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Flow Cytometry
Topics covered INTRODUCTION
METHODOLOGY◦ Fluidics◦ Generation of Scatter and Fluorescence◦ Optical System◦ Data Analysis
APPLICATIONS OF FLOW CYTOMETRY
INTRODUCTION
Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light.
The properties measured include a particle’s:
Relative size
Relative granularity or internal complexity, and
Relative fluorescence intensity.
INTRODUCTION Flow cytometry has allowed detailed insights into the cellular biology of
◦ Normal ,◦ Reactive and◦ Neoplastic tissues.
In addition clinical correlation has led to the establishment of FCM for ◦ Diagnosis and ◦ Prognosis in a range of disorders
Most clinically relevant flow assays deal with ◦ Leucocytes , ◦ Erythroid cells and ◦ Their precursors.
Fluidics in flow cytometry The purpose of the fluidics system is to transport particles in a fluid stream to the laser
beam for interrogation.
For optimal illumination the stream transporting the particles should be positioned in the center of the laser beam.
Only one cell or particle should move through the laser beam at a given moment.
Sample Preparation Peripheral smear
Bone marrow aspirate
Tissue
Sample is purified to remove debris and non viable cells are removed by purification by density gradient using Ficol Hypaque.
This will retain mature lymphocytes , monocytes and immature hematolymphoid precursors at the interface between plasma and Ficol layer.
Mature myeloid cells , RBC and RBCs precursors sink to the bottom along with dead/aggregated cells.
Fluidics in flow cytometry Cont…..
The sample is injected into a stream of sheath fluid within the flow chamber.
The design of the flow chamber causes the sample core to be focused in the center of the sheath fluid wherethe laser beam will then interact with the particles.
The flow of sheath fluid accelerates the particles andrestricts them to the center of the sample core.
Generation of light scatter Light scattering occurs when a particle deflects incident laser light.
The extent to which this occurs depends on the physical properties of a particle.
Factors that affect light scattering are
1. The cell's membrane,
2. Nucleus, and
3. Any granular material inside the cell.
Light Scatter Forward-scattered light (FSC) is proportional to cell-surface area or size.
FSC is a measurement of mostly diffracted light and is detected just off the axis of the incident
laser beam in the forward direction by a photodiode.
Side-scattered light (SSC) is proportional to cell granularity or internal complexity.
SSC is a measurement of mostly refracted and reflected light that occurs at any
interface within the cell where there is a change in refractive index
Light Scattering
Gra
nul a
ri ty
Size
Florescence Florescence:
Defined as the emission of light from a molecule(florochrome) that has absorbed light energy.
If the electron is excited by absorbing a photon of light , it will move to a higher orbital.
This electron will quickly reverts back to the lower orbit , during this reversion , the electron will give off some of its energy as light , which is the florescence.
Light sources Lasers are the light source of choice for clinical cytometry.
Alternative sources of excitation are available (mercury arc , halogen lamps)-
Less focused and broader spectrum of excitation –lead to decreased resolution and autoflorecence when compared to lasers.
Laser used- Argon type-488 nm wavelength.
Florochromes used-
1. Florescin IsoThioCyanate[FITC] and
2. Phycoerythrin[PE]
Optical System Consists of excitation optics and collection optics.
Excitation optics: Consist of the laser and lenses that are used to shape and focus the laser beam.
Collections optics: Consist of a collection lens to collect light emitted from the particle–laser beam interaction and a system of optical mirrors and filters to route specified wavelengths of the collected light to designated optical detectors.
Optical Filters
Optical filters are devices that selectively transmit light of different wavelengths.
Longpass filter: Attenuates shorter wavelengths and transmits longer wavelengths over the active range of the target spectrum.
Shortpass filter: Attenuates longer wavelengths and transmits shorter wavelengths over the active range of the target spectrum.
Bandpass filter: Passes wavelengths within a certain range and rejects wavelength outside that range.
