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ROLE OF FLOW CYTOMETRY
IN LEUKEMIAS
PRESENTER: DR. HAJRA K. MEHDI
MODERATOR: DR. CSBR PRASAD
SRI DEVARAJ URS MEDICAL COLLEGE, KOLAR
INTRODUCTION
• Flow –cells in motion
• Cyto – cells
• Metry - measure
• Measuring multiple physical and chemical properties of
cells while in fluid stream.
• Quantitative & qualitative analysis.
• Expensive & sophisticated technology used
increasingly from research to clinical laboratories.
1986 1995
2004
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
PRINCIPLE
• An optical-to-electronic coupling system that records
how a cell scatters incident laser light and emits
fluorescence.
• This process is performed at a rate of thousands of cells
(5000-10000) per second.
• It can detect the size of cell as small as 0.1um.
5 MAIN COMPONENTS
1) Flow cell
2) Optical system
3) Detector & Analogue to Digital conversion(ADC)
4) Amplification system
5) Computer software
INSTRUMENT OVERVIEW
FLOW CELL
• Transports cells in a fluid stream to
laser beam for interrogation.
• Monodisperse suspension
• Cells flow in single file.
SHEATH
FLUID
SAMPLE
CORE
STREAM
LASER
LIGHT SCATTERING
• Due to laser light reflecting & refracting off the cells
without alteration of wavelength.
• Forward scatter
• Side scatter
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
Y
Laser
Side Scatter (SSC)
90° deflection
~ Cell structures
Forward Scatter
(FSC)
< 10° deflection
~ Cell size
Fluorescence Intensity
Antigen Density
FORWARD SCATTER
• Light scattered in forward
direction.
• 1-10 degrees
• Proportional to size of cell
• Refractive index & absorptive
properties.
• Larger cells scatter more light
than smaller cells.
FORWARD SCATTER DETECTOR
The magnitude of the
voltage pulse
recorded for each cell
is proportional to the
cell size.
SIDE SCATTER
• Light reflected from internal
structures of the cell.
• Correlates with granularity
of the cell.
• The signals collected by the
side-scatter detector located
90 degrees from the laser’s
path.
FLUORESCENSE
• Fluorochrome – absorb light over a range of wavelength
& emit light at longer wavelength (stoke’s shift).
• Absorption of light electron to be raised to a
higher energy level.
• Excited electron quickly decays to its ground
state emitting excess energy as photon of light.
• Transition energy termed as fluorescence.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
COMMON FLUOROCHROMES
• Phycoerythrin (PE)
• Fluorescein Isothiocyanate (FITC)
• Allophycocyanin (APC)
• Peridinin-Chlorophyll Protein (PerCP)
• Tandem Dyes
• PE-Texas Red (ECD)
• PE-Cy5 (PC5)
• PE-Cy5.5 (PC5.5)
• PE-Cy7 (PC7)
• APC-Cy7
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
OPTICAL FILTERS
• Direct specified wavelengths of light to designated
optical detectors.
• Side scatter & fluorescent light collected together & then
separated in order to detect them independently.
LASER
A typical arrangement for measuring four components is
shown:
TYPES OF OPTICAL FILTERS
LONG (700nm)
550 Long Pass
(650LP)
650 Short Pass
(600SP)SHORT (500nm)
Pass Through
Filters
600/100 Band Pass
(600/100)
Transmitted <650 nm >550 nm 550 - 650 nm
(600±50)
Wavelengths
Blocked >650 nm <550 nm <550 nm & >650 nm
Short Pass Long Pass Band Pass
LONG (700nm)
550 Long Pass
(650LP)
650 Short Pass
(600SP)SHORT (500nm)
Dichroic
Filters
Transmitted <650 nm >550 nm
Diflected 90° >650 nm <550 nm
CELLULAR PARAMETERS MEASURED BY FLOW
• No reagents or probes required (Structural)
• Cell size (Forward Light Scatter)
• Cytoplasmic granularity (90 degree Light Scatter)
CELLULAR PARAMETERS MEASURED BY FLOW
• Reagents are required.
• Structural
• DNA content
• DNA ratios
• RNA content
• Functional
• Surface and intracellular receptors.
• DNA synthesis
• DNA degradation (apoptosis)
• Cytoplasmic Ca++
• Gene expression
DATA DISPLAY
• Once a data file has been saved, the data can be displayed in
a number of different plots
Histograms 2-D plots 3-D plots
-Scattergram - tomogram plot
- Density plot
- Contour plot
DOT PLOT
• Bivariate display/
"scattergram" / bitmap
• Plots one dot or point on the
display for each cell which
passes through the
instrument.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
Amount of Blue Markers
AmountofYellowMarkers
REMEMBER THIS
12
3 4
DENSITY PLOT
• Density plots simulate a
three dimensional display of
events .
• “Third" parameter being the
number of events.
• Usually coloured
• Shades of grey, indicate the
relative numbers of events.
GATING
• It is done to isolate cell subpopulations of interest.
• Eliminates results from unwanted particles/ cells.
