Laboratory specimen: collection, safe transport and biosafety

1
Laboratory specimen collection, safe transport
and biosafety
Specimen Collection
2
Learning objectives

• At the end of the presentation participants
  should be able to
• Explain the procedures, preparation, processing
  and transport of specimens

3
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

4
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 microbiologists, clinicians
  and epidemiologists

5
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)

6
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

7
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)

8
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

9
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 24
  hours

10
Serum

• Collection
• Venous blood in sterile tube
• let clot for 30 minutes at ambient temperature
• glass better than plastic
• Handling
• Place at 4-8C for clot retraction for at least
  1-2 hours
• Centrifuge at 1 500 RPM for 5-10 min
• separates serum from the clot

11
Serum

• Transport
• 4-8oC if transport lasts less than 10 days
• Freeze at -20C 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

12
Cerebrospinal fluid (CSF)

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

13
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 4-8C (if up to 48hrs or -70C for
  longer duration)

14
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

15
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

16
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 4C do not freeze
• store at -15C - for Ag detection, polymerase
  chain reaction (PCR)
• Transport
• 4C (do not freeze) dry ice for (Ag detection
  and PCR)

17
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 4C if testing within 48 hours,
  -70C if longer store at -15C for Ag detection
  and PCR
• Transport
• 4C (do not freeze) dry ice for Ag, PCR
  detection

18
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 4C store at -15C for Ag
  detection and PCR
• Transport
• 4C (do not freeze) dry ice for antigen
  detection and PCR

19
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

20
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
21
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
22
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

23
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

24
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

25
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

26
Virologic Investigations
27
Bacteriologic Investigations
28
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

29
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

30
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

31
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 liters
• 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

32
Food samples

• Handling and transportation
• As fast as possible
• Keep perishable food at 2-8 C
• 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

33
Labeling specimens

• Patients name (or Patient Identifier)
• Unique ID number (Research/Outbreak)
• Specimen type
• Date, time and place of collection
• Name/ initials of collector

Patients Name/Identifier Unique ID
Number RRR-0023 001712643003 Date,
Time, Place of Collection Sept 22, 2006, 1515 hr,
Maka Community Hospital, Barton, Capiz,
Philippines Specimen Type Serum Collected by MDR
34
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

35
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 patients name, unique
  identifier, and date of collection
• Use line listing for multiple patients
• Original documents kept by investigation team

36
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

37
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

38

39
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

40
Reference
41
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

42
(No Transcript)