Impact of Sample Handling and Processing on Bioanalycial Outcome

IMPACT OF SAMPLE HANDLING AND
PROCESSING ON BIOANALYTICAL
OUTCOME
Haiko Pillu
Head Technical Operations
CPU Antwerpen
SAFETY & EFFICACY CLINICAL TRIAL SOLUTIONS
SGS Life Science Services Biopharm Day Seminar – Antwerp, October 29, 2015

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SGS LIFE SCIENCE SERVICES BIOPHARMA DAY – OCTOBER, 29 2015
INTRODUCTION
 Data from clinical assays (biomarkers, PK, PD, and
immunogenicity) are often key outcomes from clinical trials
 Implementing these endpoints in clinical trials is very :
 Costly
 time - and resource-consuming
Ensuring appropriate measures are taken from the sample
collection until the completion of laboratory testing is
paramount.

3
SAMPLES WITHIN CLINICAL TRIALS
Past
 Clinical studies  sample
treatments
 PK samples
 Low amount of PD samples
 Sample treatment
 1 step handling
 Amount of time per sample
= limited
 Techniques
 Centrifugation
 Aliquoting
 Freezing
 Occasional sample preparations
with larger sample
treatments/procedures
Present
 Clinical studies  larger/ expanded sample
treatments
 PK assesments
 PD assesments : more specific
treatments on sample preparation
 Sample treatments
 Multiple steps during treatments
 sample treatments up to 5h for each
sample /batch of samples
 Large amount of samples/aliquots
 up to 10 different assays on 1 time
point
 Specific conditions
 Time consuming treatment schedules
 New processes  new techniques
 Implementation new techniques
 Need for training, specific qualifications
 Running pilot studies
evolution

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Past
 Clinical studies  sample
treatments
 PK samples
 Low amount of PD samples
 Sample treatment
 1 step handling
 Amount of time per sample
= limited
 Techniques
 Centrifugation
 Aliquoting
 Freezing
 Occasional sample preparations
with larger sample
treatments/procedures
Present
 Clinical studies  larger/ expanded sample
treatments
 PK assesments
 PD assesments : more specific
treatments on sample preparation
 Sample treatments
 Multiple steps during treatments
 sample treatments up to 5h for each
sample /batch of samples
 Large amount of samples/aliquots
 up to 10 different assays on 1 time
point
 Specific conditions
 Time consuming treatment schedules
 New processes  new techniques
 Implementation new techniques
 Need for training, specific qualifications
 Running pilot studies
evolution

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 Lab manuals getting more specific/demanding
 Specific conditions/requests
 Timelines to centrifugation
 Timelines after centrifugation
 Timelines to storage
 Storage conditions
 Matrix used
 Additional handling steps
 ….
All this makes it more challeging to maintain good
sample quality and to plan resources

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WHAT’S ON THE OTHER END?
Why are all these parameters set?
How do we get to these (more complex) sample
treatments?
What are the thoughts/reasoning behind it?
How is it proven to be effective?
How will information passed by from lab to site?

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SETTING THE RIGHT
PARAMETERS
 Source :” The effects of anticoagulant choice and sample
processing time on hematologic values of juvenile
whooping cranes” (Joan Maurer,Betsy Reichenberg, Cristin Kelly,Barry K. Hartup)
 Case study describes collection blood and the outcome
with following factors
 2 anti- Coagulants used (K3 EDTA and LiHE)
 Slides made immediatly versus 4-6h delay
 Questions
 Does the anti coagulatant has an impact on results?
 Does the processing time have an impact on results?
 Is there any correlation of factors between anti-Coalgulant
and/or sample processing time?
1

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SETTING THE RIGHT PARAMETERS
 The total granulocyte concentration(heterophils and
eosinophils; H/E concentration) of each of the divided
samples

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 The relative (%) leukocyte counts of each of the divided
samples

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 In this specific case study no effect has been seen on
results depending on your anti-coagulant if an immediate
sample processing was feasible.
 However when having a time delay (sample processing) this
have an impact
Validation of techniques on specific parameters is key to
check if :
• goals are reached
• final results provided are fully “appropriate and correct” to
analysis demands and results

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VALIDATION –
TIME & RESOURCE
 Part 1 : Method Implementation and qualification
 Set up and testing of PBMC protocol for preparation and
testing
 Testing of PBMC stimulation procedure
 Testing labelling procedures
 Lysis, fixation, permeabilization buffer selection
 Acquisition and analysis templates/gating strategy
 Preliminary testing of method reproducibility
BioanalyticalLab
1month
2