• Eliminates the need to sort cells physically to study their
characteristics.
• Done electronically or manually.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
FLOW CYTOMETRY PROTOCOL
SPECIMEN HANDLING & SAMPLE PREPARATION
Specimen -single cell suspension
-peripheral blood, bone marrow,
effusions, CSF, urine, semen
Blood & BM - EDTA/heparin
- RBC lysis – ammonium chloride
METHODOLOGY
• Pipette 100 ul of specimen into a tube
• Add appropriate monoclonal antibody combination labelled
with fluorescent dye (5-20 microl)
• Incubate at room temperature for 15 min
• Add 1 ml of lysing solution
• Centrifuge for 5 min at 2000 rpm & discard supernatant
• Add 2ml of PBS (Phosphated buffer saline)
• Centrifuge for 5 min at 2000 rpm & discard supernatant
• Repeat washing with PBS second time
• Re-suspend the cells in 0.2 to 0.5 ml of sheath fluid (Isotoin)
• Read on flow cytometer
• Collect ,store & analyze the data
IMMUNOPHENOTYPING
• A process used to identify cells, based on the types of
antigens or markers on the surface of the cell.
• It is one of the application of flow cytometry
• The technique is called "immunophenotyping" for 2
reasons:
1) It is dependent on the activity of antibodies, which are
immunological substances
2) It is used chiefly to identify lymphoid and hematopoietic
cells, which are part of the immune system.
Because of being a fast, objective, and quantitative method,
flow cytometry has now become the preferred method for :
(1) lineage assignment.
(2) maturational characterization of malignant cells.
(3) detection of clonality.
(4) heterogeneity and aberrant features of the malignant
cell populations.
(5) quantitation of hematopoietic cells.
(6) To detect minimal residual disease.
USES
Hematological malignancies (leukemias & lymphomas)
o The diagnosis & classification
o Assessment of biological parameters associated with
prognosis
o Detection of antigen used as therapeutic targets
o Detection of residual neoplastic cells following therapy
Plasma cell neoplasms
Myelodysplastic syndrome & Myeloproliferative disorders
Paroxysmal Nocturnal Hemoglobinuria
IMMUNOPHENOTYPING IN LEUKEMIAS
Antigen expression in normal cells is tightly regulated
process resulting in characteristic pattern of antigen
acquisition & loss with maturation that is cell lineage
specific.
1) Gain of antigens not normally expressed by cell type or
lineage - Aberrant expression.
2) Abnormally increased or decreased levels of
expression.
3) Asynchronous antigen expression.
4) Abnormal homogenous expression of one or more
antigens.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
NORMAL PATTERN OF ANTIGEN EXPRESSION
• All hematopoietic cells arise from hematopoietic stem
cells.
• These stem cells can be identified due to expression of:
Bright CD34, CD133
Intermediate CD45
Dim to absent CD38
Variable CD90
Dim CD123, CD117, HLA-DR, CD13, CD33
• Maturation towards lineage commited progenitors is
accompanied by:
Slight decrease in CD34 & CD45
Loss of CD13 & CD90
Increase in CD38 & HLA-DR
CD MARKERS
Lineage independent
antigen
Lineage specific/ Lineage associated
CD34-Stem cell
marker
CD 19 CD 3 CD 13
HLA-DR CD 22 CD 7 CD 33
CD10(CALLA) CytoCD22 CD 5 CD 117(C-
Kit)
CD45Leucocyte
common Ag
Cyto 79 a Cyto CD3 CD 14
(Monocytic)
CD4,CD8
B Cell T Cell Myeloid
LINEAGE ASSIGNMENT IN ACUTE LEUKEMIA
AML ALL-B cell ALL-T cell
Definitive Cytoplasmic MPO Cytoplasmic CD79a
Cytoplasmic CD22
CytoplasmicCD3
Surface CD3
Strongly
associated
CD117 CD 19
CD10
CD7
Moderately
associated
CD13
CD33
TdT CD5
CD2
FCM AND COMMON MARKERS
All lymphoid cells: CD45+ (LCA)
Myeloid cells: Anti-MPO, CD13, CD33, CD14, CD117
Megakaryocytic marker: CD41, CD42, CD61
B-cells: cCD22, CD22, CD19, CD20, FMC7, CD23,
CD79a, CD79b, CD10, SmIg, IgM, k/l
T-cells: cCD3, CD3, CD2, CD5, CD7, CD4 or CD8,
TCR-α/β, TCR-γ/δ
NK cells: CD16, CD56, CD57
Plasma cells: CD38, CD138, kappa and lambda light chains
Blasts: CD34, TdT
Others: HLA-DR, cyclin D1, CD55, CD59,
glycophorin. A
PANELS FOR ACUTE LEUKEMIA
A) Primary panel :
B-cells - CD10, CD19
T-cells - CD3, CD7, CD4, CD8
Myeloid - CD13, CD33, CD117
Non-lineage - HLADR, CD34
Positive Control: CD45 (LCA)
Negative Control: Isotype IgG1
B) Secondary panel:
B-lineage specific - cytoCD22 / cytoCD79a
T-lineage specific - cytoCD3
Myeloid lineage specific - Anti-MPO
Other Markers – Tdt, CD99, CD41, CD61, SmIg & CD56
Principle Example Implementation
At least one reagent for
population identification
CD45 for general cell type,
Lineage associated antigen for
