Laboratory specimen: collection, safe transport and biosafety

Successful laboratory investigations

* advance planning
* collection of appropriate and adequate specimens
* labeling and documentation of laboratory specimen
* storage, packaging and transport to appropriate laboratory
* the ability of the laboratory to accurately perform the diagnostic tests
* biosafety and decontamination procedures to reduce the risk of further spread of the disease
* 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
o consultation between microbiologists, clinicians and 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)

Some tips

* Laboratory investigation should start as early as possible
* Specimens obtained early, preferably prior to antimicrobial treatment likely to yield the infective pathogen
* Before doing anything, explain the procedure to patient and relatives
* When collecting the specimen, avoid contamination
* Take a sufficient quantity of material
* Follow the appropriate precautions for safety

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 24 hours

Serum Collection
Venous blood in sterile tube
+ let clot for 30 minutes at ambient temperature
+ glass better than plastic
Handling
Place at 4-8°C for clot retraction for at least 1-2 hours
Centrifuge at 1 500 RPM for 5-10 min
+ separates serum from the clot
Transport
4-8oC if transport lasts less than 10 days
Freeze at -20°C 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

Collection
o Lumbar puncture
o Sterile tubes
o Aseptic conditions
o Trained person

Cerebrospinal fluid (CSF)

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 4-8°C (if up to 48hrs or -70°C for longer duration)

Rectal swabs
Advantage
o convenient
o adapted to small children, debilitated patients and other situations where voided stool sample not feasible

Drawbacks
o no macroscopic assessment possible
o less material available
o not recommended for viruses

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

Stool samples for viruses
Timing
o within 48 hours of onset
Sample amount
o 5-10 ml fresh stool from patients (and controls)

Methods
o fresh stool unmixed with urine in clean, dry and sterile container

Storage
o refrigerate at 4°C; do not freeze
o store at -15°C - for Ag detection, polymerase chain reaction (PCR)

Transport
o 4°C (do not freeze); dry ice for (Ag detection and PCR)

Stool samples for bacteria
Timing
o during active phase
Sample amount and size
o fresh sample and two swabs from patients, controls and carriers (if indicated)

Method
o Cary-Blair medium
o For Ag detection/PCR – no transport medium

Storage
o refrigerate at 4°C if testing within 48 hours, -70°C if longer; store at -15°C for Ag detection and PCR

Transport
o 4°C (do not freeze); dry ice for Ag, PCR detection

Stool samples for parasites
Timing
o as soon as possible after onset

Sample amount and size
o at least 3 x 5-10 ml fresh stool from patients and controls

Method
o mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative
o unpreserved samples for Ag detection and PCR

Storage
o refrigerate at 4°C; store at -15°C for Ag detection and PCR

Transport
o 4°C (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
WHO/CDS/EPR/ARO/2006.1

Nasopharyngeal aspirate
Tilt head slightly backward
Instill 1-1.5 ml of VTM /sterile normal saline into one nostril
Use aspiration mucus trap
Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril
WHO/CDS/EPR/ARO/2006.1

Sputum
Collection
Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container

o avoid saliva or postnasal discharge
o 1 ml minimum volume

Respiratory samples
Handling and Transport
All respiratory specimens except sputum are transported in appropriate media

o bacteria: Amie’s or Stuart’s transport medium
o 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
o ambient temperature for bacteria
o 4-8°C for viruses

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
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

Specimen Transport
media
Storage condition
Purpose/ Lab investigation
Transport
Pending test
Throat swab
VTM
Isolation
Isolation, serology
Stool
Isolation
Urine
Clotted blood
Whole blood
Virologic Investigations
Specimen
Transport
media
Storage condition
Purpose/ Lab investigation
Transport
Pending test
Throat swab
Amie’s or Stuart’s TM
Isolation Visualization
Bacteriologic Investigations
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

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

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

Food samples
Collect suspect food earliest
Collect aseptically - sterile tools, containers

Solid Food
o cut 100-200 grams from centre with sterile knife
o raw meat or poultry - refrigerate in a sterile plastic jar

Liquids
o shake to mix, use sterile tube
o water used for cooking -- 1-5 liters

Contact surfaces (utensils and/or equipment) for food processing
o moisten swab with sterile 0.1% peptone water or buffered distilled water; put the swab in an enrichment broth

Food samples
* Handling and transportation
o As fast as possible
o Keep perishable food at 2-8 °C
o Cool hot food rapidly - put containers under cold running water
o Pack samples to prevent spillage
o Contact the laboratory regarding method of transport and anticipated time of receipt
o Seek help from environmental/veterinary microbiologist

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

Patient’s Name/Identifier Unique ID Number
RRR-0023 001712643003
Date, Time, Place of Collection
Specimen Type
Serum
Collected by:
MDR

Glass slides for microscopy
Label slides individually
o use glass marking pencil
o ensure markings don’t interfere with staining process

Each slide should bear:
o patient name
o unique identification number
o date of collection

Some Tips
* Pre-print labels
* Permanently affix label to the specimen container.
* Glass slides for microscopy labeled individually
* One specimen – one lab request
* Each slide should bear the patient’s name, unique identifier, and date of collection
* Use line listing for multiple patients
* Original documents kept by investigation team


Case investigation form
Epidemiologist sends:
Patient information
o age (or date of birth), sex, complete address

Clinical information
o 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
o acute or convalescent specimen
o 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

Criteria for rejecting samples
Mismatch of information on the label and the request
Inappropriate transport temperature
Excessive delay in transportation
Inappropriate transport medium
o specimen received in a fixative
o dry specimen
o sample with questionable relevance

Insufficient quantity
Leakage
Reference

Most of the slides used in this presentation are 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

References

* Communicable Disease Toolkit: Iraq Crisis. Guidelines for the collection of specimens for laboratory testing. WHO, 2003
* 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

Successful laboratory investigations.ppt