Validation Criteria vs. Biology

Transcription

1 Flow Cytometry Biomarker Assayss Validation Criteria vs. Biology Jennifer Hincks 2015 Envigo 1 envigo.com

2 What is Flow Cytometry? Measure the fluorescence properties of particles (normally cells) in a stream of fluid 2

3 What do we measure? blood leukocytes ll lt Discovery Pre-clinical cell cultures Clinical + Standard immunophenotyping + Cellular activation + Rare cell populations + Receptor Occupancy lavage fluid processed tissues

4 What guidelines do we work to? No Flow specific guidelines Lee et al. Not flow specific O Hara et al. (2011) Outlines standard Validation recommendation 4

5 Standard Validation Design Test Purpose Design Acceptance criteria Intra-assay Determine precision n=3 to 6 animals n=3 replicates n=2 occasions %CV 25% Rare cell events (<5% parent population) %CV 30% Inter-analyst Deterine variability between analysts n=3 to 6 animals n=3 replicates n=2 analysts %CV 25%; %Bias between analysts 25% Rare cell events (<5% parent population) %CV 30%; %Bias between analysts 30% Generate concordance and Inter-analyser determine variability between instruments n=3 to 6 animals n=3 replicates n=2 analysers %CV 25%; %Bias between analysers 25% Rare cell events (<5% parent population) %CV 30%; %Bias between analysers 30% Inter-animal Generate a normal range of data N=10 male, n=10 female animals Range of values defined Stability Determine fresh and processed stability n=3 to 6 animals n=3 replicates Fresh T0hr Fresh and processed T24hr Fresh and processed T48hr %CV 25%; %Bias from T0hr 25% Rare cell events (<5% parent population) %CV 30%; %Bias from T0hr 30% 5

6 Simple immunophenotyping flow assay + Basic immunophenotyping in blood samples + Simple sample preparation + Abundant cell populations + Extracellular markers + Population well defined + T lymphocytes + B lymphocytes y + NK cells + Monocytes + Neutrophils 6

7 Sample preparation Aliquot samples Incubate with Antibodies Lyse and wash 3 hours Offline-data 2t to 3hours 7

8 Complex data SS S Lin No Gate SS S Lin % WBC Lymphocytes CD45-FITC FS Lin 38.6% CD2 20-PC Lymphocytes B cell 10 Tcell CD3-PC7 19.5% 65.8% in SS Li WBC Lymphocytes No Gate % Neutrophil 3.25% Monocyte CD14-ECD PE CD NK Cells % n FS Li CAL beads CD3-PC7 PE Channel

9 Large data-sets Animal ID Replicate WBC Lymphocytes T cells B cells NK cells Neutrophils Monocytes % Cells/µL % Cells/ % Cells/ Cells/ Cells/ Cells/ Cells/ % % % % µl µl µl µl µl µl Mean SD %CV Mean SD %CV Mean SD %CV

10 Definition of a clarifying criteria + What happens when not all animal/donors pass a given test? + Has the test has passed or failed? + Defined a new clarifying criteria that 2/3 of tests must pass to be considered to have passed the acceptance criteria + Increase animal number to improve confidence in the result + n=6 animals 10

11 Summary so far and more complex assays + Criteria generally appropriate for simple assay however need a few more decision making tools: + 2/3 samples passed at acceptance criteria + Simple! + But complex assays are more challenging + Complex sample preparation + Rare cells (CD34+ stem cells in blood) + Intracellular markers (Ki67, proliferation) + Intracellular markers that are rare (FoxP3, regulatory T cells) + Intracellular markers that are only expressed upon stimulation (pstat, activation) + Intracellular markers that are rare and only expressed upon stimulation 11

12 Rat Th17 and Treg assessment + Remit: + Measure proportion of Th17 and regulatory T cells (Treg) in rat PBMC (peripheral blood mononuclear cell) preparations + Challenges + PBMC preparation p challenging g with low volumes of blood + Tregs + Th17 + rare cell population + uses an intracellular marker (FoxP3) + rare cells population + uses IL-17 as a marker, which is only present upon stimulation and suppression of excretion intracellularly + Not so simple! 12

