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Module 5
Laboratory Diagnostics,
Specimen Collection,
and Biosafety Issues
1
Learning Objectives
• Prepare and maintain collection kits
• Collect and transport specimens safely and
correctly
• Manage laboratory specimens safely
• Interpret laboratory data
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Session Overview
• Specimen collection
• Specimen storage, handling, shipping
• Disposing infectious waste in the field
• Safety precautions in the laboratory
• Working with reference laboratories
• Managing laboratory data
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The Specimen
Collection Kit
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Specimen Collection Kit
• Viral transport medium (VTM)
• Preparing the specimen collection kit
• Managing the specimen collection kit
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What is Viral Transport Medium?
• Used in the collection of samples for
viral isolation and testing
• Prevents specimen from drying out
• Prevents bacteria and fungi growth
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How to choose VTM
• Can be made in a lab or purchased
• Different types of VTM:
– For collection of animal specimens
– For viral isolation
– For molecular testing
• (Do not use phosphate-based media)
• If VTM is not available, 100% ethanol can be
used for molecular testing
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Storing VTM
• Sterile collection vials
containing 2-3 ml of VTM
• Vials can be stored in a
freezer at -20 ºC until use
• Vials can be stored for
short periods of time
at 4 - 6 ºC
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Managing Media Stock
• Keep records of when the VTM was
made
• Do not use vials if the liquid becomes
cloudy
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Specimen Collection Kit
• Collection vials with
VTM
• Polyester fiber-tipped
applicators
• Sterile saline
(0,85% NaCl)
• Sputum or mucus trap
• Tongue depressors
• Specimen collection
cups or Petri dishes
• Transfer pipettes
• Secondary container
• Ice packs
• Items for blood
collection
• Personal protective
equipment
• Field collection forms
• A pen or marker for
labeling samples
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Sputum Trap
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Polyester Fiber-Tipped
Applicator
• Should be drayon,
rayon, or polyesterfiber swabs
• Do not use calcium
alginated or cotton
swabs nor ones
with wooden sticks;
they inhibit PCR
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Tongue Depressors
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15 ml Conical Centrifuge Tubes
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Specimen Collection Cups
and Petri Dishes
collection cups
petri dishes
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Transfer Pipettes
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Phlebotomy Supplies
• Tourniquet
• Disposable needles
• Vacuum tubes with
EDTA
• Plastic needle holder
• Alcohol and iodine
swabs
• Gauze
• Band-aids
• Biohazard sharps
container
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Personal Protective
Equipment
• Gloves
• Mask
• Gown
• Eye protection
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How to Manage Kits
• Store specimen collection kits in a dry,
cool place
• Store specimen collection kit where it
will be accessible after hours and on
weekends
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How to Safely And Correctly
Collect Samples
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Clinical Specimen
Collection Responsibility
• Usually a hospital staff function
• May be a rapid responder function if
hospital staff are unavailable
• Designate at least one member of the
team trained to collect specimens
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Clinical Specimen Sources
Be prepared to collect specimens
before you leave for the field
• Suspected cases
– Symptoms consistent with influenza
• Contacts
– Including people living or working with
suspected cases
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What to Collect
Preferred specimens
• Nasal swabs
• Throat swabs
• When possible: Nasopharyngeal specimens
Other specimens
• Posterior pharyngeal swabs
• Nasal washes
• Acute and convalescent serum
Collect the sample on several different days
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What to Collect
From an Ambulatory patient
• Nasal swab and
• Throat swab
• Can be collected into the same VTM
From an Intubated patient
• Lower respiratory aspirate
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When to Collect
Respiratory Specimens
• As soon as possible after symptoms begin
• Before antiviral medications are
administered
• Even if symptoms began more than one
week ago
• Collect multiple specimens on multiple days
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Respiratory Specimens
•
•
•
Nasopharyngeal aspirate or swabs
Posterior pharyngeal swabs
Nasal swabs or nasal wash
Nasopharyngeal aspirate and
