Document

Report
Hong Kong’s “Children of 1997”
Biobank Project
「九七的兒女」出生隊列生物庫
- Sample collection, processing and storage
Cindy Lin 林詩琳
School of Public Health, The University of Hong Kong
3-Nov-14
“Children of 1997” Birth Cohort
「九七的兒女」出生隊列
8,327 infants born in April and May 1997, Hong Kong
7,154 active follow-up in Hong Kong currently
3-Nov-14
2
“Children of 1997” Birth Cohort
「九七的兒女」出生隊列
8,327 infants born in April and May 1997, Hong Kong
7,154 active follow-up in Hong Kong currently
3-Nov-14
3
“Children of 1997” Birth Cohort
「九七的兒女」出生隊列
8,327 infants born in April and May 1997, Hong Kong
7,154 active follow-up in Hong Kong currently
生物庫
3-Nov-14
4
Biobank Project
What ?
• A bank of “biomaterials” (samples of blood, urine, stool and etc.)
Why ?
•
•
•
•
Chronic disease pattern
Biomarkers
Genetics and epigenetics
Microbiome
How ?
• Preparation work
• Sample collection
• Sample processing and storage
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5
Preparation work
前期準備
基礎設施
1. Infrastructure
• Facilities for
sample processing
• Storage center
• Setting up clinics
• Inventory system
for tracking
samples
• Stock
management
system
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標準作業程序
2.Protocol
• Standard
operating
procedure (SOP)
• Sample transfer
protocol
問卷設計
3. Questionnaire
design
• ALSPAC cohort
• Validated
instrument in local
population
時間表
招募參與者
4. Schedule &
Participant
Recruitment
• Targeted area and
number
• Invitation letter
• Follow-up calls
6
Pilot study
試驗性研究
There are always some problems you can not anticipate …
Time
•Streamline the
overall procedures
Utility
•Maximize the utility of
available resources
Response
•Improve the participant
response rate
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7
(Aug 2013 – Oct 2014)
We have 4 clinics,
currently 3 operating in parallel
Blood sample
processing site
PWH
YMT
HKU
•
•
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NP
Other biomaterials processing site (Lab in School of Public Health, SPH)
Storage center (Center for genomic sciences, CGS)
8
Stock inventory
物資庫存管理
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9
SOP
標準作業程序
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10
Principles for SOP
標準作業程序制定原則
Future proofing
• Avoid processing approach that inherently preclude future analysis
• Serve as wide a range of scientific investigation as can be anticipated
Quality control and assurance
•Consistency
•Centralize and standardize processing methodology
Sample security
• Ultra low temperature to ensure long term stability and integrity
• Protect samples from freeze thaw degradation by storing multiple aliquots
Cost
• Cost effectiveness
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Questionnaires
問卷
Child’s questionnaire
• Diet
• Physical activity
• General health
• Moods and feelings
• Future development
• Microbiota
• Sleeping habit
• Respiratory health
Parents’ questionnaire
• Physical health
• Social skills
• Moods and feelings
• Infant diet
• Parents’ sleeping habit
• Socio-economic status
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Physical assessment forms
體格檢查表
•
•
•
•
•
•
