Payvand
Clinical Specialty Lab.
FLOWCYTOMETRY
Behzad Poopak, DCLS PhD
bpoopak@yahoo.com
What Is Flow Cytometry?
 Flow ~ cells in motion
 Cyto ~ cell
 Metry ~ measure
 Measuring properties of cells while in a fluid
stream
Cytometry vs. Flow Cytometry
Cytometry
 Localization of antigen
is possible
 Poor enumeration of
cell subtypes
 Limiting number of
simultaneous
measurements
Flow Cytometry.
 Cannot tell you where
antigen is.
 Can analyze many
cells in a short time
frame.
 Can look at numerous
parameters at once.
Applications of Flow Cytometry.
• Cell size.
• Cytoplasmic granularity.
• Cell surface antigens (Immunophenotyping).
• Apoptosis.
• Intracellular cytokine production.
• Intracellular signalling.
• Gene reporter (GFP).
• Cell cycle, DNA content, composition, synthesis.
• Bound and free calcium.
• Cell proliferation
• Cell sorting, single cell cloning
Flow cytometry & Hematopathology
1. Distinction between neoplastic and benign
conditions,
2. Diagnosis and characterization of lymphomas
and leukemias,
3. Assessment of other neoplastic and
preneoplastic disorders such as plasma cell
dyscrasias and MDS,
4. Detection of MRD in patients with acute
leukemia or chronic lymphoid malignancies.
5. In some groups of lymphoid neoplasms, FCM
study also provides prognostic information.
Principle of Flow Cytometry
Fluidics
• Cells in suspension
• Cells flow in single-file
• Intercepted by light source(s) (laser)
Optics
• Scatter light and emit fluorescence
• Signal collected, filtered and
• Converted to digital values
Electronics
• Storage on a computer
Data display and analysis
Basic Principles of Flow Cytometry
 Single cell or particle
suspension
 Fluorescent dyes or
Abs that can be
attached to an antigen
or protein of interest
 Flow cell, sheath fluid
and a focused laser
beam
The Flow Cell
Sheath
Cell
Sample Stream
The introduction of a
large volume into a
small volume in such a
way that it becomes
“focused” along an axis
is called Hydrodynamic
Focusing.
Sample
Sample
Sheath
Sheath
Laser Focal Point
Sheath
Sample
Core
Stream
Incoming Laser
Low Differential
High Differential
Sample Differential
10 psi
10 psi
10 psi
10.2 psi
10.4 psi
10.8 psi
Difference in pressure between sample and sheath
This will control sample volume flow rate
The greater the differential, the wider the sample core.
If differential is too large, cells will no longer line up single file
Results in wider CV’s and increase in multiple cells passing
through the laser at once. No more single cell analysis!
Basic Principles cont’d
 Light is either scattered or
absorbed when it strikes a
cell
 Light scatter is dependent
on the internal structure,
size and shape.
 Forward scatter = size of
the cell
 Side Scatter = complexity
of the cell
Forward Scatter
Laser Beam
FSC
Detector
Side Scatter
Laser Beam
FSC
Detector
Collection
Lens
SSC
Detector
Side scatter
Granulocytes
Monocytes
Lymphocytes
Forward scatter
Why Look at FSC v. SSC
 Since FSC ~ size and SSC ~ internal structure, a
correlated measurement between them can
allow for differentiation of cell types in a
heterogeneous cell population
Granulocytes
SSC
Lymphocytes
Monocytes
RBCs, Debris,
Dead Cells
FSC
Low
Medium High levels of forward scatter
Side scatter
----> increasing cell size
Forward scatter
Side scatter
Medium
Low
Forward scatter
Increasing levels of side scatter ---->
increasing cell granularity
High
BY “GATING” EACH OF THE AREAS IN
2 DIMENSIONS, YOU CAN ADAPT FLOW CYTOMETRY TO PERFORM DIFFERENTIAL COUNTS!
Side scatter
Granulocytes
Monocytes
Lymphocytes
Forward scatter
FLOW CYTOMETRY
- Cells are labeled with fluorescent antibodies
directed against cell surface molecules
- Using different color fluorochromes allows
counting of many markers simultaneously
and allows identification of several markers
on the same cell ( Multiparameter Flow)
- In the instrument, cells pass one-by-one past
a laser to excite the fluorochromes and
there are detectors for each type of
fluorochrome
- cells are labeled with fluoresence antibodies
Flouresent tag
Surface of a cell, e.g., a lymphocyte
(in solution)
Fluorescence
Photon emission as an electron returns from an excited state to
ground state
What Happens in a Flow
Cytometer (Simplified)
Fluorochrome
Basic Principles cont’d
 Fluorescent dyes absorb
light of a specific
wavelength and reemit
light of a different
wavelength
 Fluorescent signals are
detected by PMT and
amplified
 Optical filters are used to
steer light of specific
wavelengths to the photo
detector
Reflected
Dichroic
Filter
Passed
Short or Long Pass
Filter
Band Pass Filter
Adsorbed
Absorption
Filter
Electronics
 Electrical pulses are
digitized, the data is
stored (‘list mode data’),
analysed and displayed
through a computer
system.
