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Laboratory Training for Field Epidemiologists
Specimen collection
Sample collection and shipping
May 2007
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Laboratory Training for Field Epidemiologists
Learning objectives
At the end of the presentation, participants should
understand the:
Procedures, preparation, processing andtransport of specimens
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Laboratory Training for Field Epidemiologists
Successful laboratory
investigations
Advance planning
Collection of adequate and appropriate specimens
Sufficient documentation
Biosafety and decontamination
Correct packaging
Rapid transport
Choice of a laboratory that can accurately perform the tests
Timely communication of results
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Laboratory Training for Field Epidemiologists
Specimen collection:
key issues
Consider differential diagnoses
Decide on test(s) to be conducted
Decide on clinical samples to be collected to conduct
these tests
consultation between microbiologist, clinicians and
epidemiologist
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Laboratory Training for Field Epidemiologists
Transport medium
Allows organisms (pathogens and contaminants) to
survive
Non-nutritive - does not allow organisms to proliferate
For bacteria i.e., Cary Blair
For viruses - virus transport media (VTM)
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Laboratory Training for Field Epidemiologists
Blood for smears
Collection
Capillary blood from finger prick
make smear
fix with methanol or other fixative
Handling and transport
Transport slides within 24 hours
Do not refrigerate (can alter cell morphology)
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Blood for culturesCollection
Venous blood
infants: 0.5 2 ml
children: 2 5 ml
adults: 5 10 ml
Requires aseptic technique
Collect within 10 minutes of fever
if suspect bacterial endocarditis: 3 sets of blood culture
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Blood for cultures
Handling and Transport
Collect into bottles with infusion broth
change needle to inoculate the broth
Transport upright with cushion prevents hemolysis
Wrap tubes with absorbent cotton
Travel at ambient temperature
Store at 4oC if cant reach laboratory in 24h
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Serum
CollectionVenous blood in sterile test tube
let clot for 30 minutes at ambient temperature
glass better than plastic
Handling
Place at 4-8o
C for clot retraction for at least 1-2 hours
Centrifuge at 1 500 RPM for 5-10 min
separates serum from the clot
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Serum
Transport
4-8oC if transport lasts less than 10 days
Freeze at -20oC if storage for weeks or months
before processing and shipment to referencelaboratory
Avoid repeated freeze-thaw cycles
destroys IgM
To avoid hemolysis: do not freeze unseparated
blood
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Collection
Lumbar puncture
Sterile tubes
Aseptic conditions
Trained person
Cerebrospinal fluid (CSF)
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CSFHandling and transportation
Bacteria
preferably in trans-isolate medium,
pre-warmed to 25-37C before inoculation
OR
transport at ambient temperature (relevant pathogens do
not survive at low temperatures)
Viruses
transport at 4-8oC (if up to 48hrs or -70oC for longer
duration)
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Stool samples
Collection:
Freshly passed stool samples
avoid specimens from a bed pan
Use sterile or clean container
do not clean with disinfectant
During an outbreak - collect from 10-20 patients
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Rectal swabs
Advantage
convenient
adapted to small children, debilitated patients and
other situations where voided stool sample not feasible
Drawbacks
no macroscopic assessment possible
less material available
not recommended for viruses
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Laboratory Training for Field Epidemiologists
Stool samples for virusesTiming
within 48 hours of onset
Sample amount
5-10 ml fresh stool from patients (and controls)
Methods fresh stool unmixed with urine in clean, dry and sterile container
Storage
refrigerate at 4oC; do not freeze
store at -15oC - for Ag detection,polymerase chain reaction (PCR)
Transport
4oC (do not freeze); dry ice for (Ag detection and PCR)
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Laboratory Training for Field Epidemiologists
Stool samples for bacteriaTiming
during active phaseSample amount and size
fresh sample and two swabs from patients,
controls and carriers (if indicated)
Method
Cary-Blair medium
For Ag detection/PCR no transport medium
Storage
refrigerate at 4oC if testing within 48 hours, -70oC if longer;
store at -15oC for Ag detection and PCR
Transport
4oC (do not freeze); dry ice for Ag, PCR detection
