1. 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

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
3-Nov-14

4. “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

5. 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
3-Nov-14

6. 4. Schedule & Participant Recruitment
Preparation work 前期準備
基礎設施
標準作業程序
時間表
招募參與者
問卷設計
1. Infrastructure
Facilities for sample processing
Storage center
Setting up clinics
Inventory system for tracking samples
Stock management system
2.Protocol
Standard operating procedure (SOP)
Sample transfer protocol
3. Questionnaire design
ALSPAC cohort
Validated instrument in local population
4. Schedule & Participant Recruitment
Targeted area and number
Invitation letter
Follow-up calls
3-Nov-14

7. Pilot study 試驗性研究
There are always some problems you can not anticipate …
Response
Improve the participant response rate
Utility
Maximize the utility of available resources
Time
Streamline the overall procedures
3-Nov-14

8. currently 3 operating in parallel
(Aug 2013 – Oct 2014) NP
YMT
HKU
PWH
We have 4 clinics,
currently 3 operating in parallel
Blood sample processing site
Other biomaterials processing site (Lab in School of Public Health, SPH)
Storage center (Center for genomic sciences, CGS)
3-Nov-14

9. Stock inventory 物資庫存管理
3-Nov-14

10. SOP
標準作業程序
3-Nov-14

11. Principles for SOP 標準作業程序制定原則
Avoid processing approach that inherently preclude future analysis
Serve as wide a range of scientific investigation as can be anticipated
Future proofing
Consistency
Centralize and standardize processing methodology
Quality control and assurance
Ultra low temperature to ensure long term stability and integrity
Protect samples from freeze thaw degradation by storing multiple aliquots
Sample security
Cost effectiveness
Cost
3-Nov-14

12. 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
Infant diet
Parents’ sleeping habit
Socio-economic status
3-Nov-14

13. Physical assessment forms 體格檢查表
Height
Weight
Sitting height
Head circumference
Arm circumference
Waist circumference
Hip circumference
Grip strength
Blood pressure
Body composition
Pubertal stage
3-Nov-14

14. Schedule 時間表 Clinics open at Saturday & Sunday
Friday during summer holidays
Public holidays
3-Nov-14

15. Preparation work before clinics2
Registration 預約 3
Confirmation letter & stool pack
確認信&糞便樣本收集包裹 1
Invitation letter
邀請信
Preparation work before clinics 5
Reminder call
提醒備忘 4
Fieldwork documents (barcode labels, lab forms)
文書 (條形碼標籤，實驗室送檢表格)
3-Nov-14

16. Sample collection at clinics1
Anthropometric measurements
體格檢查
Height
Weight
Head, arm , waist, hip circumference
Blood pressure
Pubertal stage
Grip strength 3
Lung function test
肺功能測試 2
Blood taking 抽血
30 ml
FEV1
FVC
Sample collection at clinics 4
Sample collection
生物樣本
Stool
Urine (9 ml)
Saliva (2-5 ml)
Hair (0.15g)
Toenails (10) 5
Questionnaires 問卷
Child’s questionnaire
Parent’s questionnaire
Coupons
$200 禮券
Examination report
檢查報告
3-Nov-14

17. On site procedures - anthropometric measurements
Bio-impedance analysis (BIA)
生物電阻分析
Blood pressure 血壓
3-Nov-14

18. On site procedures - blood taking抽血
3-Nov-14

19. On site procedures - lung function
肺功能測試
3-Nov-14

20. On site procedures - sample collection
Stool sample
糞便樣本
Hair sample
頭髮樣本
3-Nov-14

21. 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.
3-Nov-14

22. Examination report
3-Nov-14

23. Sample processing and storage
Clinics:
Original samples from participants
診所：原始樣本 1
Laboratory :
Processed samples in aliquots
實驗室：處理、等分後樣本 2
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 3
3-Nov-14

24. (tubes in close contact with ice cubes/ ice packs)
Blood sample transport
Biohazard Bag c
Biohazard Bag a
1 x 5mL Clotted (Orange)
1 x 2mL Fluoride-oxalate
1 x 3mL EDTA
Department of Chemical Pathology lab form
Serum cryolabels
Department of Hematology lab form
Biohazard Bag b
4 x 3mL EDTA
Whole blood cryolabels
Blood spot general labels
Buffy coat cryolabels
Plasma cryolabels
Biohazard Bag d
1x 5mL Clotted (Red)
Chilled in ice during temporary storage
Cooler Box
PWH-2
(4ºC)
(with few ice packs)
Cooler Box
PWH-3
(<4ºC, chilled)
(tubes in close contact with ice cubes/ ice packs)
Cooler Box
PWH-1
(<25ºC)
(with few ice packs)

25. Taxi from Clinic to PWH, arrive at around 12:00pm
(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)
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)
Confirm the sets of samples given to the PWH staff
(Applies to all)
Blood analysis and storage
Further processing
Blood analysis
Blood analysis and storage

26. Saliva, urine and stool sample transport
Cooler Box (<25ºC)
(with few ice packs)
1 x saliva (microbiome) sample
Cryolabels for saliva
Biohazard Bag e(1)
Public transport from clinic to SPH-HKU
Further processing at SPH lab
1 x stool sample
Cryolabels for stool
Biohazard Bag e(2)
1 x urine sample
Cryolabels for urine
SPH: School of Public Health

