PBS 7413 FLOW CYTOMETRY LABORATORY

COURSE OBJECTIVES 1.UNDERSTAND CLINICAL AND RESEARCH APPLICATIONS OF FLOW CYTOMETRY TECHNOLOGY 2.PERFORM STAINING TECHNIQUES 3.ANALYZE DATA 4.INTERPRET DATA AS PRESENTED IN BASIC SCIENCE AND CLINICAL SCIENCE LITERATURE

Flow Cytometry Principles And Applications

FLOW CYTOMETRY Fluorescence activated cell sorting (FACS) is a technology that allows simultaneous measurement of multiple physical and chemical characteristics of a single cell. These measurements are made on a per cell basis at routine rates of 500 to 70,000 cells per second in a moving stream.

FACS COMPONENTS 1.LASER 2.OPTICS 3.FLUIDICS 4.ELECTRONICS

LASER 1.ARGON 2.EMISSION LINE OF 488 nm 3.VISIBLE LIGHT

OPTICS 1.BEAM SPLITTERS 2.DICHROIC MIRRORS 3.LONG / SHORT PASS FILTERS 4.BAND PASS FILTERS

FLUIDICS 1.SAMPLE FLUID 2.SHEATH FLUID 3.FLOW CELL 4.NOZZLES, 70, 100, 400 um

ELECTRONICS 1.PHOTOMULTIPLIER TUBES (PMTs) 2.PHOTODIODE 3.MACINTOSH G3 COMPUTER

DATA PARAMETERS 1.FORWARD LIGHT SCATTER 2.90 o OR SIDE SCATTER 3.FLUORESCENCE EMISSION

FORWARD SCATTER 1.LIGHT SCATTER USED IS LOW ANGLE 2.SENSITIVE TO CELL SIZE AND SURFACE AREA 3.LIVE / DEAD DISCRIMINATION

SIDE SCATTER 1.LIGHT SCATTER USED IS 90 o 2.SENSITIVE TO INTERNAL STRUCTURES

FORWARD ANGLE LIGHT SCATTER RIGHT ANGLE LIGHT SCATTER

BEST ANALYSIS USING FORWARD AND SIDE SCATTER TOGETHER; ANALYSIS OF HETEROGENEOUS POPULATIONS

PERIPHERAL BLOOD MONONUCLEAR CELLS FSC LYSED PERIPHERAL WHOLE BLOOD SSCSSC SSCSSC FSC SSCSSC SSCSSC

FLUORESCENCE 1.DETECT BINDING OF LABELED LIGAND 2.LIVE/DEAD DETERMINATION 3.DNA/RNA CONTENT

FLUORESCEINTEXAS RED PHYCOERYTHRINALLOPHYCOCYANIN PROPIDIUM IODIDERHODAMINE WAVELENGTH (NM) EXCITATION OR EMISSION

SINGLE BEAM EXCITATION THREE COLOR EMISSION

UNCOMPENSATED COMPENSATED RED FLUORESCENCERED FLUORESCENCE GREEN FLUORESCENCE CD3 CD19 CD3 CD19

APPLICATIONS 1.IMMUNOPHENOTYPING 2.CELL CYCLE ANALYSIS 3.APOPTOSIS 4.CELL FUNCTION 5.CELL SORTING

CELL SURFACE STAINING SINGLE CELL SUSPENSION MONOCLONAL ANTIBODY FLUOROCHROME CONJUGATED MONOCLONAL FLUORESCENCE ANTIBODY CONJUGATED SECOND REAGENT FACS ANALYSIS / CELL SORTING

FSC SSC IgG1 FITC ISOTYPE CONTROL IgG2a PE IgG1 FITC IgG2a PE UNGATEDR1

IMMUNOPHENOTYPING CD4 FITC CD8 PECD8 PE 16% CD4+ 55% CD8+

ADVANTAGES OF FACS 1.PRECISION 2.SENSITIVITY 3.NO PHOTOBLEACHING 4.VOLUME 5.SINGLE CELL ANALYSIS 6.SAMPLING STATISTICS 7.SORT CELLS IN SEPARATE TUBES

