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 cant 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 25-37C 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)
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: 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
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
Specimen
Throat swab NPA/ swab CSF Stool Urine Serum/

Transport media
VTM VTM No No No No

Storage condition Transport

2-8 0C 2-8 0C 2-8 0C 2-8 0C 2-8 0C 2-8 0C

Pending test
-20 0C -20 0C -20 0C -20 0C -20 0C -20 0C

Purpose/ Lab investigation

Isolation Isolation Isolation, serology Isolation Isolation Isolation, serology Isolation, serology

Clotted blood
Whole blood No 2-8 0C

2-8 0C
2-8 0C

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
Patients 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 dont 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 Soak contaminated non-disposable equipment/material in 1% household bleach for 5 minutes

Disinfect working areas for future use - 1% household bleach daily

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 precautions

Droplet 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 Barrier to stop infections spread by large (>5 microns), moist droplets produced by people when they cough, sneeze or speak Patients known or suspected to have serious illnesses transmitted by airborne droplet nuclei

Gloves Gown

Contact precautions Well-fitting mask Eye protection Contact precautions Droplet precautions N95 mask Isolation room (In hospital)

Airborne precautions

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