nasopharyngeal swabs have greatest
sensitivity in detecting influenza virus
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Serological Samples
Paired serum samples are most useful
Acute sample
Within 7 days after symptom onset
Convalescent sample
More than 21 days after symptom onset
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Alternative Diagnostic Testing
If avian influenza is not the cause of the
illness, consider the following alternative
diagnostic testing:
– Culturing for other respiratory viruses
– SARS testing
– Rapid influenza test
– PPD for tuberculosis
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Personal Protective
Equipment
•
•
•
•
•
•
Masks (N-95 or N/P/R-100)
Gloves
Protective eye ware (goggles)
Hair covers
Boot or shoe covers
Protective clothing (gown or apron)
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How to Collect Specimens
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Field Data Collection Form
• Patient name
• Unique identification number
• Patient demographic information
• Patient’s health status
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Specimen Tracking System
Maintain a database to track:
• Identification number
• Subject information
• Specimen collection date
• Specimen collection location
• Diagnostic test results
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Nasopharyngeal Swab
1. Insert dry swab into
nostril and back to
nasopharynx
2. Leave in place for a
few seconds
3. Slowly remove
swab while slightly
rotating it
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Nasopharyngeal Swab
continued
4. Use a different swab
for the other nostril
5. Put tip of swab into
vial containing VTM,
breaking
applicator’s stick
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Nasopharyngeal Swab
Demonstration
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Posterior Pharyngeal Swab
• Ask the subject to
open his or her mouth
• Depress the tongue
• Swab the posterior
pharynx
• Avoid the tonsils
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Posterior Pharyngeal Swab
Demonstration
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Nasopharyngeal Aspirate
Collection Process
1.
2.
3.
4.
Attach mucus trap to vacuum source
Place catheter into nostril parallel to palate
Apply vacuum
Slowly remove catheter while slightly
rotating it
5. Repeat with other nostril using the same
catheter
6. After collection, flush catheter with 3 ml
VTM and return VTM to a plastic vial
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Nasopharyngeal Aspirate
Demonstration
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How to Collect Blood
1. Put tourniquet on subject
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Photos: CDC/ Jim Gathany
How to Collect Blood
2. Clean area with iodine and alcohol
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How to Collect Blood
3. Attach assemble needle, needle
holder, and collection tube
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How to Collect Blood
4. Insert needle into vein
Collect at least 2 ml blood
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How to Collect Blood
5. Remove needle and apply pressure
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How to Collect Blood
6. Dispose of needle in biohazard
sharps container
Do not reuse a needle
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Patient Care
• After blood draw,
apply light
pressure
• After removing
gauze, place a
sterile Band-Aid
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How to Label Samples
Use pre-printed barcode
labels:
– On the specimen
container
– On the field data
collection form
– On the log book
Label each specimen
with:
– Subject’s name
– Subject’s unique
identification number
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Personal Protective
Equipment
•
•
•
•
•
•
Masks (N-95 or N/P/R-100)
Gloves
Protective eye ware (goggles)
Hair covers
Boot or shoe covers
Protective clothing (gown or apron)
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Antecubital Venipuncture
(Blood Sample) Demonstration
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Specimen Storage, Handling,
and Transportation
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How to Store Specimens
For specimens in VTM:
• Transport to laboratory as soon as possible
• Store specimens at 4 °C before and during
transportation within 48 hours
• Store specimens at -70 °C beyond 48 hours
• Do not store in standard freezer – keep on ice or in
refrigerator
• Avoid freeze-thaw cycles
– Better to keep on ice for a week than to have repeat freeze
and thaw
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How to Store Specimens
For sera:
• Store specimen at 4 °C
• For both VTM specimens and sera,
avoid repeated freeze-thaw cycles
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Handling Infectious Materials
in the Field
• Always wear personal protective
equipment
• Be careful with sharp objects
• Treat all clinical samples as potentially
infected with avian influenza
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Packing Specimens for