specific cell lineage,e.g. CD19 for
B cell
Multiple antigens of same
lineage & maturational stage to
identify inappropriate expression
levels
Use of
CD2,CD3.CD4,CD5,CD7,CD8
simultaneously to evaluate mature
T-cells
Multiple antigens of same
lineage but different
maturational stage to identify
normal maturation &
dyssynchronous expression
Use of CD13 & CD16
simultaneously to demonstrate
neutrophilic maturation
PANEL DESIGN STRATEGIES
Principle Example Implementation
Separation of different cell
lineage
Use of CD11b & CD15
simultaneously to separate
monocytic & neutrophilic
maturation
Demonstration of clonality Use of kappa & lambda in
combination with a B cell lineage
reagent,e.g.CD19
Identify frankly aberrent antigen
expression
Use of T or NK cell associated
antigens such as CD7 / CD56 in
combination with CD34
PANEL DESIGN STRATEGIES
FLOW CHART - APPROACH
TO ACUTE LEUKEMIA
Provisional diagnosis:Acute leukemia
ACUTE LEUKEMIA
AUER RODS + VE AUER RODS -VE
MPO AND/OR NSE +VE MPO/ NSE -VE
FCM
AML
FCM with first line panel
CD10, CD19, HLADR CD3 CD13, CD33, CD117 HLADR, CD34
Lineage established
Blasts, no auer rods, MPO/NSE negative
However, if lineage not established
FCM with second line panel
cCD22 cCD3, CD4, CD8 anti MPO, CD41, CD61
Lineage established
• FCM is a must
- All cases of ALL,
- AML M0/M7, and
- Undifferentiated, Bi-phenotypic leukemia
- MRD detection
For diagnostic laboratories, at least three, preferably four
color FCM is required
• As bone marrow cells express CD45, when CD45 is
combined with side scatter, which separates lineages based
on cytoplasmic complexity, the bone marrow sample is
readily separated into its cellular constituents.
• Infiltration of marrow by immature cells or blasts is more
easily recognized on a CD45 versus side-scatter plot than on
traditional forward side-scatter gating.
LEUCOCYTE ANALYSIS
Neutrophils
Monocytes
Basophils
Lymphocytes
RBC’s Myeloblasts
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ACUTE MYELOID LEUKEMIA WITH
DIFFERENTIATION
Blasts (red) showing abnormal expression of the myeloid
associated antigens
• CD33 (dim)
• CD13 (bright)
• CD15 (dim partial)
• immature antigens CD34 and CD117.
• CD11b- Negative. The blasts do not express more mature
neutrophilic antigens.
There are background maturing granulocytes (green) that are
normal and not part of the leukemic population
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ACUTE PROMYELOCYTIC LEUKEMIA
• The neoplastic cell population - promyelocytes (red):
CD33 (bright) and CD13 (intermediate).
• High side scatter indicating abundant cytoplasmic
granularity.
• Lacks expression of CD34 and HLA-DR and retains
expression of CD117 as is seen on a subset of normal
promyelocytes.
• However, in contrast to normal promyelocytes, the
abnormal cells lack significant expression of CD15, a
characteristic and common abnormality in this disorder.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ACUTE MYELOMONOCYTIC LEUKEMIA.
• Blasts (red) with a larger population of cells showing
monocytic differentiation (violet).
• The monocytic differentiation is reflected in the acquisition of
early monocyte antigens CD64 (bright), and CD36
(intermediate to bright) along with other more mature
myelomonocytic antigens CD15 (intermediate) and CD11b
(low to intermediate)
• Without significant acquisition of the mature monocyte
antigen CD14 (absent) or marked gain in the expression of
CD45 as is seen in mature monocytes.
• This finding suggests differentiation to the promonocyte
stage, a population usually included in morphologic blast
counts. In addition, a lesser degree of neutrophilic
differentiation (green) is present.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
CD5+
CD5-
CD23+ Cyclin D1+
CD10+ CD10-
SLL MCL
FL,
ALL, BL MZLHCL
CD25+ CD25-
B-cells (CD19 +)
CLL/SLL
The neoplastic cells show B cell antigens
• CD19 (intermediate) and
• CD20 (low) with
• surface lambda light chain expression (low),
• coexpression of CD5 and CD23 (intermediate).
The combination of CD5 coexpression, low-level light chain
restriction, low-level CD20 and CD23 without FMC7 is
diagnostic for CLL. The important differential is with
mantle cell lymphoma.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
PLASMA CELL NEOPLASM
The neoplastic cell population (green)with
• bright expression of CD38, but shows
• abnormal expression of CD45 (low),
• CD19 (absent) and
• cytoplasmic lambda light chain restriction with
• aberrant expression of CD56 (bright).
