Sample collection and shipping

Specimen collection

May 2007

Laboratory Training for Field Epidemiologists

 Learning objectives
At the end of the presentation, participants should
understand the:

• Procedures, preparation, processing and

transport of specimens

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

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

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)

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)

Laboratory Training for Field Epidemiologists

 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

Laboratory Training for Field Epidemiologists

 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

Laboratory Training for Field Epidemiologists

 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

Laboratory Training for Field Epidemiologists

 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
Laboratory Training for Field Epidemiologists
 Cerebrospinal fluid (CSF)

Collection

Lumbar puncture

Sterile tubes

Aseptic conditions

Trained person

Laboratory Training for Field Epidemiologists

 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)
Laboratory Training for Field Epidemiologists
 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

Laboratory Training for Field Epidemiologists

 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

Laboratory Training for Field Epidemiologists

 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)

Laboratory Training for Field Epidemiologists

 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
Laboratory Training for Field Epidemiologists
 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
Laboratory Training for Field Epidemiologists
 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

Laboratory Training for Field Epidemiologists

 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

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 secretion
gently by suction in each
nostril
WHO/CDS/EPR/ARO/2006.1

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

Laboratory Training for Field Epidemiologists

 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

Laboratory Training for Field Epidemiologists
 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

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

Laboratory Training for Field Epidemiologists

 Virologic Investigations
Transport Storage condition
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,
Clotted blood 2-8 0C serology

Whole blood No 2-8 0C 2-8 0C Isolation,

serology

Laboratory Training for Field Epidemiologists

 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
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 plastic

bottles

• tight screw capped lid

• securely fitting stoppers/caps

• an overhanging rim

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

Laboratory Training for Field Epidemiologists

 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

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 and

anticipated time of receipt

Seek help from environmental/veterinary microbiologist

Laboratory Training for Field Epidemiologists

 Labeling specimens

Patient’s name

Clinical specimen

Unique ID number
(Research/Outbreak)

Specimen type

Date, time and place of collection

Name/ initials of collector

Laboratory Training for Field Epidemiologists

 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

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 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
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

Laboratory Training for Field Epidemiologists

Laboratory Training for Field Epidemiologists
 Biosafety: protect the patient

Use single use equipment

Disinfect

Work in a clean, dedicated area

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

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 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

Laboratory Training for Field Epidemiologists

 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)

Laboratory Training for Field Epidemiologists

 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
Laboratory Training for Field Epidemiologists
 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)
Laboratory Training for Field Epidemiologists
 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

Laboratory Training for Field Epidemiologists