Optical Filters
Signal Detection Light signals are generated as particles pass through the laser beam in a fluid stream.
These light signals are converted to electronic signals (voltages) by photodetectors.
There are two types of photodetectors:
1. Photodiodes and
2. Photomultiplier tubes (PMTs).
Voltage pulse A voltage pulse is created when a particle enters the laser beam and starts to scatter
light or fluoresce.
The highest point of the pulse occurs when the particle is in the center of the beam and the maximum amount of scatter or fluorescence is achieved.
As the particle leaves the beam, the pulse comes back down to the baseline.
Data Analysis Flow cytometric data is stored according to a standard format, the Flow Cytometry Standard (FCS) format, developed by the Society for Analytical Cytology.
A single cell analyzed for four parameters :
FSC[Forward Scattered Light]
SSC[Side Scattered Light],
Fluorescence of Fluorescein isothiocyanate and Phycoerythrin.
Generate 8 bytes of data.
Data display Once a data file has been saved, cell populations can be displayed in several different
formats.
Each event is placed in the channel that corresponds to its signal value.
Signals with identical intensities accumulate in the same channel.
Data display-ParametersSingle parameter-Histogram
Dual parameter data can be displayed in two dimensions using dot, density or contour plots.
Gating A gate is a numerical or graphical boundary that can be used to define the characteristics of particles to include for further analysis.
Ex: Blood sample-Containing mixed populations-Gate restricts analysis only to lymphocytes.
Based on FSC or cell size, a gate can be set on the FSC vs SSC plot to allow analysis only of cells the size of lymphocytes.
Quadrant marker A quadrant marker divides two-parameter plots into four sections to distinguish populations that are considered: negative, single positive, or double positive.
The lower-left quadrant displays events that are negative
for both parameters.
Quadrant Marker
Upper-left quadrant contains events that are positive for the y-axis parameter (CD19 PE)
The lower-right quadrant contains events that are positive for the x-axis parameter (CD3 FITC)
The upper-right quadrant contains events that are positive for both parameters (CD19+/CD3+)
Applications of Flow cytometry
1.Diagnosis of prognosis of leukemia and lymphoma
2.Defnation of prognosis , stage and the need of therapeutic intervention in HIV patients.
3.DNA ploidy and proriliferation fraction in certain neoplasms.
4.Enumeartion of reticulocytes
5.Detection of auto antibodies to platelets and neutrophils.
Applications of Flow cytometry
Immunophenotyping: Most important application is to provide detailed antigen profiles in various hematolymphoid neoplasms.
The widespread availability of monoclonal antibodies directly conjugated with different florochromes has increased the precision of immunophenotyping.
As a general rule immunophenotyping is also useful in defining the undifferentiated/poorly differentiated malignancies (carcinoma versus leukemia /lymphoma versus sarcoma versus melanoma)
Chronic lymphoid leukemia Immunophenotyping by flow cytometry helps in differentiating between:
1. Early stage of lymphoid leukemia and a persistent reactive lymphocytosis
2. Clinically significant subtypes of mature lymphoid leukemias.
B cell clonality: Determined by population expression of single type of surface or cytoplasmic immunoglobulin light chains ( kappa and lambda)
T cell clonality: Difficult: Currently , there is no reliable analog of kappa and lambda light chain expression for the T cell receptor protein.
Hence indirect method: Lack of expression of Pan-T cell antigens[CD2,3,4,5,7]
Acute leukemias A good general rule is to perform immunophenotyping analysis if the acute leukemia is undifferentiated or is probably ALL.
Approximately 1-5% of cytomorphologic AML are undifferentiated (M0) or megakaryocytic(M7).