This immunophenotype is characteristic of that seen in a
variety of plasma cell neoplasms including multiple
myeloma, plasmacytoma and monoclonal gammopathy of
uncertain significance (MGUS). Definitive classification
requires clinical and laboratory correlation.
Case 4 : 22 year old man with fever for past 3 months.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
SCORING SYSTEM (EGIL GROUP) FOR ACUTE
BIPHENOTYPIC LEUKEMIAS (BENE ET AL)
Score B-lymphoid T-lymphoid Myeloid
2 CD79a,
cytoplasm
CD22,
cytoplasm IgM
CD3,
TCR-α/β,
TCR-γ/δ
Anti-MPO
1 CD19,
CD20,
CD10
CD2,
CD5,
CD8,
CD10
CD117,
CD13,
CD33,
CD65
0.5 TdT,
CD24
TdT,
CD7,
CD1a
CD14,
CD15,
CD64
Biphenotypic acute leukemia (EGIL) is defined when scores for the
myeloid and one of the lymphoid lineages are > 2 points.
Case 5 : 5 year old boy with fever – 1 month
MPO NEGATIVE ACUTE LEUKEMIA
Flow cytometer
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
Provisional diagnosis:Acute leukemia
Case 6 : 45 male, bone marrow
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
CD13+, CD33+, CD117+, CD34+
CASE 7
• The patient is a 52 year old man who presented with
cellulitis on his elbow and was noted to have a high white
count. A bone marrow was done and sent to a local
laboratory for phenotyping. When the laboratory called the
result back to the oncologist who had sent the sample, they
were told that there must be some mistake, and a second
opinion was sought.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
CASE 8
• A 1 year old child was brought to the emergency room by
his parents who noticed that he was extremely irritable. A
white blood count was found to be 90,000, and he was
admitted to the hospital. A peripheral blood was sent for
phenotyping, and a bone marrow examination was
performed.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
CASE 9
• The patient is a 64 year old female on methotrexate therapy
for rheumatoid arthritis. She developed anemia and a
decreasing platelet count which persisted following
discontinuation of this therapy. A bone marrow examination
was performed.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
• Diagnosis: Acute myeloid leukemia (FAB M1)
• Antigen Profile: Strongly positive for
HLADR,CD13,CD34,CD38; dimly positive for CD33,
CD71; partly positive for CD7,CD11b
CASE 10
• The patient is a 57 year old female who presented with
fatigue. She had had rheumatoid arthritis for about 5 years,
and also reported a recent upper respiratory tract infection
for which she had taken antibiotics. No blood work had been
done at that time, but she was now found to have a
hemoglobin of 10g, a white blood count of 8000 with a
relative lymphocytosis, and a platelet count of 95,000.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
CASE 11
• The patient is a 60 year old man who had pancytopenia for
several months before being diagnosed with
myelodysplastic syndrome about one year ago. About 2
months prior to this, he presented with decreasing counts
and was found to have transformed into acute leukemia. He
was treated with induction chemotherapy, and this marrow
was performed 6 weeks following this therapy.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
CASE 12
• The patient is a 72 year old female with fatigue and
anorexia for two weeks. She complained of a skin rash
which she developed about a day prior to coming to the
doctor, and on examination was found to have cellulitis on
her left arm. A bone marrow examination was performed.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
• The patient is a 49 year old man who presented to the
emergency room with confusion and disorientation. A CT
scan showed evidence of an early intracerebral bleed, and
laboratory studies showed thrombocytopenia and evidence of
disseminated intravascular coagulation. Following
heparinization and stabilization of the patient's CNS status, a
bone marrow examination was performed.
Case 13
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
• Note that the light scatter pattern is very broad, and in
particular a forward versus RALS display shows what
appears to be very large granular cells. There are no
consistent phenotypic differences between M3 and
M3v, though as shown here in the isotype control there
is often considerable FITC autofluorescence in classic
M3.
• Diagnosis: Acute promyelocytic leukemia (FAB M3)
Antigen Profile: Positive for CD71, CD33, CD9,
CD13, myeloperoxidase; partly positive for CD34,
HLADR.
CASE 14
• A 34 year old man in previously good health came to the
emergency room complaining of shortness of breath. His
hemoglobin was 9 g/dl and his white blood count was
165,000/ul.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
• In many cases of M4 AML the FALS vs RALS pattern can be
very characteristic. As shown here it frequently gives this
"forked" picture.
• This particular case is an example of M4e. Eosinophils may be
difficult to detect in flow samples. When they are numerous they
have the high RALS signal of granulocytes, but a higher
intensity of CD45 expression similar to monocytes. It may be
that some of the non-colored cells "above" the monocytes
represent eosinophils.
• Diagnosis: Acute myelomonocytic leukemia with eosinophilia
(FAB-M4eo)
Antigen Profile: Positive for CD33, HLADR and CD13;
heterogeneous positivity for CD34, CD11b, CD15, CD14; dimly
positive for CD2
CASE 15
• The patient is a 64 year old man with a known history of
anemia for the past year. About a month before this visit
he had had a viral illness, and had never fully recovered
his strength from that episode. On this visit, both his
hemoglobin and his platelet count were much lower than
they had been. A bone marrow was performed and sent for
phenotyping.