•
•
•
•
•
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Height
Weight
Sitting height
Head circumference
Arm circumference
Waist circumference
Hip circumference
Grip strength
Blood pressure
Body composition
Pubertal stage
13
Schedule
時間表
Oct
PWH
Target
48
Achieved 24
Oct
Monday
Site
YMT
48
84
NP
48
55
Total
144
163
Reg
Arr
Tuesday
Site
Reg
Arr
Wednesday Site Reg
1
Arr
Thursday
2
Site
Reg
Arr
Friday Site Reg
3
Arr
Saturday
4
6
7
8
9
10
11
13
14
15
16
17
18
20
21
22
23
24
25
27
28
29
30
31
Site
PWH
YMT
NP
PWH
YMT
NP
PWH
YMT
NP
PWH
YMT
NP
Reg
6
14
10
0
15
9
6
13
12
7
12
12
Arr
3
11
8
0
12
7
5
8
10
4
7
8
Sunday Site
5
PWH
YMT
NP
12
PWH
YMT
NP
19
PWH
YMT
NP
26
PWH
YMT
NP
Reg
9
13
10
0
14
8
0
14
0
8
14
11
Arr
8
10
7
0
10
5
0
13
0
4
13
10
Clinics open at
• Saturday & Sunday
• Friday during summer holidays
• Public holidays
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14
Registration
預約
Confirmation letter & stool pack
確認信&糞便樣本收集包裹
Invitation letter
邀請信
Preparation work before clinics
Reminder call
提醒備忘
3-Nov-14
Fieldwork documents (barcode labels, lab forms)
文書 (條形碼標籤,實驗室送檢表格)
15
Anthropometric measurements
體格檢查
Blood taking
抽血
Lung function test
肺功能測試
• FEV1
• FVC
30 ml
•
•
•
•
•
•
Height
Weight
Head, arm , waist, hip circumference
Blood pressure
Pubertal stage
Grip strength
Sample collection at clinics
Examination report
檢查報告
3-Nov-14
Coupons
Questionnaires
$200 禮券
問卷
• Child’s questionnaire
• Parent’s questionnaire
Sample collection
生物樣本
•
•
•
•
•
Stool
Urine (9 ml)
Saliva (2-5 ml)
Hair (0.15g)
Toenails (10)
16
On site procedures
- anthropometric measurements
Bio-impedance analysis (BIA)
生物電阻分析
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Blood pressure
血壓
17
On site procedures
- blood taking
抽血
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18
On site procedures
- lung function
肺功能測試
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On site procedures
- sample collection
Hair sample
頭髮樣本
3-Nov-14
Stool sample
糞便樣本
20
Instructions for stool sample collection
• Place all gel packs needed in your freezer for at least 12 hours
• Use the scoop on the cap to collect stool sample into the bottle.
• Put the stool collection bottle inside the biohazard ziplock bag and seal the bag.
• Put paper cup containing your stool specimen on top of the gel pack in the cooler bag.
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Examination report
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Clinics:
Original samples from participants
診所:原始樣本
Laboratory :
Processed samples in aliquots
實驗室:處理、等分後樣本
Sample processing and storage
Storage center:
Cryotubes & cryoboxes in -80 oC freezer
存儲中心:凍存管儲存於-80度冷凍箱
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Blood sample transport
4 x 3mL EDTA
1 x 5mL Clotted
(Orange)
1 x 2mL Fluoride-oxalate
1 x 3mL EDTA
Department of
Chemical Pathology
lab form
Serum cryolabels
Biohazard
Bag a
Biohazard
Bag a
1 x 3mL EDTA
Department of
Hematology lab form
Biohazard
Bag c
Biohazard
Bag c
Whole blood cryolabels
Blood spot general labels
Buffy coat cryolabels
Plasma cryolabels
1x 5mL Clotted (Red)
Biohazard
Bag b
Biohazard
Bag b
Biohazard
Bag d
