Transcript Flow Cytometry Interpretation

Principles and
Application of Flow
Cytometry
Rodney Stuart, MD
August 5,2005
Objectives

Basic principles of flow cytometry
Separation of cells on appearance
 Separation based on light emitting dyes
 Eliminating bleed over between colors
 Studying the cells of interest
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Examples of difficult cases
Flow Cytometry
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First labeled with fluorescent dyes.
Forced through a nozzle in a single-cell stream passing through a laser beam
The laser is focused to a known wavelength
Excitation of a specific fluorochrome
Photo-multiplier tubes detect the scattering of light and emission from the
fluorescent dye
Forward Scatter

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Cells pass through a small (50-300 µm) orifice
Detection ranges from 0.5-40 µm
the amount of light scattered in the forward direction (along the same
axis that the laser light is traveling) = size of cell
Side Scatter
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the amount of light scattered to the side (perpendicular to the axis
that the laser light is traveling) is detected in the side or 90o scatter
channel
Equates to shape and homogeneity of cells (AKAgranularity/complexity)
Forward and Side Scatter
Forward Scatter (Size)
Neutrophils
Monocytes
Lymphocytes
Side Scatter (granularity)
Fluorescence Channels

The specificity of detection is controlled by the wavelength selectivity
of optical filters and mirrors
Fluorochromes
FITC
ECFP
PE-Texas
Red
PE
DsRED
EGFP
Propidium
Iodem
PE-Cy5
APC
PerCP
EYFP
FL1
FL2
FL3
~525nm
~575 nm
~620 nm
FL4
~675 nm
One Parameter Histogram

A one-parameter histogram is a graph of cell count on the y-axis and
the measurement parameter on x-axis.
E
v
e
n
t
s
FITC
Two Parameter Histograms

A graph representing two measurement parameters, on the x- and yaxes, and cell count height on a density gradient. This is similar to a
topographical map.
-/+
+/+
-/- +/-
Compensation

