Specimen Collection: Sample Collection and Shipping

Sample collection and shipping
Specimen collection
May 2007
Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
Learning objectives
At the end of the presentation, participants should
understand the:
• Procedures, preparation, processing and

transport of specimens

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

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

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)

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)

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

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

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

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
Cerebrospinal fluid (CSF)
Collection
Lumbar puncture
Sterile tubes
Aseptic conditions
Trained person

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)
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

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

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)

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
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
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

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

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

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

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

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

Post-mortem samples
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

Virologic Investigations
Storage condition
Transport Purpose/ Lab
Specimen
media Transport Pending test 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
Clotted blood 2-8 0C
Whole blood No 2-8 0C 2-8 0C 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
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

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

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

Food samples
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

Labeling specimens
Patient’s name
Clinical specimen
Unique ID number
(Research/Outbreak)
Specimen type
Date, time and place of collection
Name/ initials of collector

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

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
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

Biosafety: protect the patient
Use single use equipment
Disinfect
Work in a clean, dedicated area

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
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

Infection control precautions
Precautions Use Requirements
Contact Patients known or suspected to have •Gloves

precautions serious illnesses easily transmitted •Gown
by direct patient contact or by
contact with items in the patient's
environment
Droplet Barrier to stop infections spread by •Contact precautions
precautions large (>5 microns), moist droplets •Well-fitting mask
produced by people when they •Eye protection
cough, sneeze or speak
Airborne Patients known or suspected to have •Contact precautions

precautions serious illnesses transmitted by •Droplet precautions
airborne droplet nuclei • N95 mask
•Isolation room
(In hospital)

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
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)
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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Sample collection and shipping

Laboratory Training for FieldEEpidemiologists

• Procedures, preparation, processing and

Laboratory Training for FieldEEpidemiologists

Laboratory Training for FieldEEpidemiologists

Decide on test(s) to be conducted

Decide on clinical samples to be collected to conduct

• consultation between microbiologist, clinicians and

Laboratory Training for FieldEEpidemiologists

Allows organisms (pathogens and contaminants) to

Non-nutritive - does not allow organisms to proliferate

For bacteria – i.e., Cary Blair

For viruses - virus transport media (VTM)

Laboratory Training for FieldEEpidemiologists

Capillary blood from finger prick

• fix with methanol or other fixative

Handling and transport

Transport slides within 24 hours

Do not refrigerate (can alter cell morphology)

Laboratory Training for FieldEEpidemiologists

Requires aseptic technique

Collect within 10 minutes of fever

Laboratory Training for FieldEEpidemiologists

Wrap tubes with absorbent cotton

Travel at ambient temperature

Store at 4oC if can’t reach laboratory in 24h

Laboratory Training for FieldEEpidemiologists

Venous blood in sterile test tube

• let clot for 30 minutes at ambient temperature

• glass better than plastic

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

4-8oC if transport lasts less than 10 days

Freeze at -20oC if storage for weeks or months

Avoid repeated freeze-thaw cycles

To avoid hemolysis: do not freeze unseparated

Laboratory Training for FieldEEpidemiologists

• preferably in trans-isolate medium,

• transport at ambient temperature (relevant pathogens do

• transport at 4-8oC (if up to 48hrs or -70oC for longer

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

Laboratory Training for FieldEEpidemiologists

• adapted to small children, debilitated patients and other

• no macroscopic assessment possible

• less material available

• not recommended for viruses

Laboratory Training for FieldEEpidemiologists

Laboratory Training for FieldEEpidemiologists

Locate areas of inflammation

Avoid swabbing soft palate;

Rub area back and forth with

Laboratory Training for FieldEEpidemiologists

Tilt head backwards

Insert flexible fine-shafted

Leave in place a few seconds

Withdraw slowly; rotating

Laboratory Training for FieldEEpidemiologists

Instill 1-1.5 ml of VTM /sterile