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Laboratory Training for Field Epidemiologists
Stool samples for parasitesTiming
as soon as possible after onsetSample amount and size
at least 3 x 5-10 ml fresh stool from patients and controls
Method
mix with 10% formalin or polyvinyl chloride, 3 parts stool to
1 part preservative
unpreserved samples for Ag detection and PCR
Storage refrigerate at 4oC; store at -15oC for Ag detection and PCR
Transport
4oC (do not freeze); dry ice for antigen detection and PCR
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Laboratory Training for Field Epidemiologists
Nasopharyngeal swab
Tilt head backwards
Insert flexible fine-shafted
polyester swab into nostril andback to nasopharynx
Leave in place a few seconds
Withdraw slowly; rotatingmotion
WHO/CDS/EPR/ARO/2006.1
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Laboratory Training for Field Epidemiologists
Naso-pharyngeal aspirate
Tilt head slightly backward
Instill 1-1.5 ml of VTM /sterile
normal saline into one nostril
Use aspiration trap
Insert silicon catheter in nostril
and aspirate the secretiongently by suction in each
nostril
WHO/CDS/EPR/ARO/2006.1
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Laboratory Training for Field Epidemiologists
Sputum
Collection
Instruct patient to take a deep breath and cough up
sputum directly into a wide-mouth sterile container
avoid saliva or postnasal discharge
1 ml minimum volume
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Laboratory Training for Field Epidemiologists
Respiratory samples
Handling and Transport
All respiratory specimens except sputum are transported in
appropriate media
bacteria: Amies or Stuarts transport medium
viruses: viral transport medium (VTM)
Transport as quickly as possible to the laboratory to reduce
overgrowth by oral flora
For transit periods up to 24 hours
ambient temperature for bacteria
4-8C for viruses
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Laboratory Training for Field Epidemiologists
Collection
Biopsy relevant tissues
place in formalin for histopathology
place in transport medium for microbiological testing
place in sterile saline for isolation of viral pathogens
Post-mortem samples
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Laboratory Training for Field Epidemiologists
Post-mortem samples
Handling and transportation
Fixed specimens can be transported at ambient
temperatures
transport specimens in transport media within 24h at
ambient temperature
transport specimens in sterile saline at 4-8oC within 48h
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Laboratory Training for Field Epidemiologists
SpecimenTransport
media
Storage conditionPurpose/ Lab
investigation
Transport Pending testThroat swab VTM 2-8 0C -20 0C Isolation
NPA/ swab VTM 2-8 0C -20 0C Isolation
CSF No 2-8 0C -20 0C Isolation,
serology
Stool No 2-8 0C -20 0C Isolation
Urine No 2-8 0C -20 0C Isolation
Serum/Clotted blood
No 2-8 0C -20 0C2-8 0C
Isolation,serology
Whole blood No 2-8 0C 2-8 0C Isolation,
serology
Virologic Investigations
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Laboratory Training for Field Epidemiologists
Water for bacteriologyPreparation
Chlorinated water - add sodium thiosulphate (0.5ml of 10%
solution or a small crystal)
Tap/ pump
remove attachments
wipe, clean and flame outlet
allow to flow (at least one minute)
Water course or reservoir - collect from a depth of at least 20
cm
Dug well - do not allow the bottle to touch the sides of the well
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Laboratory Training for Field Epidemiologists
Water for bacteriology
Collection
At least 200 ml of water sample from the source
In sterile glass bottles OR autoclavable plasticbottles
tight screw capped lid
securely fitting stoppers/caps
an overhanging rim
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Laboratory Training for Field Epidemiologists
Water for bacteriology
Handling and transportation
Test the water sample within 3 hours of collection
keep at ambient temperature
If delayed:
pack sample on ice
test refrigerated sample within 24 hours
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Laboratory Training for Field Epidemiologists
Food samplesCollect suspect food earliest
Collect aseptically - sterile tools, containers
Solid Food
cut 100-200 grams from centre with sterile knife
raw meat or poultry - refrigerate in a sterile plastic jar
Liquids
shake to mix, use sterile tube
water used for cooking -- 1-5 litres
Contact surfaces (utensils and/or equipment) for food processing
moisten swab with sterile 0.1% peptone water or buffered distilled
water; put the swab in an enrichment broth
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Laboratory Training for Field Epidemiologists
Food samples
Handling and transportation
As fast as possible
Keep perishable food at 2-8oC
Cool hot food rapidly - put containers under cold running water
Pack samples to prevent spillage