Transportation
• Use three packaging layers
• First layer should be water tight
• Use absorbent material in all layers
• No more than 500 mL should be in the
specimen container
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Transporting Specimens from
Field to Lab
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Packing Specimens for
Transportation
• Keep specimens at 4 ºC
– Fill a cooler with ice packs or coolant
packs
– Double-bag specimens if you use dry ice
• Include an itemized list of specimens
with identification numbers and
laboratory instructions
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Transporting Specimens
• Refer to WHO guidelines for the safe
transport of infectious substances and
diagnostic specimens
• Follow local regulations on the
transportation of infectious material
• Coordinate with the laboratory
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Packaging a Specimen for
Shipment Demonstration
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Waste Disposal in the Field
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Items Requiring Disposal
• Infectious blood,
body fluids, leftover
biological samples
• Disposable needles
and syringes*
• Disposable or nonreusable gloves
• Used laboratory
supplies*
• Used disinfectants
• Disposable or nonreusable protective
clothing*
* Incineration
recommended
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Building an Incinerator
1. Cut out one end of a
220 liter drum
2. Cut out 3 half-moon
openings
3. Cut 4 side holes and
insert metal rods to
form an X
4. Cut away half of other
end of drum to form lid
5. Insert first end cut as a
waste platform
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How to Burn Waste in
An Incinerator
1.
2.
3.
4.
Place waste on platform inside of the drum
Soak waste in one liter of fuel
Light the fire carefully
Watch the fire and frequently mix the waste
with the metal bars to be sure all of the
waste is burned
5. When the fire has gone out, empty the ashes
into a pit
Note: You must replace the incinerator if large holes
burn through the sides.
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Disposal Using a Pit
• Can be used for disposal of:
– Liquids, if no isolation latrine or toilet is
available
– All other flammable items such as clothing
• Building a Pit:
– Locate pit far from foot traffic flow
– Dig 2 meters deep, wide enough to hold all
contaminated waste material
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How to Burn Waste in a Pit
1.
2.
3.
4.
5.
6.
7.
8.
Place disinfected waste in the pit
Pour fuel over waste
Carefully start fire
Observe burning process
Repeat fire if waste remains after burning
Cover ashes with soil
Bury a nearly full pit with ½ meter of soil
Dig a new pit for more waste
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Disposal Safety Precautions
• Use safe handling practices for infectious
material
• Locate disposal site on health facility
grounds, away from traffic flow and public
view
• Maintain disposal site security by roping off,
posting signs, and stationing a guard
• Do not leave unburned waste in an
incinerator or pit
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Laboratory Safety
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Storing Specimens in The Lab
• Label specimens as potentially infectious
• Store specimens in a secondary container
• Store specimens in freezer at -70 °C
– Use a temperature alarm, generator, and back-up
– If a -70 °C freezer is not available, store at 4 °C
until sample can be transported to another lab
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Laboratory-Associated Risks
• An infectious sample is aerosolized
• A worker is exposed to a needle stick
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Safe Laboratory
Specimen Handling
• Follow standard precautions
• Follow Biosafety Level 2 procedures
• Minimize formation of aerosols and
droplets
• Avoid using needles
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Laboratory Safety Basics
Biosafety Levels:
• BSL 1: Wash hands, no food or drink, door that can
be closed
• BSL 2: Good microbiological techniques, protective
clothing, biological safety cabinet
• BSL 3: BSL 2 plus directional air flow, special
clothing, and controlled access
• BSL 4: BSL 3 plus isolated building, one-piece
protective suits with respirators
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Laboratory Safety Basics
BSL 3 work practices:
• Perform procedures that may generate
aerosols or droplets in a biological safety
cabinet
• Wear gloves, gowns, eye protection, and a
mask
• Decontaminate work surfaces after use
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http://www.who.int/csr/disease/avian_influenza/guidelines/handlingspecimens/en/index.htm
Guidelines for Potential Avian
Influenza Specimens
• Use BSL 2 laboratory with BSL 3
practices for:
– Diluting specimens
– Nucleic acid extractions
– Diagnostic testing that does not involve
culturing
• Use BSL 3 laboratory with BSL 3
practices for:
