ICCS e-newsletter CSI
Summer 2011
Jeannine T. Holden, MD
Emory University School of Medicine
Atlanta, GA
Case History
The patient is a 35 year old male with a history
of B lymphoblastic leukemia/lymphoma
diagnosed several years prior to the present
study and treated with chemotherapy. Both
original diagnosis and treatment were performed
at another institution. Patient is reportedly doing
well clinically, and the present study is routine
monitoring. His peripheral blood counts are all
within normal limits. The patient does report a
recent two day history of malaise and upper
respiratory symptoms that are attributed to viral
infection.
Questions…
• Is there any evidence of residual/recurrent
B lymphoblastic leukemia/lymphoma?
• Is normal hematopoietic activity intact?
• Is there any evidence of other diseases?
• Are there any other problems with this
case?
Flow cytometric
immunophenotyping
• Bone marrow aspirate
• Acquired on a FACSCanto cytometer using
Diva software, FCS2.0 formatted listmode
data
• Fourteen four-color tubes
• Tubes #3 through #14 gated at acquisition
(enriching for mononuclear cells)
• Files are labeled ICCS001 through ICCS014
Antibody panel
(FITC/PE/PerCP/APC)
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ICCS001: isotype controls
ICCS002: CD14/CD13/CD45/CD34
ICCS003: HLA-DR/CD25/CD45/CD33
ICCS004: CD3/CD4/CD45/CD8
ICCS005: CD2/CD7/CD45/CD5
ICCS006: CD10/CD19/CD45/CD34
ICCS007: CD36/CD117/CD45/CD34
ICCS008: CD15/CD11b/CD45/CD34
ICCS009: CD103/CD22/CD45/CD11c
ICCS010: CD16/CD56/CD45/CD38
ICCS011: FMC-7/CD23/CD45/CD19
ICCS012: KAPPA/CD20/CD45/CD19
ICCS013: LAMBDA/CD20/CD45/CD19
ICCS014: KAPPA/LAMBDA/CD20/CD5
Antibody panel
(FITC/PE/PerCP/APC)
Ignore high
background staining
with this reagent in
• this
ICCS001:
case! isotype controls
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ICCS002: CD14/CD13/CD45/CD34
ICCS003: HLA-DR/CD25/CD45/CD33
ICCS004: CD3/CD4/CD45/CD8
ICCS005: CD2/CD7/CD45/CD5
ICCS006: CD10/CD19/CD45/CD34
ICCS007: CD36/CD117/CD45/CD34
ICCS008: CD15/CD11B/CD45/CD34
ICCS009: CD103/CD22/CD45/CD11c
ICCS010: CD16/CD56/CD45/CD38
ICCS011: FMC-7/CD23/CD45/CD19
ICCS012: KAPPA/CD20/CD45/CD19
ICCS013: LAMBDA/CD20/CD45/CD19
ICCS014: KAPPA/LAMBDA/CD20/CD5
Antibody panel
(FITC/PE/PerCP/APC)
•
•
•
•
•
•
•
•
•
•
•
•
•
•
ICCS001: isotype controls
ICCS002: CD14/CD13/CD45/CD34
ICCS003: HLA-DR/CD25/CD45/CD33
ICCS004: CD3/CD4/CD45/CD8
ICCS005: CD2/CD7/CD45/CD5
ICCS006: CD10/CD19/CD45/CD34
ICCS007: CD36/CD117/CD45/CD34
ICCS008: CD15/CD11B/CD45/CD34
ICCS009: CD103/CD22/CD45/CD11c
ICCS010: CD16/CD56/CD45/CD38
ICCS011: FMC-7/CD23/CD45/CD19
ICCS012: KAPPA/CD20/CD45/CD19
ICCS013: LAMBDA/CD20/CD45/CD19
ICCS014: KAPPA/LAMBDA/CD20/CD5
History of B ALL
means that this tube
is probably the most
informative
So, look at the analysis pdf, scrolling to Tube
ICCS006, and look for candidate malignant
populations…
• Cells co-expressing CD19, CD34, and CD10 are readily
identifiable
• We know that normal B-cell progenitors (“hematogones”) are
likely present
• Can we identify any populations that are phenotypically
aberrant (particularly as compared to the expected normal Bcell progenitors)?
One of the dotplots for this tube
looks odd…
• B-cells and B-cell progenitors are
present, but there is also an
unexpected population.
• Can you see the unexpected
population?
• Don’t worry if you can’t, as it’s
difficult to see if you aren’t already
familiar with the normal patterns as
generated by another laboratory.
One of the dotplots for this tube
looks odd…
• So, let’s make it easier…
– The cells circled in blue are immature
B-cell progenitors. They co-express
CD34 and CD10.
– The cells circled in green are more
mature B-cell progenitors. The coexpress CD10 but not CD34.
– The cells circled in orange are mature
B-cells. They express neither CD10
nor CD34, and do express surface
immunoglobulin light chains.
– The cells circled in pink are the
aberrant population. If you backgate
you’ll find that these cells co-express
CD10 and CD34. Are these cells
recurrent/residual leukemic blasts?
Not convinced?
• Well, let’s compare it to a
normal hematogone
pattern…
– The dotplot on the bottom
shows the expected normal
hematogone pattern with
the CD19/CD45
combination.
– The dotplot on the top
definitely has another
population in there.
Not convinced?
• Well, let’s make it even
easier…
– The dotplot on the bottom
shows the expected normal
hematogone pattern with
the CD19/CD45
combination.
– Here I’ve added the colored
gates, just to make it clear.
Notice that the pink circle is
essentially empty in the
bottom dotplot.
But now it’s really confusing, because these
dotplots are both from the same analysis in the
same patient…
• Don’t believe me?
– You can go look at the
full analysis pdf and
see for yourself.
– And while you’re at it,
look at the other
CD19/CD45
combinations in tubes
12 and 13. Neither of
them shows the
aberrant population.
So, what’s going on?
• Residual/recurrent leukemia
present, but only shows up in
one tube?
– Unlikely degree of sampling
error.
– Unlikely immunophenotype,
as even if these cells do
express CD19, CD10, and
CD34, they also appear to
express very high density
CD45, and that’s not
characteristic of B ALL.
And the answer is…
• “Carry over”
– Tube 5 in this analysis is
directed at identifying
normal (and aberrant) Tcells.
– Note that the CD7/CD45
combination identifies cells
in both the orange and pink
circles. These are normal Tcells. And of course they
also express CD2 and CD7.
“Carry over”
• Already stained cells from one tube are
introduced to another tube of stained cells
• Results in overlapping dotplots
• Typically identified in sequential tubes
• May occur as a result of…
–
–
–
–
Pipetting error (unusual)
Splashing of one sample into an another
Inefficient washing/rinsing of sipper
Machine tubing: cells from first tube remain in
tubing, acquired with next tube
How do I know for sure that this
is “carry over”?
• Because I deliberately contaminated
tube 6 with cells from tube 5, that’s
why.
• But here’s another example to show
you what it looks like…
Another example of “carry over”
• Cervical lymph
node fine
needle aspirate
from a 58 year
old female
• Normal blood
cell counts
• Cytologically
benign
• Why does this patient’s sample harbor
cells that co-express CD19, CD10, and
CD34?
• Is this an unusual early presentation of
B ALL?
• And why can’t
I find a distinct
CD34+
population in
this tube from
the same
analysis in the
same patient?
Look familiar?
Conclusions
• Exploit panel redundancy to investigate
unexpected findings.
• Assume that errors will occur.
• Consider all information about a case,
including the clinical history.
• Don’t have the clinical history? Get it.