HSC enumeration of fresh samples

Transcription

1 HSC enumeration of fresh samples Claude LEMARIÉ QC Facility Medical Director Cell Therapy Facility, Centre de Thérapie Cellulaire, Département de Biologie du Cancer, Institut Paoli-Calmettes, Comprehensive Cancer Center, & Inserm CBT-1409, Marseilles, France

2 No conflict of interest to disclose Disclosure

3 Minimal QC to perform on HSC cellular therapy products viable CD34+ count : FACT-JACIE v6 : shall be performed for apheresis (D ), before and after processing procedures that alters the final CD34+ cell count (D ). TNC count and viability : FACT-JACIE : shall be performed for all cellular products (D ). before and after processing procedures that alters the final TNC count (D ).

4 Minimal QC to perform on HSC cellular therapy products (2) Colony-Forming Cell numeration : European pharmacopeia : not necessarily on all produtcs, initialy: determine correlation betweeen CD34 and CFC counts, later on: to be done periodically and if the process changes (changes could affect CD34+ cells quality). FACT-JACIE : periodically evaluate the viability and potency of cryopreserved cellular therapy products (D9.2.2) Other counts : FACT-JACIE : shall be performed for all cellular therapy products, before and after processing procedures that alters the final cell population (D ). This includes cells that perform the actual function intended by the product and cells that may cause side effects». Microbiological controls : European pharmacopeia : where justified FACT-JACIE: post processing (D8.1.1)

5 CD34+ hematopoietic cells Frequency : 1-5% of bone marrow cells. Morphology : mononuclear cells, microscopically undiferenciable. Identification : with phenotype assay : CD34 (high), CD45 (dim), Low SSC, Low to Intermediate FSC, with functional assay: Colony Forming Cells.

6 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.

7 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 sample storage : 4 C (ISHAGE) Time from sample collection to testing : < 12h (ISHAGE)

8 Sample preparation (1) No ficoll : risk of loss of cell subsets, no need of purified subset. No permeabilisation: target antigens are at the cell surface Dilution : adapt sample concentration by dilution (ex: Stem-Kit : <30 Leuk. x 10e6/mL) (Eur. Pharmacopeia, ISHAGE) Pipetting of sample: use reverse pipetting for beads & cell suspension (ISHAGE) Duplicate (ISHAGE)

9 Sample preparation (2) Appropriate antibodies: - use class III anti-cd34 + pan anti-cd45 (Eur. Pharmacopeia, ISHAGE), - use conjugated Ab (Eur. Pharmacopeia), - include a negative control (recommendation: Eur. Pharmacopeia), - choose the brightest fluorochrome for the most weakly expressed antigen: CD34 PE (Eur. Pharmacopeia, ISHAGE): Lower MFI Higher MFI

10 Sample preparation (3) RBC lysing agent : choose lysing agent that preserve light scatter characteristics and antigen expression (NH4Cl) (Eur. Pharmacopeia, 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.

11 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 : single paltform mandatory ISHAGE and FACT-JACIE : recommendation

12 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 (impaired beads) The absolute count of CD34+ cells per microliter is calculated using the following expression: Number of CD34 cell x Beads concentration Number of beads

13 Table I. Interlaboratory CV of absolute CD34 cell numbers : Comparing beads p=0.23 p=0.06 Barnett D BJH 2000 Brocklebank AM Cytometry 2001

14 Cytometer set up PMT voltage (ISHAGE, European pharmacopeia) : 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 (ISHAGE, European pharmacopeia) : Color compensation is analyzed and adjusted. These compensation are specific for sample preparation and number/type of fluorochromes. Setting up verification (ISHAGE, European pharmacopeia) : Analyze a positive control tube to verify settings

15 Function checks : Internal Controls FACT-JACIE Standards v6: Function checks shall be performed for testing instruments prior to testing donor, recipient, or cellular therapy product samples (D ), Material: fluorospheres, Results analysis : daily comparison with target value. Internal QC : FACT-JACIE Standards v6: Where available, controls shall be used each day of testing and shown to give results within the defined range established for that material (D ), Material: stabilized blood (known target value and acceptable range), Results analysis : daily : comparison with target value : validate reagents and equipments to autorize use, monthly : comparison with pairs (if results are externalized) and follow tendancies: detect deviations.

16 Acquisition Correct threshold setting (European pharmacopeia, ISHAGE) Analyzed events: Sufficient number of total events: CD45+ (European pharmacopeia), Sufficient number of target events: 100 CD34+ (ISHAGE). Correct gating (European pharmacopeia, ISHAGE) : 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.

17 Sequential gating strategy Brocklebank AM Cytometry 2001

18 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: -Gatingstrategy : 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.

19 «State of the art» in 2012 Whitby A. Clinical Cytometry 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.

20 Use commercial systems (software + reagents) Designed to numerate CD34+ cells, following international guidelines. FACT-JACIE Manual v6: «Processing Facilities are encouraged to discontinue any use of research-grade reagents and adopt diagnostic kits.» Autostandardisation: : Automatic compensations and PMT voltages set up. Daily optical and voltage controls : fluorosphere beads. Acquisition / Analysis software Automated data aquisition and patient reporting. Reagents: ex: Stem-Kit (BC) or Stem Cell Enumeration kit (BD) : CE-IVD diagnostic kits : CD45FITC, CD34PE, 7-AAD lyse no wash, single platform methods.