 The end result is
quantitative information
about every cell analysed
 Large numbers of cells
can be processed quickly
Comprehensive antibody panels
 The rationale :
(1) The lineage of the cells of interest (e.g., myeloid, B-cell,
T-cell),
(2) Their maturity status,
(3) The clonality, where appropriate,
(4) The specific subtype of hematopoietic malignancy and
(5) The status of the normal elements present.
 Appropriate isotype controls are included in the panels. The
evaluation of the FCM data also relies on internal controls,
however (e.g., T-cells serve as internal control for B-cells and vice
versa)
Abs Panel, the European Group for the
Immunological Characterization of Leukemias
(EGIL) for the diagnosis and classification of
acute leukemia
Panel of antibodies recommended by the British
Committee for Standards in Haematology (BCSH) for the
diagnosis and classification of acute leukemia
Panel of antibodies recommended by the European
LeukemiaNet, ELN for the diagnosis and
classification of acute leukaemia
Panel of antibodies recommended by the US–
Canadian Consensus Group for the diagnosis
and classification of acute leukemia
Reactivity of mAbs vs. FAB-AML
Development of a FACS histogram
45%
Negative cells
Counts
Positive cells
Fluorescent intensity
Intensity scales
are logarithmic
Note: The operator can set the “gate” by visual inspection of
the histogram. The “gate” defines negative versus positive.
THE OPERATOR SETS “GATES” DEFINING
POSITIVE AND NEGATIVE FOR EACH MARKER.
CD19
CD19
CD19+
CD3-
CD19+
CD3 -
CD3 +
CD3+
CD3
CD19-
CD19-
CD3-
CD3+
CD3
Therefore, you can define each cell counted with regard to CD19 or CD3
positivity. Note that there are not normally cells in the circulation that
express both T and B cell surface markers.
Below are the FCM results on a peripheral
blood specimen studied) at a
teaching hospital:








CD2 48% moderate
CD3 45% moderate
CD4 21% moderate
CD7 47% moderate
CD8 20% moderate
CD13 3% moderate
CD33 1% moderate
CD34 1% weak
TdT 55% moderate
CD19 47% moderate
CD20 26% moderate
CD22 47% moderate
sIgM 48% moderate
Kappa 3% moderate
Lambda 2% moderate
CD10 36% moderate
CD45 100% strong
HLA-DR 55% moderate
Interpretation
 The results indicated a proliferation of immature cells
(TdT+). The case was interpreted as ALL with a mixed
(B-cell and T-cell) lineage.
 Because of the data-reporting format, it is unclear
whether the immature cells are of B- or T-cell lineage,
however. Although fluorescence intensities were
mentioned, data interpretation in this particular
laboratory was actually based on percent positive with
an arbitrary 20% cutoff.
 When proper visual data analysis was subsequently
applied to the raw data, it became apparent that the
blood sample contained a clearly identifiable
neoplastic population of precursor B-ALL, admixed
with a high number of normal T-cells.
Steps in Flowcytometry
1. Preanalytical (specimen handling and
processing, including antibody staining),
2. Analytical (running the sample through the
flow cytometer and acquiring data), and
3. Postanalytical (data analysis and
interpretation).
 Deficiencies such as suboptimal instrument performance, poor
reagent quality (antibodies and/or fluorochromes), or poor
specimen quality can all result in inadequate resolution of
positive and negative immunofluorescence.
Preanalytical Phase
 Little control over certain factors, eg. specimen collection
and transportation, which can adversely affect the sample prior to its
arrival.
 Poor specimen collection
 The time elapsed between specimen acquisition
and delivery to the laboratory, and the environmental
conditions during transport are critical factors
 As a rule, specimens cannot be held for more than
48 hours in the fresh state after collection. This time
window is much narrower for samples harboring a tumor with
a high turnover rate (e.g., Burkitt lymphoma).
 Exposure to extreme temperatures and the presence of blood
clots (or gross hemolysis) are conditions that can render a
blood or bone marrow specimen unacceptable for analysis.