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS
ROLE OF FLOW CYTOMETRY IN LEUKEMIAS

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ROLE OF FLOW CYTOMETRY IN LEUKEMIAS

  • 1. ROLE OF FLOW CYTOMETRY IN LEUKEMIAS PRESENTER: DR. HAJRA K. MEHDI MODERATOR: DR. CSBR PRASAD SRI DEVARAJ URS MEDICAL COLLEGE, KOLAR
  • 2. INTRODUCTION • Flow –cells in motion • Cyto – cells • Metry - measure • Measuring multiple physical and chemical properties of cells while in fluid stream. • Quantitative & qualitative analysis. • Expensive & sophisticated technology used increasingly from research to clinical laboratories.
  • 5. PRINCIPLE • An optical-to-electronic coupling system that records how a cell scatters incident laser light and emits fluorescence. • This process is performed at a rate of thousands of cells (5000-10000) per second. • It can detect the size of cell as small as 0.1um.
  • 6. 5 MAIN COMPONENTS 1) Flow cell 2) Optical system 3) Detector & Analogue to Digital conversion(ADC) 4) Amplification system 5) Computer software
  • 8. FLOW CELL • Transports cells in a fluid stream to laser beam for interrogation. • Monodisperse suspension • Cells flow in single file. SHEATH FLUID SAMPLE CORE STREAM LASER
  • 9. LIGHT SCATTERING • Due to laser light reflecting & refracting off the cells without alteration of wavelength. • Forward scatter • Side scatter
  • 11. Y Laser Side Scatter (SSC) 90° deflection ~ Cell structures Forward Scatter (FSC) < 10° deflection ~ Cell size Fluorescence Intensity Antigen Density
  • 12. FORWARD SCATTER • Light scattered in forward direction. • 1-10 degrees • Proportional to size of cell • Refractive index & absorptive properties. • Larger cells scatter more light than smaller cells.
  • 13. FORWARD SCATTER DETECTOR The magnitude of the voltage pulse recorded for each cell is proportional to the cell size.
  • 14. SIDE SCATTER • Light reflected from internal structures of the cell. • Correlates with granularity of the cell. • The signals collected by the side-scatter detector located 90 degrees from the laser’s path.
  • 15. FLUORESCENSE • Fluorochrome – absorb light over a range of wavelength & emit light at longer wavelength (stoke’s shift). • Absorption of light electron to be raised to a higher energy level. • Excited electron quickly decays to its ground state emitting excess energy as photon of light. • Transition energy termed as fluorescence.
  • 17. COMMON FLUOROCHROMES • Phycoerythrin (PE) • Fluorescein Isothiocyanate (FITC) • Allophycocyanin (APC) • Peridinin-Chlorophyll Protein (PerCP) • Tandem Dyes • PE-Texas Red (ECD) • PE-Cy5 (PC5) • PE-Cy5.5 (PC5.5) • PE-Cy7 (PC7) • APC-Cy7
  • 19. OPTICAL FILTERS • Direct specified wavelengths of light to designated optical detectors. • Side scatter & fluorescent light collected together & then separated in order to detect them independently. LASER
  • 20. A typical arrangement for measuring four components is shown:
  • 21. TYPES OF OPTICAL FILTERS LONG (700nm) 550 Long Pass (650LP) 650 Short Pass (600SP)SHORT (500nm) Pass Through Filters 600/100 Band Pass (600/100) Transmitted <650 nm >550 nm 550 - 650 nm (600±50) Wavelengths Blocked >650 nm <550 nm <550 nm & >650 nm Short Pass Long Pass Band Pass LONG (700nm) 550 Long Pass (650LP) 650 Short Pass (600SP)SHORT (500nm) Dichroic Filters Transmitted <650 nm >550 nm Diflected 90° >650 nm <550 nm
  • 22. CELLULAR PARAMETERS MEASURED BY FLOW • No reagents or probes required (Structural) • Cell size (Forward Light Scatter) • Cytoplasmic granularity (90 degree Light Scatter)
  • 23. CELLULAR PARAMETERS MEASURED BY FLOW • Reagents are required. • Structural • DNA content • DNA ratios • RNA content • Functional • Surface and intracellular receptors. • DNA synthesis • DNA degradation (apoptosis) • Cytoplasmic Ca++ • Gene expression
  • 24. DATA DISPLAY • Once a data file has been saved, the data can be displayed in a number of different plots Histograms 2-D plots 3-D plots -Scattergram - tomogram plot - Density plot - Contour plot
  • 25. DOT PLOT • Bivariate display/ "scattergram" / bitmap • Plots one dot or point on the display for each cell which passes through the instrument.
