Sample collection and shipping
Specimen collection
May 2007
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Laboratory Training for FieldEEpidemiologists
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Learning objectives
At the end of the presentation, participants should
understand the:
• Procedures, preparation, processing and
transport of specimens
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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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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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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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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 cultures
Collection
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 can’t reach laboratory in 24h
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Serum
Collection
Venous blood in sterile test tube
• let clot for 30 minutes at ambient temperature
• glass better than plastic
Handling
Place at 4-8oC 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 reference
laboratory
Avoid repeated freeze-thaw cycles
• destroys IgM
To avoid hemolysis: do not freeze unseparated
blood
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Cerebrospinal fluid (CSF)
Collection
Lumbar puncture
Sterile tubes
Aseptic conditions
Trained person
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CSF
Handling and transportation
Bacteria
• preferably in trans-isolate medium,
pre-warmed to 25-37°C 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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Stool samples for viruses
Timing
• 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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Stool samples for bacteria
Timing
• during active phase
Sample 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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Stool samples for parasites
Timing
• as soon as possible after onset
Sample 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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Throat swab
(posterior pharyngeal swab)
Hold tongue away with
tongue depressor
Locate areas of inflammation
and exudate in posterior
pharynx, tonsillar region of
throat behind uvula
Avoid swabbing soft palate;
do not touch tongue
Rub area back and forth with
cotton or Dacron swab
WHO/CDS/EPR/ARO/2006.1
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Nasopharyngeal swab
Tilt head backwards
Insert flexible fine-shafted
polyester swab into nostril and
back to nasopharynx
Leave in place a few seconds
Withdraw slowly; rotating
motion
WHO/CDS/EPR/ARO/2006.1
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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 secretion
gently by suction in each
nostril
WHO/CDS/EPR/ARO/2006.1
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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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Respiratory samples
Handling and Transport
All respiratory specimens except sputum are transported in
appropriate media
• bacteria: Amie’s or Stuart’s 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
• 4-8°C for viruses
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Post-mortem samples
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
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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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Virologic Investigations
Transport
Storage condition
media
Transport
Pending test
Purpose/ Lab
investigation
Throat 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/
No
2-8 0C
-20 0C
Isolation,
serology
Specimen
2-8 0C
Clotted blood
Whole blood
No
2-8 0C
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2-8 0C
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Isolation,
serology
Water for bacteriology
Preparation
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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Water for bacteriology
Collection
At least 200 ml of water sample from the source
In sterile glass bottles OR autoclavable plastic
bottles
•
tight screw capped lid
•
securely fitting stoppers/caps
•
an overhanging rim
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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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Food samples
Collect 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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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 and
anticipated time of receipt
Seek help from environmental/veterinary microbiologist
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Labeling specimens
Patient’s name
Clinical specimen
Unique ID number
(Research/Outbreak)
Specimen type
Date, time and place of collection
Name/ initials of collector
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Glass slides for microscopy
Label slides individually
• use glass marking pencil
• ensure markings don’t interfere with staining process
Each slide should bear:
• patient name
• unique identification number
• date of collection
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Case investigation form
Epidemiologist sends:
Patient information
• age (or date of birth), sex, complete address
Clinical information
• date of onset of symptoms, clinical and immunization
history, 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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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 FieldEEpidemiologists
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Biosafety: protect the patient
Use single use equipment
Disinfect
Work in a clean, dedicated area
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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
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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 daily
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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Infection control precautions
Precautions
Contact
precautions
Droplet
precautions
Airborne
precautions
Use
Requirements
Patients known or suspected to have
serious illnesses easily transmitted
by direct patient contact or by
contact with items in the patient's
environment
Barrier to stop infections spread by
large (>5 microns), moist droplets
produced by people when they
cough, sneeze or speak
•Gloves
•Gown
Patients known or suspected to have
serious illnesses transmitted by
airborne droplet nuclei
•Contact precautions
•Droplet precautions
• N95 mask
•Isolation room
(In hospital)
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•Contact precautions
•Well-fitting mask
•Eye protection
Criteria for rejecting samples
Mismatch 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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WHO reference materials
Guidelines 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 for
the Iraqi Crisis (2003)
Costing for sampling materials and diagnostic reagents for the
Iraq crisis (2003)
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Module 3: Sample collection and shipping
Developed by the Department of Epidemic and
Pandemic Alert and Response of the World Health
Organization with assistance from:
European Program for Intervention
Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
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