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What Is Flow Cytometry?
Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in
a fluid stream
Cytometry vs. Flow CytometryCytometry
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.
• 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
Applications of Flow Cytometry.
Flow cytometry & Flow cytometry & HematopathologyHematopathology1. 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 CytometryPrinciple of Flow Cytometry
FluidicsFluidics
OpticsOptics
ElectroniElectronicscs
• Cells in suspension
• Cells flow in single-file
• Intercepted by light source(s) (laser)
• Scatter light and emit fluorescence
• Signal collected, filtered and
• Converted to digital values
• Storage on a computerData display and analysisData 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
Sample StreamCell
The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Hydrodynamic Focusing.Focusing.
Incoming Laser
Sample
Sheath Sheath
Sheath
Sample
SampleCore
Stream
Low Differential High Differential
Laser Focal Point
Sample Differential
10 psi
10.2 psi
10 psi
10.4 psi
10 psi
10.8 psi
Difference in pressure between sample and sheathThis will control sample volume flow rateThe greater the differential, the wider the sample core. If differential is too large, cells will no longer line up single fileResults in wider CV’sCV’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
FSCFSCDetectorDetector
Laser BeamLaser Beam
Side Scatter
FSCFSCDetectorDetector
CollectionCollectionLensLens
SSCSSCDetectorDetector
Laser BeamLaser Beam
Sid
e s
catt
er
Sid
e s
catt
er
Forward scatterForward scatter
Lymphocytes
Monocytes
Granulocytes
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
FSC
SSC
Lymphocytes
Monocytes
Granulocytes
RBCs, DebrisRBCs, Debris,,Dead CellsDead Cells
Sid
e s
catt
er
Sid
e s
catt
er
Forward scatterForward scatter
Low Medium High levels of forward scatter >----increasing cell size
Sid
e s
catt
er
Sid
e s
catt
er
Forward scatterForward scatter
Low
High
Incre
asin
g levels
of
sid
e s
catt
er
>--
--
incre
asin
g c
ell g
ran
ula
rity
Medium
BY “GATING” EACH OF THE AREAS INBY “GATING” EACH OF THE AREAS IN2 DIMENSIONS, YOU CAN ADAPT FLOW CYTO-2 DIMENSIONS, YOU CAN ADAPT FLOW CYTO-METRY TO PERFORM DIFFERENTIAL COUNTS!METRY TO PERFORM DIFFERENTIAL COUNTS!
Sid
e s
catt
er
Forward scatter
Lymphocytes
Monocytes
Granulocytes
FLOW CYTOMETRYFLOW CYTOMETRY
- Cells are labeled with fluorescent antibodies directed against cell surface molecules
- Using different color fluorochromes allowscounting of many markers simultaneouslyand allows identification of several markerson the same cell ( Multiparameter Flow)
- In the instrument, cells pass one-by-one pasta laser to excite the fluorochromes andthere are detectors for each type offluorochrome
- cells are labeled with fluoresence antibodies
Flouresent tag
Surface of a cell, e.g., a lymphocyte (in solution)
FluorescenceFluorescence
Photon emission as an electron returns from an excited state to ground state
What Happens in a Flow What Happens in a Flow Cytometer (Simplified)Cytometer (Simplified)
cellflash.swf
Fluorochrome
Basic Principles cont’dBasic 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
ElectronicsElectronics
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 Comprehensive antibody panelspanels 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 Abs Panel, the EEuropean uropean GGroup for the roup for the IImmunological Characterization of mmunological Characterization of LLeukemias (eukemias (EGILEGIL) ) for the diagnosis and for the diagnosis and classification of acute leukemiaclassification of acute leukemia
Panel of antibodies recommended by the Panel of antibodies recommended by the BBritish ritish CCommittee for ommittee for SStandards in tandards in HHaematology (aematology (BCSHBCSH) ) for the diagnosis and classification of acute for the diagnosis and classification of acute
leukemialeukemia
Panel of antibodies recommended by the Panel of antibodies recommended by the EEuropean uropean LLeukemiaeukemiaNNet, et, ELNELN for the diagnosis for the diagnosis
and classification of acute leukaemiaand classification of acute leukaemia
Panel of antibodies recommended by Panel of antibodies recommended by the the US–Canadian Consensus Group US–Canadian Consensus Group for for
the diagnosisthe diagnosisand classification of acute leukemiaand classification of acute leukemia
Reactivity of mAbs vs. Reactivity of mAbs vs. FAB-AMLFAB-AML
45%
Development of a FACS histogramDevelopment of a FACS histogram
Co
un
ts
Fluorescent intensity
Negative cells
Positive cells
Note: The operator can set the “gate” by visual inspection ofthe histogram. The “gate” defines negative versus positive.
Intensity scalesare logarithmic
CD
19
CD3
THE OPERATOR SETS “GATES” DEFININGTHE OPERATOR SETS “GATES” DEFININGPOSITIVE AND NEGATIVE FOR EACH MARKER.POSITIVE AND NEGATIVE FOR EACH MARKER.
