SUPPLEMENTARY MATERIAL
PREPARATION OF ANALYSIS TEMPLATE
Create the following bi-variate dot-plots as shown in Supplementary Figure 1 and
Supplementary Figure 2:
1) 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.
2) 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.
3) Page 1 upper right: Tube 1, CD19 vs SSC, R2 gated.
4) Page 1 middle left: Tube 1, CD3 vs SSC, ungated. Set R3 around the T-cell
population (CD3+SSClo).
5) Page 1 middle centre: Tube 1, FSC vs SSC, R1*R2*R3 gated.
6) Page 1 middle right: Tube 1, CD19 vs SSC, R1*R2*R3 gated.
7) 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.
8) Page 1 lower centre: Tube 2, CD5 vs kappa, R1*R2 gated. Show quadrant
statistics.
9) Page 1 lower right: Tube 2, CD5 vs lambda, R1*R2 gated. Show quadrant
statistics.
10) For page 1 layout and approximate region placement, see Supplementary
Figure 1.
11) Page 2 upper left: Tube 3, CD5 vs CD20, R1*R2 gated. Set region R5 (see
below). Show regions statistic for this plot.
12) Page 2 upper centre: Tube 3, CD38 vs CD20, R1*R2 gated. Set region R6 (see
below). Show regions statistic for this plot.
13) Show gate statistics for the page 2 upper left plot (CD5 vs CD20, R1*R2
gated).
14) Page 2 upper right: Tube 3, CD38 vs CD20, R1*R2*R5*R6 gated.
15) Page 2 middle left: Tube 4, CD5 vs CD22, R1*R2 gated. Set region R7 (see
below). Show regions statistic for this plot.
16) Page 2 middle centre: Tube 4, CD22 vs CD81, R1*R2 gated. Set region R8
(see below). Show regions statistic for this plot.
17) Show gate statistics for the page 2 middle left plot (CD5 vs CD22, R1*R2
gated).
18) Page 2 middle right: Tube 4, CD5 vs CD81, R1*R2*R7*R8 gated.
19) Page 2 lower left: Tube 5, CD5 vs CD79b, R1*R2 gated. Set region R7 (see
below). Show regions statistic for this plot.
20) Page 2 lower centre: Tube 5, CD5 vs CD43, R1*R2 gated. Set region R8 (see
below). Show regions statistic for this plot.
21) Show gate statistics for the page 2 lower left plot (CD5 vs CD79b, R1*R2
gated).
22) Page 2 middle right: Tube 5, CD79b vs CD43, R1*R2*R9*R10 gated.
23) For page 2 layout and approximate region placement, see Supplementary
Figure 2.
ADJUSTING REGIONS
1) For CELLQuest users: if automatic recalculation is on, switch it off now. If
automatic recalculation is off, recalculate now.
2) Zoom on the page 1 upper left hand plot (CD19 vs. SSC, ungated) to expand
the CD19+SSClo events (see Supplementary Figure 3).
3) Zoom in on page 1 upper middle plot (SSC vs FSC, R1-gated) to expand the
mononuclear cells (see Supplementary Figure 4).
4) Zoom on the page 1 upper right hand plot (CD19 vs. SSC, R2-gated) to
expand the CD19+ SSClo events (see Supplementary Figure 5).
5) Zoom on the page 1 middle right hand plot (CD19 vs. SSC, R1*R2*R3-gated)
to expand the CD19+ SSClo events (see Supplementary Figure 6).
6) Adjust R3 to include CD3+SSClo events (see Supplementary Figure 7)
7) Adjust R4 to include leucocytes and exclude erythrocytes and debris (see
Supplementary Figure 8).
8) Adjust gates R1 & R2:
a. 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 Supplementary Figure 9.
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 Supplementary Figure 10).
c. Repeat this process for R2 if necessary (see Supplementary Figure
11).
9) Recalculate now and repeat step (8) if necessary.
10) 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 Supplementary Figure 12.
11) 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).
12) 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 Supplementary Figure 2. Contaminating events may include Tcells, NK-cells, and Monocytes; the profiles for these potential contaminants
are shown in Supplementary Figure 13, Supplementary Figure 14, and
Supplementary 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.
More precise instruction for setting individual regions follow. Regions R5R10 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).
13) 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 Bprogenitors. 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.
14) 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 Bprogenitors. If no B-progenitors are present, the position of the upper edge of
R6 is not relevant.
15) 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 Supplementary
Figure 16.
16) 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 Supplementary Figure 17.
17) 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 Bprogenitors. 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.
18) 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.
19) 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 Supplementary Figure 18.
20) 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 Supplementary Figure 19.
21) 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 Supplementary Figure 20.
22) 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 Bprogenitors 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.
23) Adjust R10: If polyclonal B-cells are present, set the left edge of R10 to the
right of the CD5-CD43- mature B-cell population. If B-progenitors are present
(i.e. CD5-CD43+) set the lower edge of R10 to delineate CLL cells and Bprogenitors. If no B-progenitors are present, set the lower edge of R10 to
approximately the same level as the horizontal quadrant in the page 1 lower
middle plot (CD5 vs kappa, R1*R2 gated).
24) If there is a population of cells that are suspicious of having a CLL-phenotype
with respect to CD79b/CD43 expression but are not clearly separated from the
normal B-cells, adjust the gate in the CD79b vs CD43 plot from R1*R2 to
R9*R2*R1. This may improve the separation between the remaining CLL
cells and normal B-cells – if so, adjust R10 accordingly. Return the gate to
R1*R2. See Supplementary Figure 21.