Fresh specimens for FCM
 Liquid samples (peripheral blood, bone
marrow, body fluids) and
 Solid tissue (lymph nodes, tonsils/ adenoids,
spleen, bone marrow biopsies, and
extranodal infiltrates).
Specimen Type
 PB & BMA can be collected in either EDTA or
heparin.
 The volume required depends on the WBC count;
10 mL of blood is adequate in most instances.
 Store & transfer at RT (at RT in delay). Referred blood
,should be accompanied by a hemogram and a fresh blood smear
 Approximately 3 to 5 mL of BMA is usually
sufficient for a comprehensive FCM analysis,
 Degenerative changes in BMA tend to occur more
quickly than PB
Diagram of lymph node slicing and the
allocation of the slices to different studies
F, FCM analysis; H, histology; I, immunohistochemistry;
M, molecular studies.
Each slice is less than 2 mm thick.
 Preparing nucleated cell suspensions
 Cell yield and viability
 Sample staining
- Surface antigens, staining is performed on viable
unfixed cells.
 All staining is performed at 40C to minimize capping
and antigen shedding.
 Appropriate isotype controls are included.
 The usual number of cells recommended for
immunostaining is 106 cells (low cell yield, it is possible to
perform the staining with as few as 1 × 105 to 2 × 105 cells/tube)
- Intracellular antigens, the staining procedure is more
laborious than cell surface antigen staining and calls for cell
fixation and permeabilization
Case 0f ALL
Immunophenotype:
Immunophenotyping of Bone marrow aspirate by flow cytometry
shows predominant a B cell population (about 89% of the cells
analyzed) The majority of these B cells show expression of CD10,
CD19, HLA-DR. They were negative for CD2, CD5, CD7, CD13,
CD33, CD20 and Tdt. Review of BMA smear shows a predominant
lymphoblast population (65%). Dual Positive for CD10 / CD19.
Cytochemistry:
Myeloperoxidase: All leukemic blasts were MPX negative.
Interpretation / Diagnosis: Immunophenotyping results, together with
morphological findings and Cytochemistry of
BMA, are consistent with B lymphoblastic
leukemia (Early pre B-cell type).
AML-M3 , Classic or
Hypergranular type
Immunophenotype:
Immunophenotyping of BMA by flow cytometry shows
a predominant leukemic cell population (about 85%
of the cells analyzed, Gated on region 1) that is
positive for CD13, CD33 & CD117,CD45. The
majority of gated cells were negative for
CD2,CD3,CD4,CD5,CD7,CD8,CD10, CD11b, CD11c
,CD14,CD19, CD20,CD25,CD41, CD61, HLA-DR.
These cells have intermediate granularity (based on
side-scatter signal).
Cytochemistry:
All of the leukemic cells were intensely
myeloperoxidase Positive.
File: CD10.FCS Date: 09-11-2011 Time: 13:40:32
Immunophenotyping results, together
400 with morphological
Gate: R1
findings and Cytochemistry of BMA, are consistent with B
lymphoblastic leukemia (Early 320
pre B-cell type). RN1
200
File: CD19.FCS Date: 09-11-2011 Time: 13:42:24
Particles: 13036 Acq.-Time: 57 s
200
160
SSC -
250150
200
100
Gate: R1
RN1
160
R1
counts
120
150
SSC -
partec PAS
240
counts
250
50
100
50
Particles: 13134 Acq.-Time: 48 s
R1
0
0
50
80
80
0
40
100
150
FSC -
200
250
1
10
100
FL1 CD10
1000
0400
0
1000
0
50
100
150
200
250
1
10
100
1000
CD34.FCS
File: HLA.FCS Date:
Time: 13:54:48 File:
Particles:
13551Date:
Acq.-Time:
38 s Time: 13:56:48 Particles: 14047 Acq.-Time: 12 s
FSC 09-11-2011
FL109-11-2011
200
1000
Gate: R1
200
200
250
250
Gate: R1
Gate: R1
RN2
RN2
FL2 CD19
SSC counts
counts
counts
SSC -
R1
120
150
80
10 100
50
0
1
1
10
100
1000
FL2 CD19
0
50
100
150
200
250
0 Gate
FSCCount
Region
Ungated
Count/ml
200
1 Gate: R110674 10 10674
R1
<None>
- 100
FL2
RN1
R1
93
47
-
0
RN1
160
120
R1
80
10
40
80
100
100
120240
150
160100