CELL CYCLE ANALYSIS 1.FACS IS METHOD OF CHOICE FOR FAST, ACCURATE DETERMINATION OF CELL CYCLE DISTRIBUTIONS 2.DETERMINATION OF ANUEPLOIDY 3.% OF S PHASE

Normal Cell Cycle 2N4N eventsevents DNA content

TUMOR DNA STAINING SINGLE CELL SUSPENSION PERMEABILIZE WITH METHANOL DIGEST DS RNA WITH RNASE STAIN DNA WITH PROPIDIUM IODIDE

G0/G1 = 60% S = 13% G2/M = 27% Cell Cycle Analysis DNA CONTENT EVENTSEVENTS

CELL CYCLE ANALYSIS S G2/M G0/G1 TUMORTUMOR DIPLOIDDIPLOID G0/G1 = 79.5% S = 12.7% G2/M = 7.8% DNA INDEX = 1.11

MEASUREMENT OF APOPTOSIS 1.APOPTOSIS IS PROGRAMMED CELL DEATH WHERE THE CELL GOES THROUGH A HIGHLY REGULATED PROCESS OF “DYING” 2.CHARACTERISTICS ARE CONDENSATION OF THE CHROMATIN MATERIAL AND BLEBBING OF NUCLEAR MATERIAL 3.OFTEN ACCOMPANIED BY INTERNUCLEOSOMAL DEGRADATION OF DNA

CYTOMETRY IN CELL NECROBIOLOGY APOPTOSIS NECROSIS CELL DEHYDRATION APOPTOTIC BODIES CELL AND MITOCHONDRIAL SWELLING PLASMA MEMBRANE RUPTURE

DETECTION METHODS FOR APOPTOSIS 1.STRAND BREAKS IN DNA CAN BE LABELED ENZYMATICALLY IN SITU BY TdT 2.STRAND BREAKS IN DNA CAN BE LABELED DIRECTLY USING dUTP 3.MEMBRANE PHOSPHATIDYL SERINE CAN BE DETECTED WITH ANNEXIN V

APOPTOSIS PROPIDIUMIODIDEPROPIDIUMIODIDE ANNEXIN V FITC 31% Necrotic 33% Viable 35% Apoptotic

CELL FUNCTION 1.STAINING OF ACTIVATION ANTIGENS 2.INTRACELLULAR Ca 2+ LEVELS 3.INTRACELLULAR pH LEVELS 4.MEMBRANE POTENTIAL 5.CYTOKINE SECRETION

FSC SSCSSC ACTIVATED LYMPHOCYTES

CD25 FITC CD69PECD69PE ACTIVATION ANTIGENS 33%27% 7%

CELL SORTING AS LIQUID IS EJECTED INTO AIR, IT WILL FORM DROPLETS BY VIBRATING THE NOZZLE AT A DEFINED FREQUENCY, THE SIZE OF THESE DROPLETS AND THE POSITION ALONG THE STREAM WHERE THEY FORM CAN BE CONTROLLED WITH PRECISION. A DROPLET CONTAINING A CELL IS APPLIED EITHER A NEGATIVE OR POSITIVE CHARGE AND SORTED BY PASSING THROUGH AN ELECTRIC FIELD.

CHROMOSOME ANALYSIS AND SORTING INDIVIDUAL CHROMOSOMES CAN BE ANALYZED IN FLOW AFTER APPROPRIATE PRESERVATION AND ISOLATION. THE MOST COMMON METHOD IS TO USE TWO DIFFERENT DNA DYES, ONE (HEOCHST 33258) THAT BINDS PREFERENTIALLY TO AT- RICH DNA AND ONE (CHROMOMYCIN A3) THAT BINDS PREFERENTIALLY TO GC-RICH DNA.