25) Assess the lower right plot, which displays events defined as CLL cells. The
cells should appear as a single population with homogeneous antigen
expression. See Supplementary Figure 22.
26) Once all adjustments have been made, save the CELLQuest document using
the “Save As” command with a filename corresponding to the relevant sample.
This should be stored with the FCS files as a permanent record of the samplespecific gating strategy.
CALCULATING RESULTS
1) The minimum information that is required is as follows:
a. Tube 1 leucocyte events “T1Leuc”: G4 = R4
b. Tube 1 B-cell events “T1B”: G14 = R2*R1 from tube 1
c. Contaminating events “T1Con”: G15 = R3*R2*R1 from tube 1
d. Tube 3 B-cell events “T3B”: G14 = R2*R1 from tube 3
e. Tube 3 CLL-cells “T3CLL”: G11 = R5*R6*R2*R1 from tube 3
f. Tube 4 B-cell events “T4B”: G14 = R2*R1 from tube 4
g. Tube 4 CLL-cells “T4CLL”: G11 = R7*R8*R2*R1 from tube 4
h. Tube 5 B-cell events “T5B”: G14 = R2*R1 from tube 5
i. Tube 5 CLL-cells “T5CLL”: G11 = R9*R10*R2*R1 from tube 5
(N.B. the definitions above assume that the CELLQuest analysis protocol has
been used)
2) Input this data onto the automatic calculator in www.cll-mrd.org and this will
generate the result automatically. If this is not available, manual calculation is as
follows:
a. B-cells as a percentage of total leucocytes (“B%L”)
“B%L” = 100 * T1B / T1Leuc
b. Limit of detection due to total event count
= 100 * 50 / T1Leuc
c. Limit of detection due to contamination in B-cell gate
= 100 * T1Con / T1Leuc
d. Limit of detection for assay (“LOD”)
This is the greater of (b) or (c),
IF (100* 50 / T1Leuc) > (100 * T1Con / T1Leuc)
THEN LOD = (100 * 50 / T1Leuc)
ELSE LOD = (100 * T1Con / T1Leuc)
e. CLL cells as a percentage of leucocytes:
If two or more of the CLL regions contain less than 50 events, then CLL
cells are below the limit of detection for the assay. If two or more of the
CLL regions contain more than 50 events calculate the average CLL cells
as a percentage of leucocytes for files containing over 50 events in the
CLL regions. If the average is above the limit of detection, then this is
reported as the CLL cell percentage of leucocytes. Otherwise CLL cells
are below the limit of detection for the assay.
IF (T3CLL>50 AND T4CLL>50 AND T5CLL>50),
THEN CLL%Leuc = B%L*[(T3CLL/T3B) + (T4CLL/T4B) +
(T5CLL /T5B)]/3
ELSEIF (T3CLL>50 AND T4CLL>50),
THEN CLL%Leuc = B%L* [(T3CLL /T3B) + (T4CLL /T4B)]/2
ELSEIF (T3CLL>50 AND T5CLL>50),
THEN CLL%Leuc = B%L* [(T3CLL /T3B) + (T5CLL /T5B)]/2
ELSEIF (T4CLL>50 AND T5CLL>50),
THEN CLL%Leuc = B%L* [(T4CLL /T4B) + (T5CLL /T5B)]/2
ELSE CLL%Leuc=0.
IF CLL%Leuc > LOD,
THEN REPORT “CLL cells = CLL%Leuc % of leucocytes”
ELSE REPORT “CLL cells < LOD % of leucocytes”.
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1: Layout of page 1 of analysis template
Supplementary Figure 2: Layout of page 2 of analysis template
Supplementary Figure 3: Zoom in on B-cells in the upper left plot on page 1.
Supplementary Figure 4: Zoom in on lymphoid cells in upper middle plot on page 1
Supplementary Figure 5: Zoom in on B-cells in upper right plot on page 1
Supplementary Figure 6: Zoom in on B-cell area showing contamination in middle
right plot on page 1
Supplementary Figure 7: Adjust R3 to enclose CD3+SSClo events in middle left plot
on page 1
Supplementary Figure 8: Adjust R4 to enclose leucocyte events and exclude
erythrocytes/debris in lower left plot on page 1
Supplementary Figure 9: Adjust R2 to exclude erythrocytes/debris and cellular
aggregates in upper middle plot on page 1
Supplementary 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.
Supplementary 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.
Supplementary 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.
Supplementary 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.
Supplementary 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.
Supplementary 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.
Supplementary 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 Bcells as possible (b). Return the gate to R2*R1 (c).
Supplementary 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
Supplementary 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).
Supplementary 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).
Supplementary 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.
Supplementary Figure 21: If a discrete population is not apparent in the CD5 vs CD43
plot (lower middle on page 2), adjust the gate to show events in R9*R2*R1 – i.e.
events with weak CD79b and strong CD5 (a). This may provide a clear delineation
between CLL cells and polyclonal B-cells. Optimise R10 so that CLL cells are not
excluded whilst excluding as many normal B-cells as possible (b). Return the gate to
R2*R1 (c).
Supplementary Figure 22: 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 R9 and R10 accordingly.
SUPPLEMENTARY FIGURE 1
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