Acute leukemia immunophenotyping panel:
ALL:
B cell lineage-CD19, kappa , lambda, CD10, TdT (terminal deoxynucleotidedyl transferase)
T cell-CD5, CD7,CD 2,CD3
AML:
Myelomonocytic lineage-CD13,CD33,CD117, Monocytic: CD14,CD64
Blasts-CD34,HLA DR
Erythroid: Glycophorin A
Megakaryocytic:CD41,CD61
Non-Hodgkins lymphoma and Plasmacytic neoplasias
Anaplastic variants of plasma cell leukemias , multiple myeloma and small variants of Waldenstroms macroglobulinemia may not be easily recognizable.
Immunophenophenotyping allows a more definitive diagnosis in these secretory B cell malignancies.
Hodgkins disease The immunoanalyiss of Hodgkin’s is almost entirely done by in situ techniques as it is impossible to get any significant number of RS cells into a mononuclear suspension.
It maybe be difficult to identify RS cells in frozen sections. However they can be easily visualized in formalin fixed tissues.
The combination of anti CD15 , anti CD30 and anti CD 45 with a morphologic examination-Confirms the diagnosis of Hodgkins disease.
Proliferation antigens Ki67 protein-Absent in G0 , is first expressed in G1 phase and then is variably present throughout S ,G2 and M phases.
Ki 67 proliferation index has been evaluated most widely on NHLs and breast carcinomas.
Proliferating cell nuclear antigen/Cyclin-36 kD delta sub unit of DNA polymerase , found in G1 through M phases of the proliferating cycle , peaking in S phase: directly correlate with mitotic activity of breast , colon carcinomas ,NHL.
ImmunodefiencyAIDS-The virus infects cells expressing the CD4 antigen. The most prominent cell affected is the CD4 lymphocyte.
The concentration of CD4 T cell can be easily detected by flow cytometry.
Transplantation During the first months after transplantation, alterations in the CD4/CD8 ratio are predictive of the diagnosis of Acute cellular rejection(ACR) .
Hayes and associates , reported a significant decrease in blood activated T cells (CD2/DR) 3-4 days before the clinical onset of ACR , followed by an increase in this population at onset.
Most studies indicate that virtually all lymphoid cells in rejecting allografts are T cells and
increased numbers of intragraft T cells are a sensitive marker of ACR.
These parameters can be monitored by flowcytometry
Anti CD3 therapy Used in treating denovo and steroid resistant solid organ allograft rejection.
OKT3 functions by causing either destruction of T cells or inducing immune paralysis-Elimating or steric blocking the CD3 and associated T cell antigen receptor on T cell surface.
The flowcytometric evaluation of OKT3 therapy primarily deals with verifying the loss of detectable CD3 antigen on T cells and
Auto immune thrombocytopenia
The rapid detection of either circulating or platelet bound antibody is significant evidence in supporting an auto immune cause for thrombocytopenia.
Correlation with the overall clinical and laboratory picture is essential because such auto antibodies may be seen with thrombotic thrombocytopenia purpura , transfusion and pregnancy induced alloimmunization and certain drug therapy.
The primary advantage of flowcytometry is the ability to analyze rapidly and objectively a large number of platelets , despite low in concentration.
Autoimmune and Alloimmune Neutropenia
The flow cytometric method for detection of either circulating anti neutrophil or neutrophil bound immunoglobulin is very similar to that used for platelets.
The primary difference is that neutrophils are more labile necessitating more rapid and fastidious processing.
Reticulocyte quantification The reticulocyte concentration can allow the clinician to ascertain whether the marrow is responding to peripheral blood loss or is being directly suppressed .
Most cytometric assays utilize either thiazole orange or auramine O.
Primary difficulty: Inability to distinguish Howell Jolly bodies from reticulocytes
DNA ploidy and proliferation fraction
The proliferation fraction or DNA ploidy measurement may correlate with the intrinsic biologic aggressiveness of the neoplasm.
The percentage of neoplastic cells in proliferative phases of cell cycle can be approximated by many techniques.
DNA quantification methodology
After pre treatment with with RNAase , an intercalating dye(propidim iodide) infiltrates and bind to the DNA→The amount of PI florescense is proportional to the DNA content of the cells.
