BiologicalResourceCentres(BiologicalResourceCentres(BRCsBRCs))

DavidMurray

UCDClinicalResearchCentre

Overview of Talk

• What is a Biological Resource Centre ?

• Establishing a Bioresource : Key Features– Key Staff & Infrastructure

• Sample Handling– Collection– Processing– Storage– Shipping

• Bioresourcing by Application– Genomics– Transcriptomics– Proteomics

• Quality Control

• Security

• Legal Issues

What is a Biological Resource Centre (BRC) ?

• Aplacethatcollects,stores,processesanddistributesbiologicalmaterialsandthedataassociatedwiththosematerials.

• Typically,those“biologicalmaterials”arehumanbiospecimens–suchastissueorblood‐andthe“data”aretheclinicalinformationpertainingtothedonorofthatbiospecimen.

• ABRCcanalsoincludetissuesfromotheranimals,cellandbacterialcultures,andevenenvironmentalsamples

Key Features : Infrastructure

• Fridges / Freezers / Liquid N2 tanks

• Air Conditioning

• Ventilation

• Monitoring and Alarm System

• Controlled Access

• Backup Power

• 5-10% Backup Space

• Room for expansion

Key Staff

• Bioresource Centre Manager– Responsible for all operations– Controls access to resource– Supervise all staff with access to

BRC.– Training of all staff

•Research Nursing Staff

•Technicians for sampleprocessing & storage

•Data Managers

Examples of Biospecimens

• Blood– Serum, Plasma, PBMCs

• Protein, DNA, RNA– Proteomics, Sequencing, Gene Expression Analysis

• Urine– Protein, DNA, Metabolites

• Proteomics, Epigenetics, Metabolomics

• Tissue– Protein, RNA, DNA

• Proteomics, Transcriptomics

• Saliva, Hair, Nail, CSF, FNA, BAL.

Importance of Bioresources inTranslational Medicine Research

• Key resource to support truly translational studies

• Assemble large amount of material in standardisedmethod– Enables one to answer important questions pertaining to

common and rare diseases and individuals.

• Crucial for development of modern therapies.– Commercial / Innovation aspect

• Testing Hypothesis

• Biomarker Identification & Validation

• Personalised medicine & patient stratification.

• Control Biocollections

•10,000 individuals sampled from thegeneral population

•SnapShot of well being of the island.

•Control group for genetic case-controlstudies

Collection

Processing

Storage

Shipping.

Sample Handling

Sample Collection : Labeling

• Always Label samples

• Standardised approach– Biocollection ID, sample type,

date, coded personal id, barcode– Aliquot number– Storage Conditions– No donor identity

• Label quality– Withstand storage conditions ?

Sample Collection : Tissue Collection & Transport

• Surgical specimen / Biopsy

• Plan & develop SOPs with surgeons, clinical staff &pathologists

• Preservation <1 hr of excision.– Transfer to lab in closed sterile container on wet ice.– Longer distance /time : liquid N2 or dry ice.

• RNAlater solution if LN2/dry ice unavailable.– Snap freezing

• 30-60min after removal (Liquid N2 or dry ice)• No direct contact with tissue.• <0.5cm3

• Record time of collection and time of processing.

Sample Collection : Urine Collection

• Easytocollect

• SourceofDNA,Protein(incMetabolites)

• Sterilewidemouthedleakproofcontainer(>50mlcapacity)

• Storeimmediatelyoniceorrefrigerator

• Process&Store(‐80oC)within48hr.

Sample Collection : Blood Collection

• Treat at potentially infectious.

• Ideally fasting 8-12 hrs.

• EDTA, ACD (citrate), Heparin tubes

• Process within 1hr. (ACD if >1 hr)

• Heparin tubes not for proteomics, DNA Extraction,isolation lymphoblastic cell types.

• Amount ? Ethically justified.

• Transport at room temp

• Record time of collection & time of processing

Sample Collection : Blood Collection

Material Serum Plasma Plasma DNA Haematology RNA Whole blood

Vacutainer SST

PST P100

EDTA EDTA PAXgene

CPT

Tube Descriptor

Serum Separator

Tube

Plasma Separator

Tube

Proteomics Tube

EDTA Tube Full Blood Count Transcriptomics Tube

PBMC Preparation

Tube

Volume 3.5 or 5ml 8.0ml 8.5ml

3ml, 6ml or 10ml

3.0 0r 7.0ml 2.5ml 4ml or 8ml

Product Code 367956 &

367954

367964 366456 367838 &

367873 & 367525

366385

366450

762165 362781,

362782

Order of Draw 1 3 5 2 7 6 4

Local Processing

Yes Yes Yes No No Yes Yes

Transport to

Laboratory

-80C -80C -80C +4C Ambient See methods Ambient

Function Immunology&

Metabolomics (see method

for

proteomics)

Clinical

chemistry

Proteomics

DNA Full Blood Count RNA PBMCs,

DNA, High Content

Screening,

RNAi

J McPartlin, Trinity Biobank

Blood Collection & Processing

• Blood : Buffy coat– DNA Extraction

• Buffy Coat isolated & stored at-70oC indefinitely.

– RNA isolation• Within 4 hours collection.

• Blood Processing– Within 48hr– Serum / Plasma : within

2hr.– Separate before freezing.

• Proteomics nightmare!

Data Collection

• Standardised Approach

• Depends on type of study, disease and type ofspecimen.

