ICCS e-Newsletter CSI
Jonathan Fromm, MD, PhD
University of Washington
Seattle, WA USA
History and physical examination
• The patient is a 24-year-old woman presenting
with shortness of breath (of uncertain
duration). She reports episodes of chills but
denies fevers, night sweats, and weight loss.
• Physical exam is unremarkable.
• Past medical history is remarkable for an
episode of thrombocytopenia in the setting of
sepsis.
Radiographic studies
• Chest CT demonstrates an anterior
mediastinal mass measuring 9.2 x 6.8 x 5.7
cm.
• Additional abdomen and pelvis CT was
negative for lymphadenopathy or
organomegaly.
CBC data
CBC value
Normal range
WBC: 5.8x103/microliter
4.3-10
RBC: 3.89x 106/microliter
3.80- 5.00
hemoglobin: 11.9 g/dL
11.5-15.5
hematocrit: 34%
36-45
MCV: 88 fL
81-98
MCH: 30.6 pg
27.3-33.6
MCHC: 34.7 g/dL
32.2-36.5
platelet count: 279x103/microliter
150-400
Peripheral blood differential
Cell type
% from WBC
Absolute number
(103/microliter)
Reference range
(Absolute #)
Neutrophils
69
4.06
1.80-7.00
Lymphocytes
17
1.00
1.00-4.80
Monocytes
10
0.59
0.00-0.80
Eosinophils
3
0.18
0.00-0.50
Basophils
1
0.06
0.00-0.20
Immature
granulocytes
0
0
0.00-0.05
Work-up and evaluation
• The anterior mediastinal mass is biopsied.
• Flow cytometric immunophenotyping was
performed on this sample. An additional tube
is added after reviewing the initial data.
Flow cytometry tubes
Data acquisition was performed on a modified LSRII flow cytometer.
Data analysis was performed using Woodlist software (in-house software
written by Brent L Wood)
Standard immunophenotyping tubes for lymphoma (B and T cell tubes). After
Evaluation of the T cells, a tube for classical Hodgkin lymphoma (CHL) was
run.
Tube
Pac B
FITC
PE
ECD
PECy5.5
PECy7
A594
APC
APCA700
APCH7
B cell
CD20*
Κ
λ
-
CD5
CD19
CD38
CD10
-
CD45
T cell
CD8^
CD2
CD5
CD34
CD56#
CD3
CD4
CD7
CD30
CD45
Hodgkin
CD95
CD64
CD30
CD5
CD40
CD20
-
CD15
CD71
CD45
*CD20 run using V450; ^CD8 run using BV; #CD56 run using PeCy5
Evaluation of B cell tube
T cells are green; kappa and lambda restricted B cells are blue and red, respectively. The
first panel shows all cells, the second panel shows all lymphocytes, and the last three
panels show all B cells.
The lymphocytes and B cells represent 97% and 20.2% of the WBC, respectively.
B cells demonstrate polyclonal expression of surface light chains.
Evaluation of the T cell tube
CD4+ T cells are in red, CD8+ T cells are green, and NK are blue. The first panel shows all
cells and the remaining panels show all lymphocytes.
T cells comprise 77.0% of the leukocytes and 79.0% of the lymphocytes. The T cells
represent demonstrate a normal pattern of antigen expression with an increased
CD4:CD8 ratio of 10.7. The CD4+ T cells demonstrate an increase in a subset with
increased CD45 and CD7.
Presence of subset of CD4+ T cells with
increased CD45 and CD7
Expanded subset
with increased CD45
and CD7 on the CD4+
T cells.
The CD4+ T cells (left) have an expanded subset with increased CD45 and CD7. For
comparison, the CD4+ T cells from a reactive lymph node (right) do not have this
expanded subset.
Given this finding, a tube that can immunophenotype CHL was run.
Gating strategy for CHL tube
Putative Hodgkin and Reed-Sternberg (HRS) cells events must fall in all four gates and form
a distinct population. The cells must have increased side scatter (1st panel), be CD30+ and
have increased autofluorescence in the FITC channel compared to the immunoblasts (see
below) and small lymphocytes (2nd panel), express no or low CD20 (3rd panel), and
express CD40 at or greater intensity than a reactive B cell (4th panel). In practice, HRS
cells should also express CD95 (although a gate requiring expression of CD95 is typically
not drawn). Immunoblasts and B cells are identified with the oblique and vertical arrows,
respectively. For our patient, this strategy identifies a distinct HRS population.
CHL data
HRS cells are colored in red, all other events are blue. All events are shown in the top panel,
only HRS events are shown in the bottom panels. The HRS cells have expression of
CD30 (low to intermediate), CD40 (bright), CD95 (intermediate to bright), CD15 (bright),
CD45 (variable), CD5 (variable; data not shown) without expression of CD20 or CD64
(position of negative defined by isotype matched control experiments). HRS represent 0.08%
of the leukocytes.