13 Sample preparation Prepare Aliquot Stimulate 4 Incubate with Lyse, wash, PBMCs samples hours antibodies fix and permeabilise 8 hours, Day 1 Incubate overnight Offline-data 2 hours, Day 2 Wash Incubate with antibodies 5 hours, Day 2 13

14 Data for complex assay Lymphocytes y T lymphocytes CD4+ T Lymphocytes SSC-A SSC-A SSC-A FSC-A CD3 FITC CD4 PE-Cy5 FoxP3 eflu uor660 Unstimulated 403% 4.03% 001% 0.01% Treg 0 Th % 0.11% IL-17a PE FoxP3 eflu uor Stimulated % 0.13% Treg Th % 2.87% IL-17a PE 14

15 Choose the relevant populations to assess + Treg values assessed from Unstimulated + Population may have changed in stimulated samples, therefore unstimulated sample is as close as possible to original sample efluor660 FoxP Unstimulated 4.03% 0.01% 95.85% 0.11% IL-17a PE + Th17 values assessed from Stimulated Stimulated + IL-17 only yproduced in stimulated cells, therefore cannot be detected in the unstimulated sample r660 FoxP3 efluor % 0.13% % 2.87% IL-17a PE 15

16 Assay performance Treg Passed all criteria except stability at 30% Th17 Generally failed. Why? 16

17 Th17 Assessment Intra-assay 1 Intra-assay 2 Animal ID Replicate Th Mean 2.41 SD CV% 40.5 Animal ID Replicate Th Mean 2.52 SD 2.2 CV% Mean Mean SD 0.51 SD CV% CV% Mean 7.1 Mean 3.41 SD SD CV% 30.8 CV%

18 Why did Th17 fail? + Rare cell event + Also subject to: + Variability in sample preparation + Robust stimulation + Acceptable permeabilisation to allow antibody to access the antigen inside the cell + Complex data, with gating set on the unstimulated sample + Are we assessing this parameter appropriately? 18

19 Used cut-point style approach + Defined a cut-point from inter-animal assessment + Cut-point defined in unstimulated samples + Any value above this has been stimulated appropriately and can be classed as a positive + Range defined in stimulated samples + Any deviation from this value on study can be considered a change from the normal range + Need higher animal number + As this study wasn t designed for a cut-point approach insufficient animals to define a tolerance + Allows us to define a positive sample and where a change from normal has been observed in sample 19

20 Simple assay variation Aliquot samples Incubate with Antibodies Lyse and wash 3 hours Offline-data 2t to 3hours 20

21 Sources of variation for simple assay Biological Aliquots Analyst Analyser Antibodies Instrumental Instrumental Instrumental Instrumental Data Data Data Data 21

22 Sources of variability Prepare Aliquot Stimulate 4 Incubate with Lyse, wash PBMCs samples hours antibodies and permeabilise 8 hours Incubate overnight Offline-data 2 hours Wash Incubate with antibodies 5 hours 22

23 Sources of variation for simple assay Biological Aliquots Analyst Analyser Antibodies 1 Antibodies 2 Stimulation Sample preparation Instrumental Instrumental Instrumental Instrumental Instrumental Instrumental Instrumental Data Data Data Data Data Data Data 23

24 Understanding The Number + In complex analyses is there so much potential variation that the traditional replicate approach not sufficient? + Understanding the biology of the system and the sources of variation is key + We should apply this knowledge to our validation design + What are important factors to consider for flow analyses? + Biological variability and sample processing + Detection of rare cell events + Number of cells counted through the instrument, e.g. LLOQ + Gating and LOD (limit of detection) from FMO (fluorescence minus one) samples or unstimulated samples 24

25 Acknowledgements + The Biomarker, Bio and Clinical Sciences Team: + Adrian Freeman + Asha Lad + Karen Cadwallader + Tom Scott + Yulia Tingle + Usha Agarwala + Yvonne Whitehead + Emma Keen + Gareth Thomas, Statistics + Caroline Gennell, GSK 25

26 The BBC Team 26