Webinar Series Overcoming challenges in cellular analysis Multiparameter analysis of rare cells January 28, 2015 Instructions for Viewers • To share webinar via social media: • To share webinar via e ‐ mail: • To see speaker biographies, click: View Bio under speaker name Sponsored by: • To ask a question, click the Ask A Question button under the slide window
Webinar Series Overcoming challenges in cellular analysis Multiparameter analysis of rare cells January 28, 2015 Brought to Participating Experts you by the Science /AAAS Custom Publishing Andrea Cossarizza, M.D., Ph.D. Office University in Modena and Reggio Emilia School of Medicine Modena, Italy David Cousins, Ph.D. University of Leicester Sponsored by: Leicester, UK
ANDREA C ANDREA COSSARIZZA SSARIZZA Multiparam eter analysis of rare cells
OUTLINE OF THE TALK Rare cell analysis: background and keypoints •
OUTLINE OF THE TALK Rare cell analysis: background and keypoints • Main problems in the detection of such cells •
OUTLINE OF THE TALK Rare cell analysis: background and keypoints • Main problems in the detection of such cells • Possible solutions: from hardware to software •
OUTLINE OF THE TALK Rare cell analysis: background and keypoints • Main problems in the detection of such cells • Possible solutions: from hardware to software • Rare cells in the immune system: the case of iNKT •
BACKGROUND • >30 years ago: enumeration of fetal red blood cells in the maternal circulation at a frequency of 1/10,000 to 1/100,000 by Cupp.
BACKGROUND • >30 years ago: enumeration of fetal red blood cells in the maternal circulation at a frequency of 1/10,000 to 1/100,000 by Cupp. • Now: detection and quantitation of several rare cell populations in blood or bone marrow.
BACKGROUND • >30 years ago: enumeration of fetal red blood cells in the maternal circulation at a frequency of 1/10,000 to 1/100,000 by Cupp. • Now: detection and quantitation of several rare cell populations in blood or bone marrow. • Essential tool in the diagnosis and monitoring of hematological cancers and immunological disorders, as well as in the identification of Ag ‐ specific cells.
WARNING • Rare ‐ event analysis is the art of finding a needle in a haystack
WARNING • Rare ‐ event analysis is the art of finding a needle in a haystack • The frequency of the event of interest, and the signal ‐ to ‐ noise ratio of the method used to detect the event are the two most important factors.
KEY POINTS ‘‘ Rare ‐ event analysis ’’: detection of events that occur • at a frequency of 1 in 1,000 ( 0.1% ) or less, although the record claimed in the literature has long stood at 1 cell in 10,000,000 ( 0.00001% ) for tumor cells spiked into peripheral blood.
KEY POINTS ‘‘ rare ‐ event analysis ,’’: detection of events that occur • at a frequency of 1 in 1,000 ( 0.1% ) or less, although the record claimed in the literature has long stood at 1 cell in 10,000,000 ( 0.00001% ) for tumor cells spiked into peripheral blood. • Detecting an event at low frequency requires a high signal ‐ to ‐ noise ratio and the acquisition of a large number of events.
IMMUNOLOGIST'S INTERESTS • Ag ‐ specific T cells • NKT and iNKT cells Circulating endothelial cells and precursors • • Stem cells (CD34+) • Particular lymphocytes subpopulations Circulating tumor cells • • Polyfunctional assays • ..........
Open pre ‐ analytical questions • How much blood from patients?