• Examples :– Coded Personal ID– Disease or Control– Treatment Arm– Diagnosis & Date of Diagnosis– Current Age, Gender & Ethnicity– Place of Residence– Current medication / therapies– Medical History

Sample Storage

• Considerations:– Type of biospecimen– Anticipated length of storage– Biomolecules of interest– Are viable cells to be isolated ?– Application

Sample Storage

• RNAlater : Protects RNA : Buys you time

– Commercial, aqueous, non-toxic tissue storage reagent– Rapidly permeates tissues to stablise and protect cellular

RNA.– Eliminates the need to immediately process tissue

samples or to freeze samples in liquid nitrogen for laterprocessing.

– Tissue can be harvested and submerged in RNAlater forstorage for specific periods without jeopardizing thequality or quantity of RNA obtained after subsequent RNAisolation.

– Specimens, once placed in RNAlater cannot be furtherused for histomorphopathological analyses.

Sample Storage

• Cryopreservation– The recommended standard for preservation of

human biological samples for a wide range ofresearch applications.

– Cells or whole tissues are preserved by cooling to lowsub-zero temperatures, typically -80°C (freezer) or -196°C (nitrogen liquid phase)

– Halt in biological activity, including the biochemicalreactions that may damage cells and tissue bythermal stress

Cryopreservation

DNA Storage

Sample Storage : Cryopreservation

• Liquid N2 Containers– -132oC : critical temperature for

sensitive tissues, organisms and cells– Vapour phase and liquid phase

storage.– Vapour phase safer : less transmission

of infectious agents.– Level & Room monitor

• Mechanical Freezers– Variety of temperatures

• -20, -40, -70 to -80, -140 oC– Alarm at 20oC above operating

temperature– Adequate backup and standby needed

Sample Storage

• General Rules for Preservation– H&E Slides, FFPE : RT (below 27oC)– DNA : -20oC– Urine : -80oC– Whole Blood/Serum/Plasma : -80oC– Tissue in RNAlater : -80oC– Cryopreserved Tissue : Liquid N2 vapour (screwcap

cryovials)– Buffy Coat : LN2– Cells : Liquid N2

• Blood & Urine : Separate first then store

• Aliquot (Avoid repeat freeze / thawing)

Sample Shipping

• Must meet international regulations.– Labelling etc

• Human biospecimens should be considered hazardous(infectious UN class 6.2)

• Consider shipping time, distance, climate, season,method of transport, regulations, type and number ofbiospecmens and their intended use.

Sample Shipping : Packaging

1. Primary leak-proof receptacle containing the specimenpackaged with enough absorbent material to absorball fluids in case of breakage

2. Secondary packaging: a second, durable watertightpackaging to enclose and protect the primaryreceptacle.– Several primary receptacles may be placed in one

secondary packaging

3. Outer shipping packaging, cushioning material,protecting contents from outside influences while intransit.

Sample Shipping

• Use appropriate insulation & refrigerant– 8oC to -20oC use gel packs– -80oC use dry ice– -150oC use liquid nitrogen

• Enough for 24hr delay

Standards in Bioresources : Quality Control

• SOPs– Every aspect : Sample collection,

processing, storage & shipping.

• Staff Training Records– Banking samples– Safety Procedures– Emergency Response

• Infrastructure– Preventative maintenance schedule– Log books for usage & incidents– Alarm systems

• Spot checks : Random, 1%Biobank– Verification of location, labeling, data

record, type of specimen &quality/integrity of specimen.

Biobank Workflow

Bioresourcing by Application

• Genomics (DNA)– Blood

• ACD tubes preferable– DNA extraction (within 48hr)

• Filter Paper (14 yr @ rt)• Buffy coat : -70oC indefinitely

– Tissue• Process & Store (>1hr post excision)

– Freeze or Paraffin

– Storage of DNA : Aliquots at -20oC– QC : Spectroscopy : A260/A280

Bioresourcing by Application

• Transcriptomics (RNA)– Dedicated work space– Blood : Generally from PBMCs

• Most Transcriptionally Active• Isolate cells and RNA >2hr after blood

collection– Tissue

• Quickly frozen & stored at -80oC (-20oCRNAlater)

– Store RNA at -80oC, transport on dry ice.– QC : Spectroscopy & Gel Electrophoresis

– More on DNA & RNA tomorrow

Bioresourcing by Application

• Proteomics– Blood : Sodium Citrate tubes (heparin can

bind proteins)• Transport at room temperature• Process immediately• Separate components• Addition of protease inhibitors• SnapFreeze and store at -80oC

– Tissue• Process within one hour• Store -80oC or below

Bioresource IT

• Security : Must protect personal information

• Coded personal identifiers

• Defined access bioinformatics system

• Backup Regularly

• No communication to third parties of sensitivematerial (inc. E-Mails)

• Sample tracking– Log of samples : Location & Status– Sample removal / replacement

Legal Aspects

• Rights of donor must be respected– Communication regarding use of sample– Protect personal information– Disposal of sample & data if requested

• Use of samples must be ethically approved onstudy by study basis. (Control Access)

• Biobank security & access– Staff– Access to clinical data = Access to Samples ?– Valuable resource / irreplaceable

Useful Links

• ISBER Best Practices for Repositories– www.isber.org/Pubs/BestPractices.pdf

• WHO Common Minimum Standards & Protocolsfor Biological Resource Centres– http://www.iarc.fr/en/publications/pdfs-

online/wrk/wrk2/standardsBRC-1.pdf