27. Public transport from clinic to
Hair and toenail sample transport
Biohazard Bag f(1)
Ziplock bags
(<25ºC)
1 x hair (no roots) sample
1 x toenails sample
Public transport from clinic to
SPH-HKU
Storage
Biohazard Bag f(2)
1 x saliva (DNA kit), if applicable
SPH: School of Public Health

28. Blood sample processing
RNA
Whole blood
spot card
0.5ml
Clotted
5ml
Insulin,
hsCRP
(1ml)
Fluroride
Oxalate
2ml
EDTA
3ml
???
Hormones antibodies
DNA
Immortal cells
Buffy coat
0.2ml
+ glycerol
Hormone
Clot factor 1 2 3 4 5 6
Serum
0.8ml
Whole
blood
Plasma
0.25ml
+ trizol
0.75ml
4.8ml
red cells
+ trizol LS
0.6ml
1.2ml
buffy
coat
plasma
Slow spin
Fast spin
RFT,
LFT,
lipids
glucose
hbA1c
CBC 7 8 9

29. Barcode 條形碼 L: Laboratory sample BC : Birth Cohort 01: # of sample
13: year 2013
SE：sample type (serum)
100001: participant ID
01: # of sample
L: Laboratory sample
3-Nov-14

30. Blood sample storage …. 血液樣本 SE1 (#1) SE2 (#1) SE3 (#1) SE4 (#1) SE5
Processing
SE denotes sample type (Serum)
Each participant has 5 aliquots of serum,
0.8ml each (SE1-SE5)
Blood sample
from several
participants #1, #2…. ….
SE1
(#2)
SE2
(#2)
SE3
(#2)
SE4
(#2)
SE5
(#2)
Paste the corresponding cryolabel on the tube (vertical and not blocking the window)

31. First, scan Box A barcode
Select the inventory
Login to the lab system
Select freezer
First, scan Box A barcode
Select corresponding boxes for the sample type (Check label description in SOP or on the label)
Repeat the process for subsequent samples in the listed direction and press confirm when finish
SE1
(#2)
SE2 ….
Scan barcode on cryolabel
SE1
(#1)
SE2 ….

32. Put the boxes , which contains the samples,
back into the -80 oC freezer

33. Saliva sample collection and processing
DNA kit
DNA
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
Spit into Collection Funnel
Remove Collection Funnel
Add and mix the contents of the Preservative Ampoule with the collected saliva
Saliva
1 ml
Microbiome
Transport at 4oC
2-5ml in a pot
Step 1 Collection of
saliva (Microbiome) sample
Participant will
Let saliva collected in the mouth for at least 1minute.
Drool into the labeled collection pot (Yellow cap).
Repeat multiple times until collecting 2-5ml.
Step 2 Processing of
In laboratory,
Aliquot saliva from collection pot into 2 aliquots of saliva (1.0 ml each)
Store the 2 aliquots of saliva sample in cryostorage tubes
Store at -80 oC
唾液樣本
SPH: School of Public Health; PMB: Patrick Manson Bldg office

34. Transport and store at ~25 oC
Urine sample, hair and toenails collection and processing
Step 1 Collection of ‘mid-stream’ urine sample
In washroom, participant will
Wash and dry hands
Unscrew the lid of urine collection pot
Pass small quantity of urine into toilet
Pass urine into collection pot (Yellow cap) until about half full
Step 2 Processing of urine sample
In laboratory,
Aliquot urine from collection pot into 3 aliquots of urine (about 1.0 ml each)
Store the 3 aliquots of urine sample in cryostorage tubes
Transport and store at ~25 oC
SPH-PMB
Toenails
Chemical exposure
Hair
0.15 g
Urine
1/2 pot
1 ml
Store at -80 oC
Transport at 4oC
Microbiome
Collection of hair (Chemical exposure)
Research assistant will
Wear a pair of new gloves before collection
Cut few pieces of hair (0.15g) from each participant using a pair of scissors
Collection of toenails (Chemical exposure)
At home, participant will
Clip the toenails and place it inside the labeled nail collection bag and bring it to the clinic
尿液樣本
頭髮、趾甲樣本

35. Stool sample collection and processing
1/3 pot
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
Step 3 Processing of stool sample
Store at -80 oC
Transport at 4oC
1 ml
In laboratory,
Use a pre-sterilized fork and transfer a small lump of stool (200ul mark)
Store the 2 aliquots of stool sample in cryotubes
糞便樣本

36. “Children of 1997” 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)
What is special about our Biobank?
Sample storage condition
Fresh sample vs -80 ºC storage
Effect of reagent
Potential problems
Regular quality test
Combine processing site and storage center
Samples from each participant stored in 2 locations
Tablet for questionnaire …
Further improvements
3-Nov-14

37. Collaborators
3-Nov-14

38. 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
3-Nov-14

39. Thank you ! For further information: Dr. Mary Schooling: cms1@hku.hk
Dr. Cindy Lin:
3-Nov-14