How to enumerate CD34+ cells ? What do you need to consider in … · 2018-04-04 · Issues in...
Transcript of How to enumerate CD34+ cells ? What do you need to consider in … · 2018-04-04 · Issues in...
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ENSEIGNEMENT FORMATION &
How to enumerate CD34+ cells ?
What do you need to consider in the
process environment ?
Flow Cytometry and Cell Analysis Workshop
ISCT Annual Meeting
Technical Sessions #1
23rd April 2014
C. LEMARIÉ
QC Facility Medical Director
Cell Therapy Facility, Institut Paoli-Calmettes, Regional Cancer Center, Marseilles, France
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Minimal QC to perform on
HSC cellular therapy products (1)
• CD34+ count :
European pharmacopeia : for all cellular products.
FACT-JACIE : for apheresis,
before processing,
after processing for processing procedures that affect CD34 cell content.
Post-thaw CD34 counts of cellular therapy products that are thawed outside
of the Processing Facility : no,
Post-thaw CD34 counts of cellular therapy products that are thawed inside
the Processing Facility : should be performed in the context of procedure
validation, or if prefreeze CD34 count is not known.
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• TNC count and viability :
Pharmacopeia :
TNC: for all cellular products,
Viability: for thawed cellular products or fresh cellular products infused more
than 24h after collection.
FACT-JACIE : for all cellular products,
TNC count + viabilty before processing,
+ after processing for processing procedures that affect TNC content or
viability (FACT-JACIE).
Minimal QC to perform on
HSC cellular therapy products (2)
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• Colony-Forming Cell numeration :
Pharmacopeia : not necessarily on all produtcs
To be done periodically and if the process changes (mobilisation, cell
packaging, processing …)
FACT-JACIE : 0
• Other counts
FACT-JACIE : Assay of target cell population for products that have been
enriched or depleted
Other testing may be performed at the discretion of the Processing Facility
Director
• Sterility tests :
Pharmacopeia : where justified
FACT-JACIE: post processing
Minimal QC to perform on
HSC cellular therapy products (3)
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Slide from A. Boehmler
Hematopoiesis
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SRC
CFU-GM
CFU-G
CFU-M
BFU-E
CFU-Mk
CFU-GEMM
LTC-IC / CAFC
Morphology,
expression of
lineage-specific
surface markers
Lin + CD34 -
Lin +/- CD34 +
CD38 + CD33 +
HLA-DR high
Thy1 low
Lin - CD34 +(?)
CD133 +
CD38 - CD33 -
HLA-DR low
Thy1 +
Phenotype Method of detection
Stem
cell
Multipotent
Progenitor cells
Lineage committed progenitors
Mature blood cell
Hematopoietic Stem and Progenitor Cells
Slide from A. Boehmler
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CD34+ hematopoietic cells
• Frequency :
1-5% of bone marrow cells
• Morphology :
mononuclear cells, microscopically undiferenciable
• Identification :
With phenotype assay :
CD34 (high),
CD45 (dim),
small SSC, small to medium FSC
With functional assay: Colony Forming Cells
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Issues in CD34+ cell numeration
• CD34 Ag can specificaly be used to numerate HSC and progenitor cells
• High correlation beetween blood concentration and apheresis
CD34+ cell harvest
• High correlation between number of viable CD34+ cells reinfused
and time to engraftment
• But interlaboratory CV % is > to TNC count
• To be usefull, this assay has to be well standardized.
Recommandations, ready to use reagents and software exist, are easy
to use and allow a high standardization of this assay.
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Sample handling
• Mixing of pack before sampling:
samples shall be representative of the cellular therapy product to be
evaluated (FACT-JACIE)
• Traceability:
identification of donor and sample source (FACT-JACIE)
• Temperature of storage:
4°C (ISHAGE)
• Time from collection to testing:
< 12h (ISHAGE)
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Sample preparation (1)
• No ficoll :
risk of loss of cell subsets, no need of purified subset.