Biohazard
Bag d
Chilled in ice
during
temporary
storage
Cooler Box
PWH-1
(<25ºC)
(with few ice
packs)
Cooler Box
PWH-2
(4ºC)
(with few ice
packs)
Cooler Box
PWH-3
(<4ºC, chilled)
(tubes in close
contact with ice
cubes/ ice packs)
Taxi from Clinic to
PWH, arrive at around
12:00pm
Confirm
the sets
of
samples
given to
the PWH
staff
(Applies
to all)
(Biohazard bag a)
Deliver to
Department of
Chemical Pathology,
PWH
(Sample reception,
3/F, New Clinical
Building, PWH)
(Biohazard bag c)
Deliver to
Department of
Hematology,
PWH
(Room 34051, 1/F,
Clinical and Sciences
Building, PWH)
Blood analysis and storage
Deliver to
Department of
Pediatrics,
PWH
(Room 903, 9/F, Li Ka
Shing Medical
Sciences Building,
PWH)
Deliver to
Department of
Chemical Pathology,
PWH
(Sample reception,
3/F, New Clinical
Building, PWH)
Further processing
Blood analysis
Blood analysis and storage
Saliva, urine and stool sample transport
1 x urine sample
1 x saliva (microbiome) sample
Cryolabels for saliva
Cryolabels for urine
1 x stool sample
Cryolabels for stool
Biohazard
Bag e(1)Biohazard
Bag e(1)
Cooler Box
(<25ºC)
(with few ice
packs)
Further processing at
SPH lab
Public transport from clinic to
SPH-HKU
SPH: School of Public Health
Biohazard
Bag e(2)Biohazard
Bag e(2)
Hair and toenail sample transport
1 x hair (no roots) sample
1 x toenails sample
1 x saliva (DNA kit), if applicable
Biohazard
Biohazard
Bag f(1)
Bag f(1)
Ziplock bags
(<25ºC)
SPH: School of Public Health
Biohazard
Biohazard
Bag f(2)
Bag f(2)
Public transport from clinic to
SPH-HKU
Storage
Blood sample processing
Clotted
5ml
Clotted
5ml
Fluroride
Oxalate
2ml
EDTA
3ml
EDTA
3ml
EDTA
3ml
EDTA
3ml
EDTA
3ml
EDTA
3ml
7
8
Slow spin
9
1
5
2
3
4
6
4.8ml
red cells
RFT,
LFT,
lipids
glucose
hbA1c
1.2ml
buffy
coat
CBC
3ml
plasma
0.25ml
0.25ml
plasma
0.25ml
plasma
0.25ml
plasma
plasma
Fast spin
Buffy coat
Buffy
0.2mlcoat
0.2ml
0.25ml
0.25ml
plasma
0.25ml
plasma
0.25ml
plasma
plasma
2ml
plasma
Insulin,
hsCRP
(1ml)
Whole
Whole
blood
Whole
blood
Whole
0.5ml
blood
0.5ml
blood
0.5ml
0.5ml
Hormones
antibodies
???
Buffy coat
Buffy coat
0.2ml
0.2ml
+ glycerol
+ glycerol
0.2ml
0.2ml
Whole blood
spot card
0.5ml
DNA
Plasma
Plasma
0.25ml
Plasma
0.25ml
Plasma
+ trizol
0.25ml
+ trizol
0.25ml
0.75ml
+ trizol
0.75ml
+ trizol
0.75ml
0.75ml
Buffy coat
0.2mlcoat
Buffy
+ trizol
LS
0.2ml
0.6ml
+ trizol
Immortal
cells
RNA
Hormone
Clot factor
Barcode
條形碼
L: Laboratory sample
BC : Birth Cohort
13: year 2013
01: # of sample
SE:sample type (serum)
100001: participant ID
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Blood sample storage
….
血液樣本
SE1
(#1)
Processing
Blood sample
from several
participants #1, #2….
SE2
(#1)
SE3
(#1)
SE4
(#1)
SE5
(#1)
SE denotes sample type (Serum)
Each participant has 5 aliquots of serum,
0.8ml each (SE1-SE5)
….
SE1
(#2)
SE2
(#2)
SE3
(#2)
SE4
(#2)
SE5
(#2)
Paste the corresponding cryolabel on the tube (vertical and not blocking the window)
Select the inventory
Login to the lab system
https://labsystem.influenza.hk/
Select freezer
First, scan Box A barcode
Select corresponding
boxes for the sample type
(Check label description
in SOP or on the label)
….
SE1
(#2)
….