From this DNA ploidy and proliferation fraction can be directly determined.
Two color techniques , using PI and a FITC conjugated antibody against cytokeratin , leucocyte common antigen-
Significantly separates benign from neoplastic cells.
DNA ploidy and proliferation fraction in Gastro intestinal tumors
DNA ploidy and proliferative fraction have been extensively studied in gastrointestinal tumors.
Currently –Only in colonic carcinomas ,DNA analysis have well documented value.
Lower proliferative fraction in early-stage disease –Decreased recurrence and increased survival.
Prognostic role of aneuploidy in colon carcinoma is controversial .
DNA ploidy and proliferation fraction in Genitourinary tumors
DNA aneuploidy and increased proliferation fraction :
Increased recurrence/decreased survival in:
Transitional cell carcinoma bladder
Renal cell carcinoma
Metastatic testicular neoplasia
Ovarian carcinoma
Uterine adenocarcinoma
DNA ploidy and proliferation fraction in Breast carcinoma
Multiple studies have shown that proliferation fraction in stage 1 and 2 breast carcinomas is inversely proportional to recurrence of the disease.
A similar trend is seen with DNA aneuploidy , though this may reflect its association with higher grades , which usually have larger proliferation fractions.
Functional Assays Oxidative burst: The ability of an individual’s neutrophils oxidative burst can be measured by oxidation of dichloroflorescein diacetate to dichloroflorescein.
The degree of fluorescence by dichloroflorescein compared to normal control is used in diagnostic evaluation of granulomatous disease.
Phagocytosis: Rare congenital immune deficiencies due to defects in neutrophils and monocyte phagocytosis.
Such disorders can be estimated by ingestion of FITC labelled bacteria as measured by flow cytometry.
Other Uses Cytogenetics
Tumor drug resistance
Recent Advances in Flow Cytometry
Recent Advances in Flow Cytometry Cont….
Instrument Improvements
There have been great improvements in the hardware and software for flow cytometers in the past 10-15 years, but the existence of increasing numbers of fluorochromes in combination with use of various laser beams greatly enhances the versatility of flow cytometry.
For instance, a 3-color flow cytometer can detect kappa and lambda staining in a single-cell suspension tube. A 5-color flow cytometer may identify kappa, lambda, CD19, and CD5, and 6-color flow cytometry will demonstrate kappa, lambda, CD19, CD5, and CD23 in a single specimen.
Therefore, a small specimen is now sufficient to do a large panel, and a large panel is able to characterize the tumor cell population more accurately for a specific diagnosis.
Recent Advances in Flow Cytometry Cont….
New Antibodies
A relatively new antibody, clusterin, can help diagnose ALCL and can help distinguish the systemic form from the primary cutaneous form
The latter does not show clusterin in the tumor cells to the same degree.
Evaluation of Cytoplasmic and Nuclear Antigens
Prognostication
CD38 and Zap 70 in CLL - unfavorable prognosis
Monitoring of Therapeutic Effect
Rituximab (anti-CD20) for the treatment of B-cell neoplasms. Alemtuzumab (anti-CD52) and BL22 (antiCD22) are also used for B-cell lymphomas/leukemias, and gemtuzumab (anti-CD33) is for AML.
Recent Advances in Flow Cytometry Cont….
T-Cell Clonality
B-cell population: Clonality identification is easy
But for T Cell –Difficult:
Recently, a set of T-cell receptor beta chain gene (TCR-Vbeta ) antibodies have become available to be used in clonal identification
References 1. David S Weinberg ,John L Carey , Flow and imaging cytometry.In Andersons Pathology, ed 10.pp 258-276
Flourescent microbead standards , Research triangle Park, NC , 1988 ,Flow Cytometry standards Corp.
Robinson Jp,editor:Handbook of flowcytometry methods , ed 2,New York,1993,Wiley-Liss
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