Evidence of T cell rosetting supports
the diagnosis of CHL
T cell rosetting is a well characterized phenomena observed in Hodgkin lymphoma. T cells
ring or rosette around HRS cells (to which they are bound). A composite
immunophenotype comprised of the HRS and T-cells can be observed in many cases. Here
rosetting is observed where most of the CD45 expression and all of the CD5 expression
comes from the T-cell. The first panel shows all events, while the second panel shows only
HRS cells. Note the diagonal relationship between CD45 and CD5 which is characteristic of
CHL. Unrosetted HRS cells are noted in the ellipse.
Diagnosis? Classical Hodgkin
lymphoma
CHL
• CHL is a unique type of B cell lymphoma where
the neoplastic HRS cells usually make up less than
1% of the leukocytes.
• The neoplastic cells are usually embedded in a
reactive infiltrate including lymphocytes,
eosinophils, plasma cells, and histiocytes.
• Diagnosis historically confirmed by paraffin
section immunophenotyping: the HRS cells are
CD30+, CD15+, Pax-5+, CD3-, CD20-, CD45-.
CHL
• The binding of T cells to HRS is characteristic of
Hodgkin lymphoma, forming HRS-T cell rosetting.
• This interaction is mediated by CD54 and CD58
on the HRS cells binding to LFA-1 and CD2 on the
T cells, resulting in the composite
immunophenotype observed by flow cytometry.
• The degree of rosetting varies between cases. In
our case, many of the events have brighter CD45
(and CD5) than the T cells, suggesting more than
1 T cell bound to each HRS cell.
Caveats regarding the analysis of CHL
cases
• HRS cells should demonstrate CD30, CD40,
and CD95, have increased forward and side
light scatter compared to small lymphocytes,
should lack intermediate to bright expression
of CD20, and should define a discrete cluster
in multi-dimensional space.
• The presence of CD15 supports a diagnosis
when present but is not required.
Caveats regarding the analysis of CHL
cases
• Other neoplasms commonly in the differential
diagnosis of CHL can usually be excluded:
– Diffuse large B cell lymphoma-usually have
expression of intermediate to bright CD20, do not
have an increase in side scatter to the extent that
HRS cells do, do not express CD15, usually express
CD30 at a lower level than an HRS population (if
CD30 is expressed), and would not show evidence
of HRS–T cell rosetting.
Caveats regarding the analysis of CHL
cases
– Nodular lymphocyte predominant Hodgkin
lymphoma is a type of Hodgkin lymphoma where
the neoplastic L&H cells express CD20, CD40, and
CD45, without CD15 or CD30 in tissue section.
While this neoplastic population cannot be
reliably detected by the flow cytometry at
present, if identified, the expression of CD20
without CD30 would strongly argue against a
population being a putative HRS population.
Caveats regarding the analysis of CHL
cases
– Anaplastic large cell lymphoma (a CD30-positive
lymphoma of T cell origin) can often have a similar
immunophenotype to CHL but CD40 is not expressed
or is expressed at a low level (lower than the reactive
small B cells).
• Note that other lymphomas (chronic lymphocytic
leukemia small lymphocytic lymphoma [CLL/SLL]
and peripheral T cell lymphoma) rarely can have
HRS cells detected by flow cytometry (and
immunohistochemistry). These cases, however,
show characteristic abnormal B or T cell
populations, strongly suggesting those diagnoses.
T cells in CHL
• CD4+ population with increased expression of
CD45 and CD7 seen in 82% of CHL, less than
5% of reactive cases.
• Can be used to:
– Screen for CHL, to prompt evaluation of CHL by
flow cytometry.
– Supports a diagnosis of CHL.
Summary
• Evaluation of T cells can suggest a diagnosis of
CHL.
• The HRS cells of CHL can be directly detected
by flow cytometry.
• Rosetting of T cells by HRS cells can be directly
detected by flow cytometry.
• The flow cytometry assay can be used to
distinguish CHL from similar entities that are
frequently in the differential diagnosis of CHL.
References
• J.R. Fromm, S.J. Kussick, B.L. Wood, Am. J. Clin.
Pathol. 126 (2006) 764–780.
• J.R. Fromm, A. Thomas, B.L. Wood, Am. J. Clin.
Pathol. 131 (2009) 322–332.
• J.R. Fromm, A. Thomas, B.L. Wood, Cytometry
B Clin. Cytom. 78 (2010) 387–388.
• J.R. Fromm, B.L. Wood, Cytometry B Clin.
Cytom. 78B (2010) 395.
References
• J. Fromm, D. Wu, United States & Canadian
Academy of Pathology 101st Annual Meeting,
Vancouver, BC, Canada, 2012.
• R. Schmitz, J. Stanelle, M.L. Hansmann, R.
Kuppers, Annu. Rev. Pathol. 4 (2009) 151–174.
• H. Stein, G. Delsol, S.A. Pileri, L.M. Weiss, S.
Poppema, E.S. Jaffe, in: S.H. Swerdlow, E.
Campos, N.L. Harris, E.S. Jaffe, S.A. Pileri, H. Stein,
J. Thiele, J.W. Vardiman (Eds.), WHO Classification
of Tumours of Haematopoietic and Lymphoid
Tissues, IARC Press, Lyon, 2008, pp. 326–329.