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Laboratory Training for Field Epidemiologists

Specimen collection

Sample collection and shipping

May 2007


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Learning objectives

At the end of the presentation, participants should

understand the:

Procedures, preparation, processing andtransport 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 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



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



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



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



Collection

Lumbar puncture

Sterile tubes

Aseptic conditions

Trained person

Cerebrospinal fluid (CSF)



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)



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


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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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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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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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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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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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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: 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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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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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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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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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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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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Water for bacteriology


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 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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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 andanticipated time of receipt

Seek help from environmental/veterinary microbiologist


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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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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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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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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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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

Biosafety: protect others the


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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 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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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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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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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

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