The document discusses the impact of sample handling and processing on bioanalytical outcomes in clinical trials, emphasizing that proper measures from sample collection to laboratory testing are essential due to the complexity and resource demands involved. It highlights the importance of validating techniques and implementing appropriate sampling conditions to ensure accurate results. Moreover, it underscores the need for effective communication and training between bioanalytical labs and clinical sites to avoid pitfalls in the process.

1. 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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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.

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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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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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SAMPLES WITHIN CLINICAL TRIALS
 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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SETTING THE RIGHT PARAMETERS
 The relative (%) leukocyte counts of each of the divided
samples

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SETTING THE RIGHT PARAMETERS
 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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