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 Part 2 : In-vitro feasibility testing of the effect of
product on CD4 Th1/Th2 and Treg response
 Set-up of culture conditions and stimulation (reagents,
stimulus, duration of culture, of stimulation)
 Dose- and time effect of product (n= up to 6 subjects)
 Test in-vitro effect on both CD4 Th1/ Th2 and Treg responses
 Culture conditions in duplicate
 FACS testing in duplicate
 Data processing and statistical analysis
BioanalyticalLab
1month
VALIDATION –
TIME & RESOURCE

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 Part 3 : Validation of a FACS method for analysis of
CD4 Th1/ Th2 subset (CD3, CD4, IFN-Gamma, IL-4) and
T Regulatory (CD3, CD4, CD25, FoxP3, CD127)
 Sensitivity
 Reproducibility:
• Between replicates;
• Between runs ;
• Between analysts;
• Between donors;
• Between two FACS systems.
 Stability testing (e.g. storage of PBMC and effect of
cryopreservation, stability of reagents)
 Robustness
BioanalyticalLab
1month
VALIDATION –
TIME & RESOURCE

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 Part 4 : transition of the method to the clinical site
 Providing procedure
 Providing training
• Handling steps
• Conditions
• Go’s don’t go’s
 Running pilot study  qualification staff
• Between replicates Analyst evaluation
• Between analysts  method/training validation
 Reviewing results
 Running clinical trial
ClinicalSite
1month
VALIDATION –
TIME & RESOURCE

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VALIDATION –
TIME & RESOURCE
 Part 5 : Testing of CD4 Th1/ Th2 subset (CD3, CD4,
IFN-g, IL-4) and T Regulatory (CD3, CD4, CD25, FoxP3,
CD127) in clinical study samples
 PBMC stimulation / 230 samples
 CD4 Th1/ Th2 (CD3, CD4, IFN-Gamma, IL-4) FACS analysis/
230 samples
 T Regulatory (CD3, CD4, CD25, FoxP3, CD127) FACS
analysis/ 230 samples
BioanalyticalLab
Xmonths

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OTHER EXAMPLES
 Urine collections : adding of additional products as Tween
or BSA to avoid interference on tubes
 Determination of Cytokines : use of a non standard blood
collection tube such as Tru Culture tubes
 Sputum induction: effect of using Sputolysin during
handling on end parameters
 Use of matrix : effect of presence of binding factors on
specific compounds/ parameters
 Storage conditions : use of snapfreezing samples, storage
at -20°C or -70°C
 …

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CONCLUSION
 Choose wise on parameters such as
 Sampling tubes (anti coagulant)
 Conditions
 Sample processing times
 Methods
 …..
 Validation of techniques is crucial
 Implementation  time consuming
 Communication/training between bioanalytical lab and site
is key to :
 get good final results
 understand potential pitt falls for bioanalytical outcome

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HOW DO WE ASSIST YOU IN THIS PROCESS?
 Validation and choosing the right method is key for good
results
 How do we achieve a good technique to deliver very good
Data when running a clinical trial?
 Specialists on the bioanalytical part, Set up/validation
techniques
 Specialists on the site part  excecution
 Running pilot studies between site and the BAN lab
• Handling of samples?
• Conditions?
• Experience
• Communication flow

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PHARMACOLOGY SYNERGIES
- FROM LAB TO CLINICAL (AND VICE VERSA) -
 A Clinical Pharmacology Unit with
 GMP pharmacy
 Class II GCP laboratory
 Fahmp & mec agreement
 Mass Spectrometry & Immunoassays Experts
 4 GLP/GMP Bioanalytical Laboratories
 25 years experience
 700 methods validated
 31 LC-MS/MS
 Services for small and large molecule
testing in TK, PK and PD
 Online clinical samples dosing with CPUs
 Discovery biomarkers translated in clinical research
 Immune function testing
• Immunogenicity, flow cytometry, cytokine multiplexed ELISA
 Biopharmaceuticals - Cell characterization
 Metabolite profiling and mass balance studies (14C-labelled drug)

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Life Science Services Pillu Haiko
Head Technical Operations
SGS Belgium NV Phone: + 32 3 217 25 77
Clinical Pharmacology Unit Fax: +32 (0) 3 217 25 81
Lange Beeldekensstraat 267 E-mail : haiko.pillu@sgs.com
2060 Antwerpen
Belgium Web : www.sgs.com/lifescience
THANK YOU FOR YOUR ATTENTION
+ 41 22 739 9548
+ 1 866 SGS 5003
+ 65 637 90 111
+ 33 1 53 78 18 79
+ 1 877 677 2667
+ 33 1 41 24 87 87

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QUESTIONS ?

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