settings Fiona Robins Flow Cytometry Department Haematology - - PowerPoint PPT Presentation

Standardisation of Application settings

Fiona Robins Flow Cytometry Department Haematology PathWest, QEII

PMT voltage applied to photomultiplier tube rCV dim beads, rSD dim beads, Optimal PMTV = 10 x rSDEN

Optimisation of Voltages to be used for patient samples (lymphocytes)

• The CS+T bead report can be used to

calculate targets and ranges that are then used to determine the optimal voltages for running human blood cells.

Record the electronic noise robust SD and the linearity maximum channel from the baseline report to use for creating the application settings Minimal rSD dimmest cells equals the rSD electronic noise x 2.5

Creating Application Settings

• Determine the minimum PMT for each

detector based on 2.5 x rSD of electronic noise

• Make sure there is a good use of dynamic

range

• Check that high positive cells will be within

the linear range (MFI is below the maximum linearity value).

• Save to catalogue as application settings

Baseline data

CS+T beads

Used to monitor the instruments performance and adjust voltages to maintain standardised settings over time in the same instrument. Application settings are automatically updated in a catalogue when the CS+T beads are run (daily) and can be applied to an open experiment in Diva

Alexa Fluor for 3 dates

14-Sep 19-Sep 21-Sep 27-Sep 14-Sep 19-Sep 21-Sep 27-Sep 482 484 486 488 490 492 494 496 498 PMT values CS+T Application settings

PE for 3 dates

14-Sep 19-Sep 21-Sep 27-Sep 14-Sep 19-Sep 21-Sep 27-Sep 413 414 415 416 417 418 419 420 421 422 PMT values CS+T Application Settings

CS+T AS CST gain Total Gain

FITC

PMTV PMTV

• n PR

for AS 14-Sep 496 490 Baseline 19-Sep 493 487

• 3

minus 3 21-Sep 497 491 1 plus 1 27-Sep 497 491 1 plus 1 FITC CST baseline 496 CS+T AS CST gain Total Gain

PE

PMTV PMTV

• n PR

for AS 14-Sep 418 420 1 Baseline 19-Sep 416 418

• 1

minus 2 21-Sep 419 421 2 plus 1 27-Sep 419 421 2 plus 1 PE CS+T baseline 417

Application settings

• PMT voltages saved as Application settings for

Lymph/Leuk analysis as 6 and 8 colour experiments, and

• ther applications such as FMH,PNH and stem cell

analysis have specific application settings

• Application settings are updated daily in the catalogue

when the CS+T beads are run

• Diva 6 does not automatically update Application settings in

the saved Experiment templates so this must be done manually.

• Compensation settings remain the same when App settings

are updated.

Importing App settings

• AML/MDS App settings are imported/applied every

time the experiment is run (8 colour settings).

• Lymphoma settings are updated when compensation

is run monthly and are linked to the templates.

• All other experiment and panel templates have

settings updated manually at the beginning of each month.

• Diva 7 will update the settings on templates

automatically when the CS+T beads are run (daily)

Note: When importing App settings into an experiment or panel that has been saved as a template there is an option to keep the saved compensation settings.

Tracking

Aim is to establish standardised Instrument settings that allow reproducible measurements (identical or at least highly comparable)

CS+T lot changes

• The old lot must first be run for a performance

check to obtain current voltage settings.

• The current voltages must be manually

adjusted when running the baseline for the new lot of CS+T beads.

• This is essential to maintain consistency in

the Application settings over multiple lots.

Consistency - At different times in the same instrument

• Requires reference beads
• Rainbow particles and CS+T (bright beads)

used by QEII to adjust voltages.

• CS+T bright beads can be used to monitor

the App settings.

• 8 peak rainbow beads. (Euroflow protocols)

Consistency -In different instruments in same lab or at another site

Protocol

• Beads (run 10 to 20 times then calculate

the average MFI) on Cytometer 1 to create defined scatter and MFI values

• Beads run on Cytometer 2

The voltages are adjusted to give the same scatter and MFI values and then saved as App settings in the catalogue

An example of a global worksheet when CS+T beads are acquired to create target values. The CS+T bright beads are gated in the FSC SSC plot The target value for each parameter is displayed in the statistics view showing the median. Record bead lot on the template

Monitoring

• Target for each detector based on the mean value for

20 runs with our 8 colour application settings.

• Monitor settings daily
• Excel spreadsheet with MFI for all detectors used to

monitor our 8 colour settings.

• Results for both instruments in Levey-Jennings

charts stay within 15% of MFI target.

• Euroflow Standardisation of Flow Cytometry Protocols Kalina T et al, Leukaemia2012.
• Compare the mean and CV for both instruments

FITC Sept 6- Nov 5 2012

19000 20000 21000 22000 23000 24000 25000 26000 27000 28000 Upper Limit Target Lower Limit TIM DataPoint TAM Data Point

FIXED DATA SET 15% Target 3548 Upper Limit 27203 Target 23655 Lower Limit 20107 TIM DATA 2SD Set 3211 Upper 2SD 27999 Mean 24788 Lower 2SD 21577 TAM DATA 2SD Set 1373 Upper 2SD 26131 Mean 24758 Lower 2SD 23385

FITC MFI for both instruments in Levey-Jennings charts

TIM TAM % CV % CV FSC 5.55 2.45 SSC 5.30 2.92 FITC 6.48 2.77 PE 6.95 2.94 PerCP-CY5.5 5.86 3.51 PE-Cy7 6.17 4.57 APC 2.52 1.99 APC-H7 2.79 3.91 V450 2.23 1.44 V500 2.18 1.61

Monitor the CV for each detector on both instruments

TIM TAM