Contact the laboratory regarding method of transport andanticipated time of receipt
Seek help from environmental/veterinary microbiologist
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Laboratory Training for Field Epidemiologists
Labeling specimens
Patients name
Clinical specimen
Unique ID number
(Research/Outbreak)
Specimen type
Date, time and place of collection
Name/ initials of collector
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Laboratory Training for Field Epidemiologists
Glass slides for microscopy
Label slides individually
use glass marking pencil
ensure markings dont interfere with staining process
Each slide should bear:
patient name
unique identification number
date of collection
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Laboratory Training for Field Epidemiologists
Case investigation form
Epidemiologist sends:Patient information
age (or date of birth), sex, complete address
Clinical information
date of onset of symptoms, clinical and immunizationhistory, risk factors or contact history where relevant,anti-microbial drugs taken prior to specimen collection
Laboratory information
acute or convalescent specimen
other specimens from the same patient
Line listing if large number of patients
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Laboratory Training for Field Epidemiologists
Case investigation form
Receiving laboratory records:
Date and time when specimen was received
Name and initials of the person receiving specimen
Record of specimen quality
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Laboratory Training for Field Epidemiologists
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Laboratory Training for Field Epidemiologists
Biosafety: protect the patient
Use single use equipment
Disinfect
Work in a clean, dedicated area
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Laboratory Training for Field Epidemiologists
Biosafety: protect yourself
Use personal protective equipment
disposable gloves
laboratory coats / gown
mask
protective eyewear / face shields if procedure is likely to
generate aerosols
If no sharps container: collect sharps immediately to prevent
needle-stick injury
Have first aid kit readily accessible
Do not reuse contaminated equipment
Biosafety: protect others the
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Laboratory Training for Field Epidemiologists
Biosafety: protect others, the
environment
Package samples appropriately for transport
Decontaminate spills - 10% bleach after wiping the surface clean
Disinfect working areas for future use - 1% household bleach dail
Soak contaminated non-disposable equipment/material in 1%household bleach for 5 minutes
wash in soapy water before re-use, sterilize if necessary
Place waste in leak-proof biohazard bags - ensure safe final
management of waste
Protect cleaning/decontamination personnel with protective coat,
thick rubber gloves
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Laboratory Training for Field Epidemiologists
Infection control precautionsPrecautions Use Requirements
Contact
precautions
Patients known or suspected to have
serious illnesses easily transmitted
by direct patient contact or by
contact with items in the patient's
environment
Gloves
Gown
Droplet
precautions
Barrier to stop infections spread by
large (>5 microns), moist droplets
produced by people when they
cough, sneeze or speak
Contact precautions
Well-fitting mask
Eye protection
Airborneprecautions
Patients known or suspected to haveserious illnesses transmitted by
airborne droplet nuclei
Contact precautionsDroplet precautions
N95 mask
Isolation room
(In hospital)
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Laboratory Training for Field Epidemiologists
Criteria for rejecting samplesMismatch of information on the label and the request
Inappropriate transport temperature
Excessive delay in transportation
Inappropriate transport medium
specimen received in a fixative
dry specimen
sample with questionable relevance
Insufficient quantity
Leakage
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Laboratory Training for Field Epidemiologists
WHO reference materialsGuidelines for the collection of clinical specimens during field
investigation of outbreaks, WHO, 2000
The role of laboratories and blood banks in disaster situations,
WHO publication, 2001
Sampling during avian influenza investigations (2006)
IDSR guidelines for specimen collection (2003)
Laboratory Needs for Emergency Situations (2003)
Overview of Laboratory Structure and Operational Needs forthe Iraqi Crisis (2003)
Costing for sampling materials and diagnostic reagents for the
Iraq crisis (2003)
M d l 3 S l ll ti d hi i
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Developed by the Department of Epidemic andPandemic Alert and Response of the World Health
Organization with assistance from:
European Program for InterventionEpidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
Module 3: Sample collection and shipping