• Dilution :
adapt sample concentration by dilution (Stem-Kit ® : <30 Leuk. x 10e6/mL),
use a buffer containing proteins, bead sticking inside tubes (ISHAGE)
• Pipetting of sample:
use reverse pipetting for beads & cell suspension (ISHAGE)
• Duplicate (ISHAGE)
• No permeabilisation: target antigens are at the cell surface
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Sample preparation (2)
• Appropriate antibodies:
- titrate Ab : not all antibodies perform optimally at the same concentration,
- use class III anti-CD34 + pan anti-CD45 (ISHAGE),
- use conjugated Ab (European pharmacopeia),
- include a negative control (European pharmacopeia),
- choose the brightest fluorochrome for the most weakly expressed antigen:
CD34 PE (ISHAGE):
Lower MFI Higher MFI
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• Viability mesure :
many factors may affect cell viability:
- overnight storage of product may lead to cell death,
- purging, T cell depletion or other manipulations may negatively impact
viability,
- unmanipulated cord blood and bone marrow contain a variable percentage
of dead cells.
Recommandation:
FACT-JACIE standards: mesure viability before processing,
+ after processing for processing procedures that affect viability.
How to mesure viability:
use a nucleic acid stain that does not cross the intact cell membrane (7-AAD)
Sample preparation (3)
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Sample preparation (4)
• RBC lysing agent :
choose lysing agent that preserve light scatter characteristics and antigen
expression (NH4Cl) (ISHAGE)
• No washing step (ISHAGE) :
- no interference of dilution buffer proteins,
- no Fc Gamma receptor on normal CD34+ progenitor cells,
- significant loss of some cell subsets.
• Incubation :
Volume: adapt incubation volume to optimize Ag-Ab reaction,
Time: incubation 15-30min,
Temperature: see Ab instructions.
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• Definitions:
- Dual Platform :
Percent CD34 from a flow cytometer multiplied by the leukocyte count
derived from a hematology analyzer.
- Single Platform :
Addition fluorescent beads of known concentration allows calculation of
absolute numbers of CD34+ cells directly from the flow cytometer.
• Which one may we choose ?
Increased cell death and debris present in cord blood + presence of
nucleated red blood cells requires single-paltform method
• What is mandatory?
European pharmacopeia and ISHAGE recommend single-paltform
FACT-JACIE: 0
Single or dual-platform ?
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Internal standard consits of calibrated fluorospheres
a known volume of cell suspension is added to a known quantity of beads
Cells and beads are acquired in the same time (same tube)
Count total beads Check % singulet
(old beads stick together)
The absolute count of CD34+
cells per microliter is
calculated using the following
expression:
Number of CD34 cell
x Beads concentration
Number of beads
How does single-platform works ?
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Comparing beads
Brocklebank AM Cytometry 2001
Barnett D BJH 2000
Table I. Interlaboratory CV of absolute
CD34 cell numbers :
p=0.23
p=0.06
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Cytometer set up
• PMT voltage :
Set up PMT voltage to be able to distinguish negative cells from positive
cells of moderate Ag density.
Voltages are reviewed and adjusted periodically.
• Compensation:
Color compensation is analyzed and adjusted.
These compensation are specific for sample preparation and number/type
of fluorochromes.
• Setting up verification:
Analyze a positive control tube to verify settings
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Daily Control
• Verify alignement:
the system optical alignment shall be verified, prior to testing cellular therapy
product samples, using adapted fluorospheres (FACT-JACIE).
• Positive control:
a positive control shall be used each day and shown to give results within
the defined range established for that material, to prove that the test
antibody is functional (FACT-JACIE).
New reagent lots shall be verified to provide comparable results to current
lots (FACT-JACIE).
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Acquisition
• Correct threshold setting
• Analyzed events:
Sufficient number of total events:
≥ 60 000 CD45+ (European pharmacopeia),
Sufficient number of target events:
≥ 100 CD34+ (ISHAGE).
• Correct gating:
Use sequencial gating strategy to select the population of interest and
minimise interference from debris, dead cells and mature cells which can
bind antibodies non specifically.
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Flow cytometric CD34+ cells
characteristics
• Positive CD34 Expression
• dim CD45 Expression
• Low to Intermediate Forward Scatter
• Low Side Scatter
Sutherland Journal of Hematotherapy 1996
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Brocklebank AM Cytometry 2001
Sequential gating strategy
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Which factor influence the most ? Levering W. Clinical Cytometry 2007
• Retrospective analysis of 9 years EQA in Benelux Countries
• Comparing influence of different factors, using a multivariate analysis
• Factors influencing absolute CD34 cell counts:
- Gating strategy : use of ISHAGE protocol,
- Laboratory expertise : participation to EQA or interlaboratory exchange,
- Single platform method better than dual,
- Class III CD34 Ab better than class I,
- PE conjugated CD34 Ab: better than FITC,
- Higher results were obtained using Lyse no Wash methods vs Lyse and
Wash.