  • 27. Amount of Blue Markers AmountofYellowMarkers REMEMBER THIS 12 3 4
  • 28. DENSITY PLOT • Density plots simulate a three dimensional display of events . • “Third" parameter being the number of events. • Usually coloured • Shades of grey, indicate the relative numbers of events.
  • 29. GATING • It is done to isolate cell subpopulations of interest. • Eliminates results from unwanted particles/ cells. • Eliminates the need to sort cells physically to study their characteristics. • Done electronically or manually.
  • 31. FLOW CYTOMETRY PROTOCOL SPECIMEN HANDLING & SAMPLE PREPARATION Specimen -single cell suspension -peripheral blood, bone marrow, effusions, CSF, urine, semen Blood & BM - EDTA/heparin - RBC lysis – ammonium chloride
  • 32. METHODOLOGY • Pipette 100 ul of specimen into a tube • Add appropriate monoclonal antibody combination labelled with fluorescent dye (5-20 microl) • Incubate at room temperature for 15 min • Add 1 ml of lysing solution • Centrifuge for 5 min at 2000 rpm & discard supernatant • Add 2ml of PBS (Phosphated buffer saline) • Centrifuge for 5 min at 2000 rpm & discard supernatant • Repeat washing with PBS second time • Re-suspend the cells in 0.2 to 0.5 ml of sheath fluid (Isotoin) • Read on flow cytometer • Collect ,store & analyze the data
  • 33. IMMUNOPHENOTYPING • A process used to identify cells, based on the types of antigens or markers on the surface of the cell. • It is one of the application of flow cytometry • The technique is called "immunophenotyping" for 2 reasons: 1) It is dependent on the activity of antibodies, which are immunological substances 2) It is used chiefly to identify lymphoid and hematopoietic cells, which are part of the immune system.
  • 34. Because of being a fast, objective, and quantitative method, flow cytometry has now become the preferred method for : (1) lineage assignment. (2) maturational characterization of malignant cells. (3) detection of clonality. (4) heterogeneity and aberrant features of the malignant cell populations. (5) quantitation of hematopoietic cells. (6) To detect minimal residual disease.
  • 35. USES Hematological malignancies (leukemias & lymphomas) o The diagnosis & classification o Assessment of biological parameters associated with prognosis o Detection of antigen used as therapeutic targets o Detection of residual neoplastic cells following therapy Plasma cell neoplasms Myelodysplastic syndrome & Myeloproliferative disorders Paroxysmal Nocturnal Hemoglobinuria
  • 36. IMMUNOPHENOTYPING IN LEUKEMIAS Antigen expression in normal cells is tightly regulated process resulting in characteristic pattern of antigen acquisition & loss with maturation that is cell lineage specific. 1) Gain of antigens not normally expressed by cell type or lineage - Aberrant expression. 2) Abnormally increased or decreased levels of expression. 3) Asynchronous antigen expression. 4) Abnormal homogenous expression of one or more antigens.
  • 38. NORMAL PATTERN OF ANTIGEN EXPRESSION • All hematopoietic cells arise from hematopoietic stem cells. • These stem cells can be identified due to expression of: Bright CD34, CD133 Intermediate CD45 Dim to absent CD38 Variable CD90 Dim CD123, CD117, HLA-DR, CD13, CD33 • Maturation towards lineage commited progenitors is accompanied by: Slight decrease in CD34 & CD45 Loss of CD13 & CD90 Increase in CD38 & HLA-DR
  • 39. CD MARKERS Lineage independent antigen Lineage specific/ Lineage associated CD34-Stem cell marker CD 19 CD 3 CD 13 HLA-DR CD 22 CD 7 CD 33 CD10(CALLA) CytoCD22 CD 5 CD 117(C- Kit) CD45Leucocyte common Ag Cyto 79 a Cyto CD3 CD 14 (Monocytic) CD4,CD8 B Cell T Cell Myeloid
  • 40. LINEAGE ASSIGNMENT IN ACUTE LEUKEMIA AML ALL-B cell ALL-T cell Definitive Cytoplasmic MPO Cytoplasmic CD79a Cytoplasmic CD22 CytoplasmicCD3 Surface CD3 Strongly associated CD117 CD 19 CD10 CD7 Moderately associated CD13 CD33 TdT CD5 CD2
  • 41. FCM AND COMMON MARKERS All lymphoid cells: CD45+ (LCA) Myeloid cells: Anti-MPO, CD13, CD33, CD14, CD117 Megakaryocytic marker: CD41, CD42, CD61 B-cells: cCD22, CD22, CD19, CD20, FMC7, CD23, CD79a, CD79b, CD10, SmIg, IgM, k/l T-cells: cCD3, CD3, CD2, CD5, CD7, CD4 or CD8, TCR-α/β, TCR-γ/δ NK cells: CD16, CD56, CD57 Plasma cells: CD38, CD138, kappa and lambda light chains Blasts: CD34, TdT Others: HLA-DR, cyclin D1, CD55, CD59, glycophorin. A
  • 42. PANELS FOR ACUTE LEUKEMIA A) Primary panel : B-cells - CD10, CD19 T-cells - CD3, CD7, CD4, CD8 Myeloid - CD13, CD33, CD117 Non-lineage - HLADR, CD34 Positive Control: CD45 (LCA) Negative Control: Isotype IgG1 B) Secondary panel: B-lineage specific - cytoCD22 / cytoCD79a T-lineage specific - cytoCD3 Myeloid lineage specific - Anti-MPO Other Markers – Tdt, CD99, CD41, CD61, SmIg & CD56