RN1
160
200
200
FL2 -
160
counts
320
80
partec PAS
50
40
40
1 00
0
10
100
1000
0
50
100 - 150
200
250
FL1
1
10
100
1000
FSC1FL1
HLA
100
%Gated
Mean-x
CV-x%
Mean-y
1000
Gate:
R1
1000
R1
81.88 Gate:
72.44
16.39 1
39.74
0.44 Q1: 3.33%
2.96
22.79 Q2: 22.52%
-
1
10
100
FL1 CD34
1000
CV-y%
Gate: R1 100
10
31.43
0.00%
FL1Q1:CD10
-
1000
1000
Q2: 0.14%
Patient Report
Format
Patient Report-2 Format
Hematogone vs.Leukemic Lymphoblast
Diagnosing Multiple Myeloma
Three Diagnostic Criteria Required
for a Positive Diagnosis of Multiple Myeloma
1
• Monoclonal plasma cells present in the bone
marrow ≥10%
• Presence of a documented plasmacytoma
2
• Presence of M component in serum and/or urine*
• One or more of the following (CRAB criteria):
3
Calcium elevation (serum calcium >11.5 mg/dL)
Renal insufficiency (serum creatinine >2 mg/dL)
Anemia (hemoglobin <10 g/dL or 2 g/dL <normal)
Bone disease (lytic lesions or osteopenia)
*Monoclonal M spike on electrophoresis IgG >3.5 g/dL, IgA >2 g/dL, light chain >1 g/dL in 24-hour urine
sample.
Durie et al for the International Myeloma Working Group. Leukemia. 2006:1-7.
53
Diagnostic Evaluation
of Multiple Myeloma
Test
Finding(s) With Myeloma
CBC with differential counts
↓ Hgb, ↓ WBC, ↓ platelets
Electrolytes
↑ Creat, ↑ Ca+, ↑ Uric acid, ↓ Alb
Serum electrophoresis with quantitative
immunoglobulins
↑ M protein in serum, may have ↓ levels of normal
antibodies
Immunofixation
Identifies light/heavy chain types M protein
β2-microglobulin
↑ Levels (measure of tumor burden)
C-reactive protein
↑ Levels (marker for myeloma growth factor)
24-hour urine protein electrophoresis
↑ Monoclonal protein (Bence Jones)
Bone marrow biopsy
≥10% plasma cells
Skeletal imaging
Osteolytic lesions, osteoporosis
Serum free light chain
↑ Free light chains
MRI
Evaluation of involvement of disease
Alb = albumin; CBC = complete blood count; Creat = creatinine; Hgb = hemoglobin;
MRI = magnetic resonance imaging; WBC = white blood cell
Abella. Oncology News International. 2007;16:27; Barlogie et al. In: Williams Hematology. 7th ed. 2006:1501; Durie et al. Hematol J. 2003;4:379; MMRF.
54
Multiple Myeloma: Disease Overview. 2006. www.multiplemyeloma.org; Rajkumar et al. Blood. 2005;106(3):812.
Peripheral blood - rouleaux
B.Poopak
Malignant Plasma Cells in
Marrow
Myeloma: A Cancer of Plasma Cells
in the Bone Marrow
Patients with multiple myeloma show a
"spike" in special regions of the
serum protein electrophoresis
Serum Protein Electrophoresis
Normal
Monoclonal Protein
in Myeloma
Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004
Figure 8. Quantitative immunoglobulins were within normal limits
Maslak, P. ASH Image Bank 2001;2001:100211
Copyright ©2001 American Society of Hematology. Copyright restrictions may apply.
Figure 8. Immunofixation electrophoresis showing a monoclonal IgA lambda light chain
restricted band
Lazarchick, J. ASH Image Bank 2001;2001:100185
Copyright ©2001 American Society of Hematology. Copyright restrictions may apply.
Immunofixation to Determine
Type of Monoclonal Protein
IgG kappa M protein
Lambda Light Chains
Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004
CASE STUDY – MULTIPLE
MYELOMA
 Serum free light chains
 Free kappa
 Free lambda
 Ratio
16.2
3754.1
0.0
3.3 – 19.4
5.7 – 26.3
0.3 – 1.7
mg/L
mg/L
 Interpretation
 Free lambda light chain monoclonal gammopathy
 Radiology
 Diffuse osteolytic lesions in thoracic and lumbar regions with
several compression fracturres
Normal serum
Immunofixation
Report:
Polyclonal
Pattern
serum
Immunofixation
Report:
Mono-Clonal
IgA – Kappa
Pattern
serum
Immunofixation
Report:
Mono-Clonal
IgM – Kappa
Pattern
Thank you, any question?