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
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.
Minimal QC to perform on
HSC cellular therapy products (2)
• 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 (3)
• 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
Hematopoiesis
Slide from A. Boehmler
Hematopoietic Stem and Progenitor Cells
Phenotype
Method of detection
Lin- CD34+(?)
CD133 +
CD38 - CD33HLA-DRlow
SRC
Stem
cell
Thy1+
LTC-IC / CAFC
Lin+/- CD34 +
Multipotent
Progenitor cells
CD38+ CD33 +
HLA-DRhigh
Thy1low
Lineage committed progenitors
Lin+ CD34Mature blood cell
CFU-GEMM
CFU-GM
CFU-G
CFU-M
BFU-E
CFU-Mk
Morphology,
expression of
lineage-specific
surface markers
Slide from A. Boehmler
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
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.
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)
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
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
Sample preparation (3)
• 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 (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.
Single or dual-platform ?
• 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
How does single-platform works ?
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
Comparing beads
Table I. Interlaboratory CV of absolute
CD34 cell numbers :
p=0.23
p=0.06
Barnett D BJH 2000
Brocklebank AM Cytometry 2001
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
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).
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.
Flow cytometric CD34+ cells
characteristics
•
•
•
•
Positive CD34 Expression
dim CD45 Expression
Low to Intermediate Forward Scatter
Low Side Scatter
Sutherland Journal of Hematotherapy 1996
Sequential gating strategy
Brocklebank AM Cytometry 2001
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.
« State of the art » in 2012
Whitby A. Clinical Cytometry 2012
•
•
•
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):
Lymphocytes
•
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.
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.
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.
Autostandardization
Tube 1
Flow set fluorospheres
Tube 2
Tube 3
Tube 4
Tube 5
20 mL
20 mL
500 mL
20 mL
CD45 FITC
20 mL
CD45 PE
7AAD
20 mL
Mix CD45 + 34
100 mL
Cytocomp cells
100 mL
100 mL
Immunotrol cells
100 mL
Stem trol
20 mL
2 ml
PBS
2 ml
2 ml
2 ml
1x NH4Cl lysing reagent
100 mL
Stem count
PMT
voltage
settings
Compensation
settings
Validation
tube
Autostandardization

Tube 2

Tube 3

Tube 4
27
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.
Stem-kit : 3 tubes
Test Tube Label
Reagents/ Ssamples
CD45-FITC/CD34-PE
45/34/7-AAD #1
45/34/7-AAD #2
20 µL
20 µL
45/CTRL/7-AAD
20 µL
CD45-FITC/CTRL-PE
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
Solution
2 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
3 flow pages: tube 1 & 2:
24h fresh apheresis
tube 3: negative control
Expected concentration:
<10 % positive tubes
Flow pages can be « customized »
« Customized » patient report
Other cellular products (1)
Frozen and
washed
apheresis
24h fresh
Marrow
(total)
Other cellular products (2)
Fresh cord
blood (total)
Thawed and
Diluted CB
(no wash)
Item to verify for results validation (1)
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 (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.
Any questions ?