  • 43. Principle Example Implementation At least one reagent for population identification CD45 for general cell type, Lineage associated antigen for specific cell lineage,e.g. CD19 for B cell Multiple antigens of same lineage & maturational stage to identify inappropriate expression levels Use of CD2,CD3.CD4,CD5,CD7,CD8 simultaneously to evaluate mature T-cells Multiple antigens of same lineage but different maturational stage to identify normal maturation & dyssynchronous expression Use of CD13 & CD16 simultaneously to demonstrate neutrophilic maturation PANEL DESIGN STRATEGIES
  • 44. Principle Example Implementation Separation of different cell lineage Use of CD11b & CD15 simultaneously to separate monocytic & neutrophilic maturation Demonstration of clonality Use of kappa & lambda in combination with a B cell lineage reagent,e.g.CD19 Identify frankly aberrent antigen expression Use of T or NK cell associated antigens such as CD7 / CD56 in combination with CD34 PANEL DESIGN STRATEGIES
  • 45. FLOW CHART - APPROACH TO ACUTE LEUKEMIA
  • 47. ACUTE LEUKEMIA AUER RODS + VE AUER RODS -VE MPO AND/OR NSE +VE MPO/ NSE -VE FCM AML
  • 48. FCM with first line panel CD10, CD19, HLADR CD3 CD13, CD33, CD117 HLADR, CD34 Lineage established Blasts, no auer rods, MPO/NSE negative
  • 49. However, if lineage not established FCM with second line panel cCD22 cCD3, CD4, CD8 anti MPO, CD41, CD61 Lineage established
  • 50. • FCM is a must - All cases of ALL, - AML M0/M7, and - Undifferentiated, Bi-phenotypic leukemia - MRD detection For diagnostic laboratories, at least three, preferably four color FCM is required
  • 51. • As bone marrow cells express CD45, when CD45 is combined with side scatter, which separates lineages based on cytoplasmic complexity, the bone marrow sample is readily separated into its cellular constituents. • Infiltration of marrow by immature cells or blasts is more easily recognized on a CD45 versus side-scatter plot than on traditional forward side-scatter gating.
  • 55. ACUTE MYELOID LEUKEMIA WITH DIFFERENTIATION Blasts (red) showing abnormal expression of the myeloid associated antigens • CD33 (dim) • CD13 (bright) • CD15 (dim partial) • immature antigens CD34 and CD117. • CD11b- Negative. The blasts do not express more mature neutrophilic antigens. There are background maturing granulocytes (green) that are normal and not part of the leukemic population
  • 57. ACUTE PROMYELOCYTIC LEUKEMIA • The neoplastic cell population - promyelocytes (red): CD33 (bright) and CD13 (intermediate). • High side scatter indicating abundant cytoplasmic granularity. • Lacks expression of CD34 and HLA-DR and retains expression of CD117 as is seen on a subset of normal promyelocytes. • However, in contrast to normal promyelocytes, the abnormal cells lack significant expression of CD15, a characteristic and common abnormality in this disorder.
  • 59. ACUTE MYELOMONOCYTIC LEUKEMIA. • Blasts (red) with a larger population of cells showing monocytic differentiation (violet). • The monocytic differentiation is reflected in the acquisition of early monocyte antigens CD64 (bright), and CD36 (intermediate to bright) along with other more mature myelomonocytic antigens CD15 (intermediate) and CD11b (low to intermediate) • Without significant acquisition of the mature monocyte antigen CD14 (absent) or marked gain in the expression of CD45 as is seen in mature monocytes. • This finding suggests differentiation to the promonocyte stage, a population usually included in morphologic blast counts. In addition, a lesser degree of neutrophilic differentiation (green) is present.
  • 61. CD5+ CD5- CD23+ Cyclin D1+ CD10+ CD10- SLL MCL FL, ALL, BL MZLHCL CD25+ CD25- B-cells (CD19 +)
  • 62. CLL/SLL The neoplastic cells show B cell antigens • CD19 (intermediate) and • CD20 (low) with • surface lambda light chain expression (low), • coexpression of CD5 and CD23 (intermediate). The combination of CD5 coexpression, low-level light chain restriction, low-level CD20 and CD23 without FMC7 is diagnostic for CLL. The important differential is with mantle cell lymphoma.
  • 64. PLASMA CELL NEOPLASM The neoplastic cell population (green)with • bright expression of CD38, but shows • abnormal expression of CD45 (low), • CD19 (absent) and • cytoplasmic lambda light chain restriction with • aberrant expression of CD56 (bright). This immunophenotype is characteristic of that seen in a variety of plasma cell neoplasms including multiple myeloma, plasmacytoma and monoclonal gammopathy of uncertain significance (MGUS). Definitive classification requires clinical and laboratory correlation.
