right plot, which displays events defined as CLL cells. The cells should appear as a single population with homogeneous antigen expression. See Figure 20.
Figure 20: The events in the middle right plot should appear as a single homogeneous population. If discrete event clusters, as seen in the (a), are present, re-adjust R7 and R8 accordingly.
23) Adjust R9: If polyclonal B-cells are present, set the lower right edge of R9 immediately to the left of the CD79bbright mature B-cell population. The upper right edge of R9 should be to the midline or left of the mature B-cell CD79b expression level. If B-progenitors are present (i.e. CD5-CD79bdim/neg) set the lower edge of R9 to delineate CLL cells and B-progenitors. If no B-progenitors are present, set the lower edge of R9 to approximately the same level as the horizontal quadrant in the page 1 lower middle plot (CD5 vs kappa, R1*R2 gated). Alternatively, set the plot to show the data from file tag.001 using CD3 expression to define the lower limit of positivity for CD5 APC. If there is no clear population by CD5/CD79b, this tube is unlikely to be informative.