21 External Quality Assessment FACT-JACIE Standards v6: D For tests performed within the Processing Facility, there shall be documentation of ongoing proficiency testing as designated by the Processing Facility Director. The results shall be reviewed by the Processing Facility Director or designee and outcomes reviewed with the staff. Expected value : unknown when sample analyzed Result analysis : done by supplier, punctual analysis and follow tendencies.

22 Item to verify for results validation (1) Before using flow cytometer : Daily optical and voltage controls conform Daily positive control between expected ranges Cytometry and handling validation (all diagnostic kits) : No errors reported by software, Gate position, Correct data input in cytometer software (sample dilution, patient weight and cellular product volume correct), 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).

23 Item to verify for results validation (2) Additional cytometry and handling validation items if available (ex: Stem-kit) : Beads quality : < 20% doublets, CD34+ count in negative control tube < 10% mean of positive tubes (Beckman Coulter specification), CD45+ difference between positive tubes (duplicates) < 20% (specification to be calculated by each facility after accuracy evaluation). HSC processing specific items : 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.

24 Analytical procedures validation Minimally manipulated HSC («Routine» processing for HSCT) : - FACT-JACIE Standards: There shall be the establishment of appropriate and validated assays and test procedures for the evaluation of cellular therapy products (D8.1.3). There shall be a process for monitoring the reliability, accuracy, precision, and performance of laboratory test procedures and instruments (D ). - National regulations : French regulation for cellular therapies (Décision du ): «validate methods and choose methods with adapted accuracy». More than minimally manipulated cells (European Regulation No 1394/2007) : - European ATMP autorisation : Perform QC with validated analytical procedures, following international (ICH Q2 R1) or national guidelines. - National regulations for hospital exemption : French regulation (Arrêté du ): «Describe validation of analytical methods».

25 ICH Q2(R1) International Conference on Harmonisation (ICH) published guidelines, to standardize technical requirements for registration of pharmaceuticals. Guideline Q2 (R1) = Validation of analytical procedures. (1) in cases where reproducibility has been performed, intermediate precision is not needed. (2) lack of specificity of one analytical procedure could be compensated by other supporting analytical procedure(s). (3) may be needed in some cases.

26 French requirements Frensh Comitee for Accreditation (COFRAC) published technical guides for ISO15189 implementation. SH GTA 04 = Technical guide for method validation. Parameters to verify for a quantitative method used following supplier manual Intra assay precision (repeatability) + Intermediate precision + Reproducibility + Accuracy + Inter assay comparison (intra laboratory) Linearity, detection and quantitation limit. Inter sample contamination Interferencies Normal values + (if possible) if necessary if necessary if necessary if necessary

27 Measure performance of analytical procedures Intermediate precision: Definition : Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. How : 2 or 3 concentration levels measured several times in different conditions. Result : CV (%). Reproducibility : Definition : precision between laboratories (collaborative studies). How : IQC ou EQC results compared to pairs. Result : Bias (% or absolute). Accuracy : Definition : expresses the closeness between the value which is accepted as a true value and the value found. How: several methods ; combine intermediate precision and reproducibility results. Result : % or absolute value (define an interval around the value found). Value found +/- accuracy includes true value in 95% of cases.

28 Measure performance of analytical procedures (optional 1/2) Quantitation limit : Definition : minimum level at which the analyte can be quantified with an acceptable accuracy. How : several methods. ex: calibration curve (several measures of successive dilutions). Result : choose the lower dilution with an acceptable CV (%). 40% CD34+ cell numeration with FACS Canto II cytometer and SCE kit 35% 30% Inaccuracy (%) 25% 20% 15% 10% 5% 0% CD34 concentration (/ µl)

29 Performance characteristics use for clinical decisions 40% CD34+ cell numeration with FACS Canto II cytometer and SCE kit 35% 30% Inaccuracy (%) 25% 20% 15% 10% 5% 0% CD34 concentration (/ µl) Quantitaion limit Thawed CB Peripheral Blood (treshold to start apheresis) Other HSC products

30 Measure performance of analytical procedures (optional 2/2) Inter assay comparison (intra laboratory) : Definition : compare values between 2 analytical procedures (if available in the laboratory). How : 1st: measure intermediate precision of each assay (used to calculate acceptable discordance limits), 2 nd : analyse several samples with both assays. Result : analyse discordances (causes and consequences) Diagramme des di di Difference Limite Uper limit sup Lower Limite limit inf

31 Acknowledgements Paoli Calmettes Institute Cell Therapy Unit: C Chabannon C Malenfant G. Bouchet J. Couquiaud J. Gaude P. Lignee B. Makni L. Regimbaud S. Ouffai I. Sielleur O. Vicari Beckman Coulter Immunotech : JY. Delovinfosse O. Jaen A. Pacheco Becton Dickinson: F. Montoya O. Munoz S. Novault