ANTIBODY PANELS T CELLB CELLMYELOIDOTHER CD7CD19CD14GLYCOPHORIN CD2CD10CD33CD41 CD3CD20CD34CD61 CD5 TdTCD11c sIg

IMMUNOPHENOTYPING 1.IMMUNE MODULATION IN INFECTIOUS DISEASES 2.LEUKEMIAS AND LYMPHOMAS 3.STEM CELL QUANTITATION

ACUTE LYMPHOBLASTIC LEUKEMIA SSCSSC FCS CD13 PECD13 PE CD10 FITC CD19 PECD19 PE CD34 FITCIntra TdT FITC FL2FL2 WBC 13,200 %69 1% 73%4% 70%

MALIGNANT LYMPHOMA FSC SSCSSC CD5 FITC CD20 PECD20 PE NEG KAPPA LAMBDA A 64% 27% 4.6% 61% 1%

FORWARD SCATTER SIDE SCATTERSIDE SCATTER LYMPHOMA 92%

CD3 FITC CD4 PECD4 PE LYMPHOMA IMMUNOPHENOTYPING CD3+CD4+ = 76%

CD3 FITC CD8 PECD8 PE LYMPHOMA IMMUNOPHENOTYPING CD3+CD8+ = 0.8%

B220 FITC SIDESCATTERSIDESCATTER LYMPHOMA IMMUNOPHENOTYPING 2.7%

CD34+ SUBPOPULATIONS 1.HEMATOPOIETIC STEM CELLS (CD34+THY1+HLADR-CD38-Lin-) 2.MULTIPOTENT PROGENITOR CELLS (CD34+HLADR+CD38+Lin-) 3.LINEAGE COMMITTED PROGENITOR CELLS (CD34+HLADR+CD38+Lin+)

PERIPHERAL BLOOD STEM CELL HARVEST PRE APHERESISAPHERESIS FCS SSC SSCSSC SSCSSC CD34 PECD34 PE CD34 PECD34 PE CD34+ = 0.79%CD34+ = 4.58%

ASSESSMENT OF PLATELET FUNCTION 1.CD41-RESTING AND ACTIVATED PLATELETS 2.CD61-RESTING AND ACTIVATED PLATELETS 3.CD62p-(p-SELECTIN)

PLATELET FCXM ADVANTAGES 1.SELECTION OF PLATELET POPUATION FROM A HETEROGENOUS SUSPENSION USING ELECTRONIC GATING 2.PERFORMANCE IMPROVES IN PATIENTS WITH PROVEN ALLOIMMUNIZATION BY LYMPHOCYTOTOXIC ANIBODY SCREENING 3.DETECT LOW TITER HLA ABS AND PLATELET SPECIFIC ABS

ACTIVATED PLATELET AGGREGATES 1.OBSERVED IN PATIENTS WITH TTP 2.RISES WITH RELAPSES, DECREASES FOLLOWING PLASMA INFUSIONS, NORMALIZED DURING REMISSIONS 3.FLOW CYTOMETRY APPEARS TO BE USEFUL IN MONITORING ACTIVITY OF THE DISEASE AND ASSESSING EFFICACY OF TREATMENT IN TTP PATIENTS

DETECTION OF ALLOANTIBODIES IN TISSUE TRANSPLANTATION 1.COMPLEMENT-DEPENDENT CYTOTOXICITY (CDC) 2.FACS CROSSMATCH

TRANSPLANTATION FCXM PB DONOR LYMPHOCYTES LN + SPLEEN PATIENT’S SERUM ANTI-HUMAN IgG (IgM)-FITC ANTI-CD3-PE MFI DETERMINED BY FACS

FLOW CYTOMETRY CROSSMATCH IN KIDNEY TRANSPLANTATION NUMBEROFCELLSNUMBEROFCELLS IgG - FITC PRE-TRANSPLANT POST-TRANSPLANT POSITIVE CONTROL

HLA FCXM ADVANTAGES 1.FASTER TURN AROUND TIME 2.LOWER INCIDENCE OF AMBIGUOUS RESULTS DUE TO POOR CELL VIABILITY 3.DIRECT DETECTION OF IgG ABS WHICH AVOIDS FALSE + RXS DUE TO IgM AUTOABS.

RELEVANCE OF FCXM IN TRANSPLANTATION 1.KIDNEY--- USUALLY DONE AS AN ADDITIONAL TEST IN PRESENSITIZED OR RETRANSPLANT CANDIDATES 2.BONE MARROW--- METHOD OF CHOICE TO DETECT ANTIBODIES BEFORE TRANSPLANTATION 3.HEART, LIVER, LUNG AND PANCREAS--- SELDOM PERFORMED