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« State of the art » in 2012 Whitby A. Clinical Cytometry 2012
Lymphocytes
• 255 participants of the UK NEQAS CD34+ Stem Cell Enumeration
program (EQA)
• 83% of participants don’t use commercial kits
• 57% of participants perform correct gating (ISHAGE):
• Participants using “single platform ISHAGE incorrectly” and “dual
platform ISHAGE incorrectly” were 1.7 and 2 times more likely to fail
an EQA exercise, respectively.
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StemCXP System
(Beckman Coulter)
Designed to numerate CD34+ cells, following international guidelines.
• Software:
Automated set-up,
Automated Data Aquisition,
Automated Patient Reporting.
• Reagents:
Stem-Kit : CE-IVD Diagnostic Kit.
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CXP sofware
• Autostandardisation by CXP software :
Assay specific PMT voltages,
Color compensation settings,
Set up control with StemTROL positive control cells.
• Automated Data Acquisition and Patient Reporting.
• Daily optical and voltage controls with FLOW CHECK
and FLOW set fluorosphere beads.
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Autostandardization
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
Flow set fluorospheres 500 mL
CD45 FITC 20 mL
CD45 PE 20 mL
7AAD 20 mL 20 mL
Mix CD45 + 34 20 mL
Cytocomp cells 100 mL 100 mL 100 mL
Immunotrol cells 100 mL
Stem trol 20 mL
PBS 2 ml 2 ml 2 ml
1x NH4Cl lysing reagent 2 ml
Stem count 100 mL
PMT
voltage
settings
Compensation
settings Validation
tube
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27
Tube 2
Tube 3
Tube 4
Autostandardization
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Stem-kit reagents
• Uses CD45FITC / CD34PE
and an CD45FITC / PE conjugated isoclonic control.
• Allows viability measure using 7-AAD.
• Lyse no wash method
• Stem-Count beads : single platform method.
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Stem-kit : 3 tubes
Reagents/ Ssamples Test Tube Label
45/34/7-AAD #1 45/34/7-AAD #2 45/CTRL/7-AAD
CD45-FITC/CD34-PE 20 µL 20 µL
CD45-FITC/CTRL-PE 20 µL
Specimen 100 µL 100 µL 100 µL
7-AAD 20 µL 20 µL 20 µL
Vortex – Incubate at room temperature for 20 minutes. Protect from light
1X NH4Cl Lysing
Solution2 mL 2 mL 2 mL
Vortex – Incubate at room temperature for 10 minutes. Protect from light
Stem-Count 100 µL 100 µL 100 µL
Vortex – Wait for at least 5 minutes and no more than 1 hour on melting ice prior to acquisition. Analyze
immediately the thawed samples. Protect from light
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3 flow pages: tube 1 & 2:
24h fresh apheresis
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tube 3: negative control
Expected concentration:
<10 % positive tubes
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Flow pages can be « customized »
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« Customized » patient report
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24h fresh
Marrow
(total)
Frozen and
washed
apheresis
Other cellular products (1)
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Fresh cord
blood (total)
Thawed and
Diluted CB
(no wash)
Other cellular products (2)
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Before using flow cytometer:
• Daily optical and voltage controls conform
• Daily positive control between expected ranges
After sample analysis:
• Gate position on 3 flow pages
• Beads quality: <20% doublets
• CD34+ in tube 3 (negative control) <10% mean of tubes 1 & 2
(Beckman Coulter specification).
• CD45+ difference between tubes 1 & 2 (tests) < 20%
(specification to be calculated by each facility after accuracy evaluation).
• Difference beetween CNT numeration perform on a hematology analyzer
and CD45 numeration perform on flow cytometer < 20%
(specification to be calculated by each facility after accuracy evaluation).
Item to verify for results validation (1)
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Item to verify for results validation (2)
• Sample dilution, patient weight and cellular product volume correct
• Result correspond to an expected value for the sample type:
viability, absolute numbers, concentrations and %
• Result is coherent with other associated ones :
blood/ apheresis, before / after processing, consecutive apheresis.
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Any questions ?