dr. claude lambert challenges, solutions and innovations in modern flowcytometr

Analysis of Cell populationsBy Flow cytometry

Claude LAMBERTLabo Immunologie CHU Saint-Etienne

European Society of Clinical Cell analysis

Fluorochrome detection

1 parameter

FluorochromeBest conjugateCompensationsNeg controls fmoartifacts

525 575 620 675 695 767

FITC

530/30

SignalSignal

Not all fluorescence is captured by your PMT

One fluorochrome one spectrum

spectraviewer

Instrument

1 - Know your optical bench

NAVIOS Laser 405 nm, 40mW Laser 488nm, 22 mW Laser 638 nm, 25mWFL9 FL10 FITC PE PE-TEXAS RED PE-Cy5 PE-Cy7 APC ALEXA FL 500 APC-Cy7

V450 V550 B525 B575 B620 B675 B780 R660 R755 R785Filtres 450/40 550/40 525/40 575/30 620/30 695/30 755 LP 660/20 725/20 755 LP

Optical band 430 à 470 530 à 570 505 à 545 560 à 590 605 à 635 680 à 710 >755 650 à 670 715 à 735 755Mirors 480 SP 550 SP 595 SP 655 SP 730 SP 710 SP 750 SPBand finale 430 - 480 530-570 505 à 545 560-590 605-635 680-710 >755 650-710 715 à 735 755

Canto II Laser 405 nm, 40mW Laser 488nm, 13 mW Laser 633 nm, 11mWHoriz V450

Horiz V500 FITC PE PerCP PE-Cy7 FL5 FL6

V450 V510 B530 B585 B670 B785 R660 R780filtres 450/50 510/50 fi

530/30 585/42 670 LP 780/60 660/20 780/60

Band finale 425 - 475 502-535 Ba

515-545 564-606 670 - 735 750-810 650-670 750-810

Canto

Navios

Canto

Navios

Leaking

2 - Know your fluorochromes

Compensations

525 575 620 675 695 767

530+30585+42

670 LP780+60

FITC

PE

PE-TxR

PE-Cy5

PerCP

PerCP-Cy5.5

PE-Cy7

530/30

= 530/30 -

525 575 620 675 695 767

530+30585+42

670 LP780+60

FITC

PE

PE-TxR

PE-Cy5

PerCP

PerCP-Cy5.5

PE-Cy7

Undercompensated

Compensations

Correct compensations

3 – Mind the compensations

Compensations

One representative conjugateStrong labellingStable fluorescencePossible surrogates

4 – Optimize your compensation settings

Inter laser Compensations

Inter laser Compensationsexception

Blue laser

Red laser

5 – Mind inter laser excitation

Optimum labelling

Fluorescence Intensité

Always be saturated

Highest signal/noise ratioSignal correlated to antigen density

SATURATION

6 - Consider the number of cells you analyze

Cell number

0.25 0.5 1 2 4 8 16 32 64 µl of Antibody

Fluorescence intensity

Saturation

Too much saturation Rise non specific labelling

Optimum labelling intensity

0.25 0.5 1 2 4 8 16 32 64 µl of Antibody

Fluorescence intensity

Find the best (minimal) saturationTITRATION

7 – ALWAYS at SATURATION

0.25 0.5 1 2 4 8 16 32 64 µl of Antibody

Fluorescence intensity

WASH

8 – Wishing reduce noise

Optimize your conjugate

JW Gratama Cytometry 1998

Antigen densityAssign copy numbers of PE molecules bound per bead

Antiboy Bound per Cell

Bikoue A et al, Cytometry 1996; 26: 137-147

Rule 4 : marker densities are not equivalents

9 – Mind marker density on cells

Best conjugate

Fluorochroms are not equivalent

Best conjugate

10 - Stronger Fluorochroms dimer staining (lower density)

Visualization of Spectral Overlap

Gallios/Navios Flow Cytometers

2 types of distortion:

‚Silent‘ Fluorochromes:• bright markers (gating)

‚Untouchable‘ Channels• dimly expressed antigens

depends on instrument configuration, and fluorochromes.

11 – dim expression : stronger and untouchable fluorochome

Optimize your Dot plots

Dot plots are more informative than histograms

3 populations

In fact more than 3 populations

2 dim graphs are most precise Graphs

Other types of graphs

Level curvesbetter definiton of valley

12 – Take advantages of plot types

Sequential selection of population

Quantification of sub groups

gives % of cells

Yes …. But % of what??

Rule 6 : Always make your gating strategy very clear

28.3%

Gating Hierarchy

Graphs ConditionningMention

Parent population (s)

28.3%

Proper labelling info

IL-17

IL-2

2

% of what??

Kamnesh R. Pradhan, et col Cytometry Part B 2011; 80B: 335–338

Gating strategy explained in a paper

13 – Clearly design & describe gating strategy

Positivity

What is positive?

Decision threshold difficult

Reproducibility among different samples even more difficult

Repeattibility between labs, operators..

14 – Be sure of positive threshold

NEGATIVITY

Cells alones(auto-fluorescence)

But does not exclude non specific labelling??

non specific deposit on cells

PC5

For each label

Exclude non specific labelling

Isotype Controls =Same providerSame isotype

Same fluorochrome

Use non specific Surrogate antibodiesThe closest to the protocol

ECD

FITC

PE/RD1PC5

Use cells in the sample that you know they are negative for the

antibody considered

ECD

FITC

PE/RD1PC5

PC5

Internal negative cell

Internal negative control

Use negative cells (red) as negative control..

Validate negativity Inside the multiple labelling

FULL FLUORESCENCE MINUS ONE (FMO)Test one color negativity while all other labelings are there

All except CD19 All except CD8

15 – Be sure on the negative

Follow acquisition

Exclude bublesTemps

Surveiller acquisition

TAB

16 – Keep clean acquisition

Good Flowcytometry analysis Knowing your Optical scheme

Good Negative controls (including FMO)

Proper settings and do not change it

Positive threshold to be validated on each sample

Label saturation…. But not too much titration

Avoid non specific labelling

Gating strategy and graph information

Optimize your conjugates

Design

Modern art