Scan barcode on cryolabel
SE1
(#1)
SE2
(#1)
SE2
(#2)
Repeat the process for subsequent
samples in the listed direction and press
confirm when finish
Put the boxes , which contains the samples,
back into the -80 oC freezer
Saliva sample collection and processing
Microbiome
Saliva
2-5ml
in a pot
Transport
at 4oC
Saliva
Saliva
1 ml
1 ml
Store at -80 oC
DNA
Step 1 Collection of
saliva (Microbiome) sample
Participant will
1. Let saliva collected in the mouth
for at least 1minute.
2. Drool into the labeled collection
pot (Yellow cap).
3. Repeat multiple times until
collecting 2-5ml.
Step 2 Processing of
saliva (Microbiome) sample
In laboratory,
1. Aliquot saliva from collection pot into
2 aliquots of saliva (1.0 ml each)
2. Store the 2 aliquots of saliva sample
in cryostorage tubes
SPH: School of Public Health; PMB: Patrick Manson Bldg office
Saliva
DNA kit
Transport at ~25 oC
and store at 4 oC at
SPH-PMB
(Only for those without
blood sample)
Collection of saliva (DNA) sample
Participant will
1. Spit into Collection Funnel
2. Remove Collection Funnel
3. Add and mix the contents of
the Preservative Ampoule
with the collected saliva
唾液樣本
Urine sample, hair and toenails collection and processing
Step 1 Collection of ‘mid-stream’ urine
sample
In washroom, participant will
1.Wash and dry hands
2.Unscrew the lid of urine collection
pot
3.Pass small quantity of urine into
toilet
4.Pass urine into collection pot (Yellow
cap) until about half full
Step 2 Processing of urine sample
In laboratory,
1.Aliquot urine from collection pot
into 3 aliquots of urine (about 1.0 ml
each)
2.Store the 3 aliquots of urine sample
in cryostorage tubes
Microbiome
Urine
1/2 pot
Transport
at 4oC
Urine
Urine
1 ml
Urine
1 ml
1 ml
Store at -80 oC
尿液樣本
Chemical
exposure
Chemical
exposure
Hair
0.15 g
Toenails
Transport and store at ~25 oC
SPH-PMB
Collection of hair (Chemical exposure)
Research assistant will
1. Wear a pair of new gloves before collection
2. Cut few pieces of hair (0.15g) from each
participant using a pair of scissors
Collection of toenails (Chemical exposure)
At home, participant will
1. Clip the toenails and place it inside the
labeled nail collection bag and bring it to
the clinic
頭髮、趾甲樣本
Stool sample collection and processing
Step 1 Collection of stool sample within 16-hour before the visit
Step 2 Packing of stool sample for delivery
Stool
1/3 pot
Transport at 4oC
Step 3 Processing of stool sample
Stool
1Stool
ml
Store at -80 oC
糞便樣本
Step 3 Processing of stool sample
In laboratory,
1. Use a pre-sterilized fork and transfer a small lump of stool (200ul mark)
2. Store the 2 aliquots of stool sample in cryotubes
“Children of 1997” Biobank
What is special about our Biobank?
• Based on a large, population representative Chinese cohort
• First generation of Chinese children to grow up in a high-income setting with a social
infrastructure similar to the Western countries
• Key developmental stage (16-18 years)
Potential problems
• Sample storage condition
• Fresh sample vs -80 ºC storage
• Effect of reagent
Further improvements
• Regular quality test
• Combine processing site and storage center
• Samples from each participant stored in 2 locations
• Tablet for questionnaire
•…
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Collaborators
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37
Funding sources
•
•
•
•
WYNG Foundation (for Biobank)
Health and Medical Research Fund
General Research Fund
Hong Kong Health Care and
Promotion Fund
• Health and Health Services Research
Fund
• Research Fund for the Control of
Infectious Diseases
• The University of Hong Kong,
University Research Council Strategic
Theme of Public Health
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38
Thank you !
For further information:
Dr. Mary Schooling: [email protected]
Dr. Cindy Lin: [email protected]
3-Nov-14
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