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The inherent overlap of emission spectra from antibody fluorescent labels makes
compensation necessary.
Use a negative control to set your High Voltages (HV) per detector. As a rule of
thumb set the High Voltages so that your negatives are in the 1st decade of the
parameters being collected. Lower high voltage settings will also result in lower
compensation settings.
PE-Texas
Red
FITC
PE
ECFP
DsRED
EGFP
Propidium
Iodem
PE-Cy5
APC
PerCP
EYFP
FL1
FL2
FL3
~525nm
~575 nm
~620 nm
FL4
~675 nm
FL1
FL2
FL3
~525nm
~575 nm
~620 nm
FL4
~675 nm
Compensation
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The amount of spectral overlap is corrected by subtracting a
percentage from the total of FL1 fluorochrome pulse
generated by the FL1 detector. The FL1 fluorochrome
“spillover” detected by the FL2 detector can be viewed as
interference. By subtracting a percentage of the total FL1
fluorochrome pulse from the total pulse generated by the FL2
detector (eg. PE + FITC interference) you can obtain a FL2
fluorochrome only pulse.
Running samples that are individually stained with the
antibody-fluorochrome components of your multi-color
samples aids in appropriate compensation.
Compensation
FL1 = FL1 - %FL2 compensation slider
FITC
PE
Compensation
-/+
+/+
-/- +/-
-/+
+/+
-/- +/-
Gating
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Allow you to view cells of interest
lets you decide which data to view and which data to ignore or discard.
Gating can subsequently be changed when you analyze your data without any
loss of information.
Forward Scatter (Size)
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Neutrophils
Monocytes
Lymphocytes
Side Scatter (granularity)
Case Scenarios
Non-Hodgkin B cell
Lymphoma/Leukemia
Case 1
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31 year-old African American HIV+
male who presents with a rapidly
enlarging neck mass
Follicular Lymphoma
(FL)
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Most common NHL in the Western World (45% of adult
lymphomas)
Middle age distribution affecting males and female equally
Morphology: partial or often complete nodal architectural
effacement by numerous closely packed follicular nodules that
most frequently homogenous with numerous small cleaved
follicular center cells/centrocytes and larger cells with open
nuclear chromatin, several nucleoli, and modest amounts of
cytoplasm, referred to as centroblasts
Bone marrow involvement in 85% of patients (paratrabecular)
Immunophenotyping
Strong CD19, CD20
 Monotypic immunoglobulin or no
surface Ig expression
 CD10 positive
 Negative for CD5 and CD43
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Diffuse Large B-cell
(DLBCL)
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20% of NHL
Median age of 60 years with a male predominance
However age range is wide (5% of childhood lymphoma)
4 variants: centroblastic, immunoblastic, T-cell/histocyte-rich,
and anaplastic
Morphology: diffuse large lymphoid cells (4-5X the size of small
lymphocytes)with vesicular chromatin. Nuclear features can
range from multilobated, cleaved, or even the appearance of
Reed-Sternberg cells
Immunophenotyping
CD20 and CD19 seen in many but not
all
 More common than FL to be sIg
negative
 Some contain cytoplasmic Ig
 Some mark like FL (CD5-, CD10+,bcl6+, bcl-2+) but can lack CD10, bcl-6,
and bcl-2
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Burkitt Lymphoma (BL)
Rapidly dividing transformed cell lymphoma
 Adolescents or young adults
 Follicular formation in a minority of cases
 Morphology: Diffuse infiltrate of intermediatesized lymphoid cells.
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Round or oval nuclei with course chromatin,
several nucleoli, and basophilic or amphophilic
cytoplasm
“Starry sky” appearance due to presence of
numerous tingible-body macrophages
Immunophenotyping
CD19, CD20+
 CD10+
 BCL6+
 Express monotypic light chains
 Cytoplasmic Ig may be present
 CD5
Immunophenotype
Comparison
Lymphoma
CD19
CD20
CD10
CD5
sIg
FL
+
+
+
-
+
DLBCL
+
+
+/-
-
+/-
BL
+
+
+
-
+
Cytogenetics
Follicular Lymphoma: t(14;18) (Ig-H
and bcl-2 gene) (90% of FL)
 Diffuse Large B cell Lymphoma:
variable, many with translocations
involving chromosome 3q27 (BCL6
locus), 10-20% contain t(14;18)
 Burkitt Lymphoma: c-myc associated
t(8;14), t(8;22), t(2;8)
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Case 2
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58 year old Caucasian male initially
presented with fatigability, weight loss,
and anorexia. He was found to have
CLL and is currently on chemotherapy.
Small Lymphocytic Lymphoma/Chronic
Lymphocytic Leukemia
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CLL: Most common Leukemia in adults in the
Western world
SLL: 4% of NHL
Older adults
Morphology: population of small lymphocytes
containing round to slightly irregular nuclei
with condensed chromatin and scant
cytoplasm, mixed with larger prolymphocytes
Smudge cell on peripheral smear
Immunophenotype
CD19 and CD20+
 CD23 and CD5+
 Low level expression of sIg and
monotypic light chain
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Rituximab
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anti-CD20 monoclonal antibody
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Rituxan may induce antibody-dependent cellmediated cytotoxicity
Rituxan may also induce complement-dependent
cytotoxicity
Causes decrease in CD20 available for
detection ab to bind
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Either through direct binding site competition
Or transient down-regulation of CD20
Case 3
14 year-old Caucasian male with previously diagnosed lymphoid
neoplasm for which he was treated with chemotherapy. He has been
clinically asymptomatic with close follow-up for the last year. Current
bone marrow biopsy obtained is shown here:
Case 3
Acute Lymphoblastic Leukemia
(ALL)
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Consists of immature, pre-cursor B (Pre-B)
(85%) or T (Pre-T) lymphocytes
Pre-B manifests in childhood (many subtypes
by European Group for the Immunological
Characterization of Leukemias)
Pre-T manifests in adolescents
Morphologically indistinguishable
Immunophenotyping needed for typing
ALL Immunophenotyping
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Terminal deoxynucleotidyl transferase
(TdT) + >95% of time
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Pre-B
Positive for: CD19, CD22, CD79a, CD24, CD34,
CD45, HLADR, CD38, CD9, and CD10
 Negative for: surface Ig expression
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Pre-T
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Positive for: CD1, CD2, CD5, CD7
Case 3
Hematogones
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Bone marrow B-cell precursors (detected on flow in
80% of bone marrow aspirates)
Cytologic features:
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Size: 10-20 microns
May appear as mature lymphocytes
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Small cell with round or oval nucleus
May be indistinguishable from neoplastic lymphoblasts
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Sometimes exhibits 1 or more indentations or shallow clefts
with condensed chromatin
Rim of deeply basophilic cytoplasm
Hematogones
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Most numerous in children (up to 21%) of bone
marrow in infants and declines to <5% after 16
Have been observed in reactive lymph nodes and in
peripheral blood
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Usually TdT negative
Other conditions can increase the number in
adults
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Regenerating marrow following chemotherapy
Autoimmune diseases
Congenital cytopenias
Lymphomas (usually stage 3)
Neuroblastomas
AIDS
Hematogone
Immunophenotype
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Always positive for:
Progenitor cell markers CD34 and CD38
 CD19
 Dim CD22
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Hematogone
Immunophenotype
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Maturation markers:
Stage 1(red):
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Stage 2(blue):
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Positive TdT and Bright CD10
Moderate CD10, dim CD20, and variable sIG
Stage 3(Yellow):
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Dim to moderate CD10, moderate to bright CD20, and variable sIG
Hematogones vs.
ALL
Red=Lymphoblasts
Yellow=Hematogones
Concurrent CD34 and CD20
Abberant over-expression of markers (eg.
CD10)
References
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http://biology.berkeley.edu/crl/flow_cytometry_basic.html
Gervasi F, Lo Verso R, Giambanco C, Cardinale G, Tomaselli C, Pagnucco G. Flow cytometric
immunophenotyping analysis of patterns of antigen expression in non-Hodgkin's B cell
lymphoma in samples obtained from different anatomic sites.
Ann N Y Acad Sci. 2004 Dec;1028:457-62.
Kroft SH, Asplund SL, McKenna RW, Karandikar NJ. Haematogones in the peripheral blood
of adults: a four-colour flow cytometry study of 102 patients.
Br J Haematol. 2004 Jul;126(2):209-12.
S Sava an1, M Büyükavc 2, S Buck1 and Y Ravindranath1 . Leukaemia/lymphoma cell
microparticles in childhood mature B cell neoplasms. Journal of Clinical Pathology
2004;57:651-653.
Maloum K, Sutton L, Baudet S, Laurent C, Bonnemye P, Magnac C, Merle-Beral H. Novel
flow-cytometric analysis based on BCD5+ subpopulations for the evaluation of minimal
residual disease in chronic lymphocytic leukaemia.
Br J Haematol. 2002 Dec;119(4):970-5.
Jilani I, O'Brien S, Manshuri T, Thomas DA, Thomazy VA, Imam M, Naeem S, Verstovsek S,
Kantarjian H, Giles F, Keating M, Albitar M.Transient down-modulation of CD20 by rituximab
in patients with chronic lymphocytic leukemia.
Blood. 2003 Nov 15;102(10):3514-20. Epub 2003 Jul 31