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.

• 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)

• 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)

Slide from A. Boehmler

Hematopoiesis

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

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

• 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)

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.

• 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 ?

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 ?

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

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

Brocklebank AM Cytometry 2001

Sequential gating strategy

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

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.

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 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

27

Tube 2

Tube 3

Tube 4

Autostandardization

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

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

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

24h fresh

Marrow

(total)

Frozen and

washed

apheresis

Other cellular products (1)

Fresh cord

blood (total)

Thawed and

Diluted CB

(no wash)

Other cellular products (2)

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)

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 ?