– Culturing avian influenza virus
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Managing Contamination
or Accidents
Contaminated work surface:
• Use 5% bleach solution for at least 5 minutes
• Make bleach solution fresh daily
Exposed laboratory worker:
• Remove infected clothing
• Wash any exposed areas
• Give post-exposure prophylaxis according to
the established emergency procedure
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Reference Laboratories
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Reference Lab and Transporter
• The referral laboratory and the
recommended transporters vary by
country
• More specific information will be
provided here
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In-country Referral Laboratories
• Provincial / District level laboratories, if
any
• National level laboratories, if any
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WHO H5N1
Laboratory Network
All viruses from human cases of avian
influenza should be shipped to a WHO
reference laboratory for:
• Vaccine development
• Antiviral susceptibility testing
• Other activities of public health
significance
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Managing and Analyzing
Laboratory Data
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Data Management Rules
• Double check data entry accuracy
• Include unique identification numbers
• Keep subject names confidential
• Track testing dates and results
• Back up the database
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Computer Software
• Epi-info (CDC)
– Free, at http://www.cdc.gov/epiinfo/
•
•
•
•
Microsoft Excel and Access
Oracle
MySQL
Filemaker Pro
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Data Analysis Measures
Prevalence
The number of cases divided by the size of the
population
Sensitivity
The number of cases identified by the test divided by
the number of true positives which were identified by
the gold standard technique
Specificity
The number of non-cases identified by the test
divided by the number of true negatives that were
identified by the gold standard technique
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How to Present Results
•
•
•
•
Report:
Time and place of the outbreak
Prevalence of infection
Clinical information about cases
Epidemic curve
Share results with local health officials
and the WHO Global Influenza Program
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Summary
• Maintain adequately stocked specimen
collection kits and store them properly when
they are not in use.
• Nasal swabs and throat swabs are the
easiest specimens to collect;
Nasopharyngeal aspirate and
nasopharyngeal swabs have greatest
sensitivity in detecting influenza virus.
• Collect multiple specimens (respiratory and
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blood) on multiple days.
Summary
• Proper specimen storage, handling, and shipping is
vital to a successful laboratory test.
• Keep track of information on the specimens
collected in a database or logbook.
• When handling infectious materials in the laboratory
or in the field, take safety precautions.
• Properly dispose of any infectious material.
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Summary
• Most countries will be able to perform some
basic influenza identification tests on a
clinical specimen, but more specific testing
may need to be done at a higher level
national or regional laboratory.
• When you report on the progress of an
outbreak investigation, share investigation
results with local health officials and the
WHO Global Influenza Program.
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Laboratory Practice
Exercises
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Glossary
Centrifuge
A machine that uses high-speed rotation to separate
materials with different densities.
Culture
The growing of microorganisms in a nutrient-rich
medium.
Nucleic acid
A component genetic material such as DNA or RNA
found in all cells in humans, animals, bacteria, and
viruses. Every species and organism has a unique
pattern.
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Glossary
Pipette
A glass or plastic tube used to measure or transfer
small amounts of liquid.
Saline
A liquid solution made of salt and water.
Viral Transport Medium (VTM)
The preservative liquid in which specimens are
stored until they are tested.
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References and Resources
• Recommended laboratory tests to identify avian
influenza A virus in specimens from humans. World
Health Organization, June, 2005.
http://www.who.int/csr/disease/avian_influenza/guide
lines/avian_labtests2.pdf
• WHO guidelines for the collection of human
specimens for laboratory diagnosis of avian
influenza infection, 12 January 2005.
http://www.who.int/csr/disease/avian_influenza/guide
lines/humanspecimens/en/index.html
• Infection control for viral haemorrhagic fevers in the
African health care setting. WHO/EMC/ESR/98.2
Section 6: Dispose of Waste Safely
http://www.who.int/csr/resources/publications/ebola/90
WHO_EMC_ESR_98_2_EN/en/index.html