  • 65. Case 4 : 22 year old man with fever for past 3 months.
  • 70. SCORING SYSTEM (EGIL GROUP) FOR ACUTE BIPHENOTYPIC LEUKEMIAS (BENE ET AL) Score B-lymphoid T-lymphoid Myeloid 2 CD79a, cytoplasm CD22, cytoplasm IgM CD3, TCR-α/β, TCR-γ/δ Anti-MPO 1 CD19, CD20, CD10 CD2, CD5, CD8, CD10 CD117, CD13, CD33, CD65 0.5 TdT, CD24 TdT, CD7, CD1a CD14, CD15, CD64 Biphenotypic acute leukemia (EGIL) is defined when scores for the myeloid and one of the lymphoid lineages are > 2 points.
  • 71. Case 5 : 5 year old boy with fever – 1 month
  • 72. MPO NEGATIVE ACUTE LEUKEMIA
  • 76. Provisional diagnosis:Acute leukemia Case 6 : 45 male, bone marrow
  • 80. CASE 7 • The patient is a 52 year old man who presented with cellulitis on his elbow and was noted to have a high white count. A bone marrow was done and sent to a local laboratory for phenotyping. When the laboratory called the result back to the oncologist who had sent the sample, they were told that there must be some mistake, and a second opinion was sought.
  • 87. CASE 8 • A 1 year old child was brought to the emergency room by his parents who noticed that he was extremely irritable. A white blood count was found to be 90,000, and he was admitted to the hospital. A peripheral blood was sent for phenotyping, and a bone marrow examination was performed.
  • 92. CASE 9 • The patient is a 64 year old female on methotrexate therapy for rheumatoid arthritis. She developed anemia and a decreasing platelet count which persisted following discontinuation of this therapy. A bone marrow examination was performed.
  • 97. • Diagnosis: Acute myeloid leukemia (FAB M1) • Antigen Profile: Strongly positive for HLADR,CD13,CD34,CD38; dimly positive for CD33, CD71; partly positive for CD7,CD11b
  • 98. CASE 10 • The patient is a 57 year old female who presented with fatigue. She had had rheumatoid arthritis for about 5 years, and also reported a recent upper respiratory tract infection for which she had taken antibiotics. No blood work had been done at that time, but she was now found to have a hemoglobin of 10g, a white blood count of 8000 with a relative lymphocytosis, and a platelet count of 95,000.
  • 102. CASE 11 • The patient is a 60 year old man who had pancytopenia for several months before being diagnosed with myelodysplastic syndrome about one year ago. About 2 months prior to this, he presented with decreasing counts and was found to have transformed into acute leukemia. He was treated with induction chemotherapy, and this marrow was performed 6 weeks following this therapy.
  • 107. CASE 12 • The patient is a 72 year old female with fatigue and anorexia for two weeks. She complained of a skin rash which she developed about a day prior to coming to the doctor, and on examination was found to have cellulitis on her left arm. A bone marrow examination was performed.
  • 111. • The patient is a 49 year old man who presented to the emergency room with confusion and disorientation. A CT scan showed evidence of an early intracerebral bleed, and laboratory studies showed thrombocytopenia and evidence of disseminated intravascular coagulation. Following heparinization and stabilization of the patient's CNS status, a bone marrow examination was performed. Case 13
  • 117. • Note that the light scatter pattern is very broad, and in particular a forward versus RALS display shows what appears to be very large granular cells. There are no consistent phenotypic differences between M3 and M3v, though as shown here in the isotype control there is often considerable FITC autofluorescence in classic M3. • Diagnosis: Acute promyelocytic leukemia (FAB M3) Antigen Profile: Positive for CD71, CD33, CD9, CD13, myeloperoxidase; partly positive for CD34, HLADR.
  • 118. CASE 14 • A 34 year old man in previously good health came to the emergency room complaining of shortness of breath. His hemoglobin was 9 g/dl and his white blood count was 165,000/ul.
  • 123. • In many cases of M4 AML the FALS vs RALS pattern can be very characteristic. As shown here it frequently gives this "forked" picture. • This particular case is an example of M4e. Eosinophils may be difficult to detect in flow samples. When they are numerous they have the high RALS signal of granulocytes, but a higher intensity of CD45 expression similar to monocytes. It may be that some of the non-colored cells "above" the monocytes represent eosinophils. • Diagnosis: Acute myelomonocytic leukemia with eosinophilia (FAB-M4eo) Antigen Profile: Positive for CD33, HLADR and CD13; heterogeneous positivity for CD34, CD11b, CD15, CD14; dimly positive for CD2
  • 124. CASE 15 • The patient is a 64 year old man with a known history of anemia for the past year. About a month before this visit he had had a viral illness, and had never fully recovered his strength from that episode. On this visit, both his hemoglobin and his platelet count were much lower than they had been. A bone marrow was performed and sent for phenotyping.