CD3- CD3+
CD
19
CD3
CD19+ CD19+ CD3 - CD3 +
CD19- CD19- 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 CD19 47% moderate CD3 45% moderate CD20 26% moderate CD4 21% moderate CD22 47% moderate CD7 47% moderate sIgM 48% moderate CD8 20% moderate Kappa 3% moderate CD13 3% moderate Lambda 2% moderate CD33 1% moderate CD10 36% moderate CD34 1% weak CD45 100% strong
TdT 55% moderate 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 FlowcytometrySteps 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.
Dr. Behzad Poopak, PhD (Hematologist)
File: CD10.FCS Date: 09-11-2011 Time: 13:40:32 Particles: 13134 Acq.-Time: 48 s
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partec PAS
Region Gate Ungated Count Count/ml %Gated Mean-x CV-x% Mean-y CV-y%R1 <None> 10620 10620 - 80.86 70.80 16.60 39.99 31.71RN1 R1 8981 7844 - 73.86 24.43 40.12 - - RN2 <None> 6965 6965 - 53.03 3.47 36.01 - -
File: CD19.FCS Date: 09-11-2011 Time: 13:42:24 Particles: 13036 Acq.-Time: 57 s
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Region Gate Ungated Count Count/ml %Gated Mean-x CV-x% Mean-y CV-y%R1 <None> 10674 10674 - 81.88 72.44 16.39 39.74 31.43RN1 R1 93 47 - 0.44 2.96 22.79 - - RN2 R1 9019 8039 - 75.31 14.17 58.89 - -
File: HLA.FCS Date: 09-11-2011 Time: 13:54:48 Particles: 13551 Acq.-Time: 38 s
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FL2
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Q1: 3.33% Q2: 22.52%
Q3: 22.60% Q4: 51.55%
partec PAS
Region Gate Ungated Count Count/ml %Gated Mean-x CV-x% Mean-y CV-y%R1 <None> 11110 11110 - 81.99 73.10 16.33 38.41 32.46RN1 R1 9102 8108 - 72.98 11.01 91.69 - - RN2 R1 3427 2776 - 24.99 29.41 118.49 - - Q1 R1 481 370 - 3.33 1.47 28.26 28.62 55.58Q2 R1 3052 2502 - 22.52 16.22 89.84 28.47 128.48Q3 R1 3837 2511 - 22.60 1.22 29.37 1.03 12.49Q4 R1 6181 5727 - 51.55 8.54 69.32 1.11 20.51
File: CD34.FCS Date: 09-11-2011 Time: 13:56:48 Particles: 14047 Acq.-Time: 12 s
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Q3: 35.93% Q4: 63.92%
partec PAS
Region Gate Ungated Count Count/ml %Gated Mean-x CV-x% Mean-y CV-y%R1 <None> 11886 11886 - 84.62 72.57 15.87 37.87 32.02RN1 R1 8202 7518 - 63.25 5.65 45.43 - - RN2 R1 84 12 - 0.10 2.15 7.78 - - Q1 R1 0 0 - 0.00 - - - - Q2 R1 97 17 - 0.14 18.44 11.75 2.08 8.23Q3 R1 5743 4271 - 35.93 1.16 28.45 1.00 0.99Q4 R1 8207 7598 - 63.92 5.58 44.96 1.02 7.61
Immunophenotyping results, together with morphological findings and Cytochemistry of BMA, are consistent with B lymphoblastic leukemia (Early pre B-cell type).
Patient Report Format
Patient Report-2 Format
Hematogone vs.Leukemic Lymphoblast
53
Diagnosing Multiple MyelomaThree 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*
3
• One or more of the following (CRAB criteria):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)
Durie et al for the International Myeloma Working Group. Leukemia. 2006:1-7.
*Monoclonal M spike on electrophoresis IgG >3.5 g/dL, IgA >2 g/dL, light chain >1 g/dL in 24-hour urine sample.
54
Diagnostic Evaluation of Multiple Myeloma
TestFinding(s) With MyelomaCBC 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
ImmunofixationIdentifies 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 imagingOsteolytic lesions, osteoporosis
Serum free light chain ↑ Free light chains
MRIEvaluation 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. Multiple Myeloma: Disease Overview. 2006. www.multiplemyeloma.org; Rajkumar et al. Blood. 2005;106(3):812 .
B.Poopak
Peripheral blood - rouleaux
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
Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004
NormalNormal Monoclonal Protein in Myeloma
Monoclonal Protein in Myeloma
Serum Protein Electrophoresis
Copyright ©2001 American Society of Hematology. Copyright restrictions may apply.
Maslak, P. ASH Image Bank 2001;2001:100211
Figure 8. Quantitative immunoglobulins were within normal limits
Copyright ©2001 American Society of Hematology. Copyright restrictions may apply.
Lazarchick, J. ASH Image Bank 2001;2001:100185
Figure 8. Immunofixation electrophoresis showing a monoclonal IgA lambda light chain restricted band
Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004
Immunofixation to Determine Type of Monoclonal Protein
Immunofixation to Determine Type of Monoclonal Protein
IgG kappa M proteinIgG kappa M protein Lambda Light ChainsLambda Light Chains
CASE STUDY – MULTIPLE MYELOMA
Serum free light chains Free kappa 16.2 3.3 – 19.4
mg/L Free lambda 3754.1 5.7 – 26.3
mg/L Ratio 0.0 0.3 – 1.7
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?
