Lyophilised Cell Standards for Flow Cytometry. Richard Stebbings, National Institute for Biological Standards and Control, UK

Transcription

1 Lyophilised Cell Standards for Flow Cytometry Richard Stebbings, National Institute for Biological Standards and Control, UK

2 NIBSC Overview WHO International laboratory for the development and production of international reference standards We have produced >95% of all international biological standards over the last 50 years The UK s Official Medicines Control Laboratory for biological medicines We became part of the UK Medicines and Healthcare Products Regulatory Agency (MHRA) in 2013 International reputation for related scientific research and provision of advice

3 Standardisation Central to NIBSC s mission > 600 WHO International Standards & Reference Materials Pharmacopoeial Standards e.g. USP, EU pharmacopeia Manufacturer s Primary and Working Standards National Standards e.g. UK European IVDs iso13485 Repositories e.g. UK Stem Ce;; Bank, Influenza Resource Centre, Centre for AIDS reagents

4 Background / Rationale (1) International Standards (IS) are the highest order standards for the calibration of measurement procedures e.g. WHO IS play a critical role in the standardization, harmonisation and quality control of diagnostic procedures and patient management IS are fundamental for the regulation of IVD tests to standardize treatment approaches IS are intended to be used for calibration of secondary standards

5 Background / Rationale (2) Secondary standards defined as: Regional or national reference preparations Working reference material used routinely with different laboratories and with different platforms Defined in units traceable to the higher order IS Controls are defined as: Manufacturer/platform or laboratory specific reference preparation Not defined in units traceable to higher order IS

6 Commutability A prerequisite for the calibration of a potential candidate material is that this material will be recognized by a wide range of tests and test platforms with nearly the same efficiency. If a material shows a significant inter-method variability in concentration then it is not suitable as a reference material. Potential reference materials should be representative of the performance with patient samples validated for the assays used.

7 Background / Rationale (3) No international standard for CD34+ stem cells used clinically to reconstitute the hematopoietic system after radiation or chemotherapy for cancer therapy exists. Enumeration of CD34+ cells is required for timing of harvest and for determining that the minimum number required for successful engraftment has been collected. A CD34+ cell standard is needed for external quality assurance of this and other cell therapies.

8 Aim: Produce an IS for CD34+ cell counting US pharmacopeia expert panel on CD34+ cell counting has been formed Chair: Nicole Provost Scientific Liaison: Fouad Atouf Panel members: Amy Cohen, Ruud Hulspas, Lori Jones, Richard Stebbings, Robert Sutherland, Lili Wang, Albertus Wognum, Gregory Hale, Michael Rosu-Myles FDA liasons: Steve Bauer, Gerald Marti

9 Remit and goals Review available methods and decide on best options for CD34+ cell enumeration Determine optimal configuration for CD34+ cell reference standard NIBSC to produce a lyophilized CD34+ cell standard Define structure for a USP General Chapter on CD34+ Cell Enumeration

10 Preparation of a lyophilised CD34+ cell standard Source: Apheresis WBC from donors treated with G-CSF to mobilise their CD34+ stem cells Fresh versus cyro-preserved Stabilise: Stabilised with mild fixation Adjust CD34+ cell count to target level Fill: Re-suspend in cryopreservation buffer, fill 10,000 vials, weigh each vial and reject ± 1% wt. lyophilise using a modified 3 day cycle.

11 Optimization: Evaluation of trial material by expert panel ISHAGE gating strategy Low -10 cells/µl Hi 30 cells/µl Definitive fill Sept. 2014

12 Collaborative study plan EU: Directive 2009/120/EC, Annex I part IV Advanced Therapy Medicinal Products A reference standard, relevant and specific for the active substance and/or the finished product, shall be documented and characterised. Recruit participants for collaborative study Looking for different countries and different platforms

13 What can you expect? 6 vials of lyophilised stem cells Trucount tubes from a single batch Protocol with ISHAGE gating strategy Publication of the collaborative study 1 co-author per participant

14 What is expected of you? Reconstitute 6 vials with distilled water 2 dilutions Stain cells for CD45 and CD34 Enumerate CD34+ cell counts Return results counts, raw data files, reagent and instrument details, estimates of uncertainty e.g. pipettes

15 Are there previous studies? CCQM BAWG pilot study for CD4+ cell counting 16 laboratories 2 manuscripts completed, 3 rd in preparation WHO IS for CD4+ cell counting Report for ECBS being prepared

16 Quantification of Cells with Specific Phenotypes P102 Pilot study jointly run by the National Institute of Biological Standards and Control (NIBSC), PTB (Physikalisch-Technische Bundesanstalt) and NIST (National Institute of Standards and Technology) Under the auspices of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM)

17 Thirdly, all CD4+ T-cell enumeration technologies need to be compatible with a form of an external quality assessment programme. Decisions regarding initiation and switching treatment regimes are based primarily on CD4 counts and clinical findings. Hence, reliable CD4 results are at the front line of care for people living with HIV/AIDS. The meeting emphasized the need for laboratory monitoring, particularly of immunological parameters, CD4 counts and percentages, to support the clinical monitoring, as advocated in the public health approach to ART scale-up.

18 Why are accurate CD4+ cell counts important? Accurate measurement of CD4+ T cells is key to ensuring that patients receive the appropriate highly active anti-retroviral therapy (HAART) and prophylactic antibiotics, depending on their CD4+ cell count falling below 350 and 200 cells per microliter, respectively. An internationally recognised reference counting standard with a defined range of CD4+ cell counts for HIV/AIDS does not exist.

19 Participant analysis of CD4+ cell counts

20 Youden diagram of CD4+ cell counts 2 outliers indicated in red (participant M) Dashed boundaries corresponding to ±15% If the outliers are dis-regarded then 90% of participant results are within a limit of ±15%

21 Sources of uncertainty

22 Mean CD4+ cell counts and uncertainties weighted uncertainty of standard number of expansion consistency mean value mean value deviation measurement factor test values C sll u(c sll ) sd(c sll ) n k c 2 µl -1 µl -1 µl -1 dilution negative dilution negative extrapolated value positive The Chi-square consistency test is negative for dilution 1 and 2, indicating that the uncertainties of the mean values of individual laboratories are underestimated. sll sll i 1 i 2 Extrapolated values were calculated according to: C0 Ci 1 i 1 i i C sll i 1 C sll i 2

23 Acknowledgements Dr Luisa Saraiva, NIBSC Dr Sandrine Vessillier, NIBSC Janet Sutherland, NIBSC Dr Lili Wang, NIST Dr Joerg Neukammer, PTB Dr Helen Parkes, LGC Dr Fouad Atouf, USP Dr Nicole Provost CD34+ cells expert panel, USP