Open pre ‐ analytical questions • How much blood from patients • Lack of available standardized method
Open pre ‐ analytical questions • How much blood from patients • Lack of available standardized method • Enriched or non enriched populations
Open pre ‐ analytical questions • How much blood from patients • Lack of available standardized method • Enriched or non enriched populations • How many markers/colors
Open pre ‐ analytical questions • How much blood from patients • Lack of available standardized method • Enriched or non enriched populations • How many markers/colours • How many cells
Open pre ‐ analytical questions • How much blood from patients • Lack of available standardized method • Enriched or non enriched populations • How many markers/colours • How many cells • Exclusion of doublets, dead cells and debris: use of a DUMP CHANNEL
Number of events to acquire CV (%) 5 1 2.5 10 20 Positive cells 10,000 1,600 400 100 25 required Frequency EVENT NUMBER TO ACQUIRE % l/n 10 10 100,000 16,000 4,000 1,000 250 1 100 1,000,000 160,000 40,000 10,000 2,500 0.1 1,000 10,000,000 1,600,000 400,000 100,000 25,000 0.01 10,000 100,000,000 16,000,000 4,000,000 1,000,000 250,000 0.001 100,000 1,000,000,000 160,000,000 40,000,000 10,000,000 2,500,000
Open analytical questions • Which instrument, and which performances
Open analytical questions • Which instrument, and which performances • Flow cytometer rates of acquisition
Open analytical questions • Which instrument, and which performances • Flow cytometer rates of acquisition • Maximize the signal ‐ to ‐ noise ratio of the cells of interest from the background
Open analytical questions • Which instrument, and which performances • Flow cytometer rates of acquisition • Maximize the signal ‐ to ‐ noise ratio of the cells of interest from the background • Data acquisition: instrument clean and the background level of noise below the threshold
Open analytical questions • Which instrument, and which performances • Flow cytometer rates of acquisition • Maximize the signal ‐ to ‐ noise ratio of the cells of interest from the background • Data acquisition: instrument clean and the background level of noise below the threshold • Spill over and carry over
Open analytical questions • Which instrument, and which performances • Flow cytometer rates of acquisition • Maximize the signal ‐ to ‐ noise ratio of the cells of interest from the background • Data acquisition: instrument clean and the background level of noise is below the threshold • Spill over and carry over • Adequate software
Our previous experience Polyfunctional analysis of Ag ‐ specific cells 2012
THE INTERBETWEENERS: INNATE ‐ LIKE LYMPHOCYTES Types of lymphocyte that blur the traditional boundaries between innate and adaptive immunity
THE INTERBETWEENERS: INNATE ‐ LIKE LYMPHOCYTES Types of lymphocyte that blur the traditional boundaries between innate and adaptive immunity Invariant Natural Killer T cells ( iNKT ) Poised to robustly produce cytokines more rapidly than conventional naïve T cells
THE INTERBETWEENERS: INNATE ‐ LIKE LYMPHOCYTES Types of lymphocyte that blur the traditional boundaries between innate and adaptive immunity Invariant Natural Killer T cells Mucosal associated invariant T cells ( iNKT ) ( MAIT ) Poised to robustly Preferentially produce cytokines localized in the more rapidly than mucosal tissues conventional naïve T cells
THE INTERBETWEENERS: INNATE ‐ LIKE LYMPHOCYTES Types of lymphocyte that blur the traditional boundaries between innate and adaptive immunity T cells Invariant Natural Killer T cells Mucosal associated invariant T cells ( MAIT ) ( iNKT ) Poised to robustly Preferentially Pre ‐ programmed to produce cytokines localized in the acquire their more rapidly than mucosal tissues effector functions conventional naïve before egress from T cells thymus
DIFFERENT CHARACTERISTICS OF INKT AND MAIT CELLS iNKT cells MAIT cells Frequency 0.01 ‐ 1% (% among human 1 ‐ 10% PBMCs) Receptors semi ‐ invariant semi ‐ invariant V α 24 ‐ J α 18 TCR , V α 7.2 ‐ J α 33 TCR , high NK receptors levels of CD161,IL ‐ 18R α . microbial antigens Antigen recognized glycolipid antigens presented by MR1 presented by CD1d Subsets CD4+, CD8+, and CD4+, CD8+, and CD4 ‐ CD8 ‐ CD4 ‐ CD8 ‐ Function Regulatory Effector ‐ memory phenotype
Gating strategy for iNKT cells and their main subsets SSC SSC SSC DUMP CHANNEL V α 24J α 18V β 11 TCR CD3 (CD14,CD19)
Gating strategy for iNKT cells and their main subsets SSC CD161 CD8 SSC SSC SSC CD4 DUMP CHANNEL V α 24J α 18V β 11 TCR CD3 (CD14,CD19) SSC CD161
NKT cells and Multiple Sclerosis (MS) Berzins SP., Nat Rev Immunol. 2011
Studies on a subset of NKT cells Polyfunctionality of iNKT cells in patients affected by different forms of Multiple Sclerosis (Relapsing ‐ Remitting RR, Primary Progressive PR, Secondary Progressive SP), in the framework of a project sponsored by the Italian Foundation for Multiple Sclerosis ‐ FISM • 3 RR patients (treated with Natalizumab) • 2 PR patiens • 5 SP patients • 5 CTR (healthy subjects)
Methods • PBMCs isolation from >30 mL of blood Stimulation with PMA (100 ng/ml) plus ionomycin (1 g/ml) for 4 hrs • • ICS with following markers: Live Dead (Aqua) CD3 PE ‐ CY5 CD4 AF700 CD8 APC ‐ CY7 iTCR (V 24 ‐ J 18) PE IFN ‐ gamma FITC IL ‐ 4 APC IL ‐ 17 BV421 TNF ‐ alpha BV605
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