Module 3 Specimen Collection (Davao)

RITM Training Manual for
Specimen
Collection, Transport
and Referral during
Infectious Disease
Outbreak Response
MODULE 3. SPECIMEN COLLECTION, HANDLING AND STORAGE
RITM Training Manual for Specimen Collection, Transport and Referral during
Infectious Disease Outbreak Response
Introduction
Specimen collection, handling and storage
the first step in the laboratory investigation of infectious diseases
1. An improperly collected, handled or stored sample may lead to false

positive or false negative results, for example:
• Contamination due to poor collection technique
• Poor specimen quality
• Wrong timing
• Insufficient quantity
• Suboptimal storage
2. Unsafe specimen collection practices may expose the health worker

and the environment to infection and harm
Objectives of Specimen Collection
Guidelines
• To standardize the procedures of specimen collection, handling
and storage;
• To ensure appropriate and high quality specimens are collected
using safe and standardized techniques and methods
• To prevent the loss of vital laboratory information due to rejection
of improperly collected, handled and stored specimens
• To ensure safety of field and laboratory health workers during
specimen collection, handling and storage
• To comply with local and international standards for quality
assurance of laboratory results
SESSION 1. General Guidelines for
Specimen Collection, Handling & Storage
Proper Specimen Collection, Handling & Storage
- characterized by safe and standard collection processes, methods and

techniques
- ensure high quality specimens for laboratory testing
Issues to consider:
• Biosafety Practices of Health Worker
• Specimen type and timing of collection
• Specimen quantity
• Specimen container considerations
• Specimen handling and Storage prior to shipment
• Documentation
Proper Specimen Collection, Handling & Storage
Health Worker Biosafety Considerations

• Ensure health worker safety first and foremost
• Practice “Universal Precautions”
• Strict aseptic techniques practiced throughout the procedure
• Hand washing before and after collection
• Appropriate PPE must be worn during collection, handling and
storage
Specimen type and timing of collection
considerations
• specimen should be representative of the infectious process

(e.g. sputum is the specimen of pneumonia and not saliva)
• suitable for the test method to be used
• It helps to know the clinical profile of the disease being
considered to guide the investigator as to the appropriate
type of specimen and timing of collection
• also important to collect the appropriate type of specimen at
the appropriate phase of the disease
Specimen quantity considerations
• specimens should be of adequate quantity for the desired tests

to be performed
• more targets or pathogens to be tested translate to more
sample volume needed for testing
• Sometimes appropriate specimen quantity
• may be difficult to achieve (e.g. infants or children, elderly)
• or the method too invasive (e.g. lumbar tap for collection of
CSF).
• important to discuss with the testing laboratory so investigator
can prioritize tests according to the most plausible pathogen
Specimen containers considerations
• If applicable, specimen should be in appropriate transport
medium (e.g. VTM for viruses, Cary Blair for bacteria )
• Liquids must be placed in a leak-proof, screw-capped
container with a capacity <1 liter
• Solids must be stored in a sift-proof container weighing
<4 kilograms
• Specimen should be placed aseptically in a sterile and/or
appropriate container
• outside of the specimen container should always be cleaned
and decontaminated
Specimen handling and Storage prior to
shipment
• Storage temperature for the specimen depends on the type of
the test that will be performed
• If the type of test requires a viable organism, then the storage
temperature should be ideal for growth of the organism
• Appropriate transport media should be used if available
organism in the specimen is required by the test procedure
• Keep the specimen from direct exposure to sun and extreme
heat
Documentation
• Complete documentation
• critical to proper specimen collection
• minimizes loss, misidentification, and other potential problems that can
affect the integrity of the outbreak investigation
• Documentation consists of labels and forms which ensure that a

specimen is identifiable and traceable
• An identifiable specimen - one that is unequivocally linked to a specific
individual with the suspected disease
• A traceable specimen - one whose location is known at all times, i.e.
under where custody is the specimen with any given time
High Quality Outbreak Specimen
• Of the type appropriate of the disease
• Suitable for the testing method
• Collected during the appropriate phase of the disease
• Of sufficient amount or volume for testing
• Placed in the proper container to prevent contamination,
deterioration, leakage and/or dessication
• Stored in optimal conditions prior to shipment to testing lab
• With complete labels and documentation for identification and
traceability
SESSION 2. Specifics on
Collection of Outbreak Specimen
Who should collect the specimens
• Epidemic Investigation and Control Team can collect specimens for
common outbreaks
• In cases where novel diseases or extremely contagious pathogens
are suspected and specialized gear, such as hazardous materials
(hazmat) suits are required, specialists must be called in
• All personnel going into the hot zone must receive proper training
and briefing on how to collect, handle, and transport the required
specimens.
• need to be trained well in safety and decontamination procedures
Which specimen to collect?
1. What is the Clinical Syndrome?
• what are the epidemiological characteristics of the
disease?
• what infections are endemic in the geographical
area?
Which specimen to collect?
2. What tests are available to identify the suspected
pathogens?
• what specimens are required for these tests?
consult the laboratory
Characteristic of the specimen (quality-consistency)
review guides and references
Who to sample?
Cases
• “typical” cases
• cases likely to carry the pathogen (e.g.
children)
• untreated patients (e.g. without antibiotics)
When to sample?
Consider timing of collection from onset of disease
• Once
• acute or convalescent phase
• depends on disease
blood culture first, then serology (typhoid)
• depends on symptoms
• fever spikes (e.g. malaria, septicaemia)
• Twice
• acute and convalescent serum
How many specimens to collect?
Ensure sufficient number of specimens
• Sampling is encouraged for outbreak cases, EXCEPT for diseases under
elimination and all suspect Emerging Infectious Diseases. These are:
• Measles
• Filaria
• Leprosy
• Malaria
• Rabies
Ensure sufficient number of specimens
• Sampling is encouraged for outbreak cases, EXCEPT for diseases under
eradication/elimination, suspect Emerging Infectious Diseases (EID) and
Vaccine Preventable Diseases (VPD). Below are the list of pathogens that
require 1 sample per case:
• For eradication: Poliovirus (AFP cases)
• For elimination: Measles, Filaria, Leprosy, Malaria and Rabies
• Other VPDs: Diphtheria, Pertussis
• EID: Ebola, Avian influenza, etc.
Repeat sampling is done for:
• to acquire proper diagnosis
• intermittent shedding
(e.g. stool microscopy for parasites, bacterial endocarditis)
• False negative due to inappropriate/ immediate sample collection
• explore chronic carriage
• acute and convalescent sera
• to determine if the patient is no longer contagious (2 consecutive negative
samples)
Example during cholera outbreak
• 10 samples, enough to confirm the outbreak
• Five samples per week during the outbreak

Session 3: Specific Collection , Handling
& Storage Guidelines per SpecimenType
Clinical samples
Transport medium
• Allows organisms (pathogens and contaminants) to
survive
• Non-nutritive - does not allow organisms to proliferate
• Media to be used is dependent on the suspected
pathogen
Examples:
• Bacteria – Cary Blair for enterics, Amies for other bacteria
• Viruses - virus transport media (VTM)
• Parasites- 10% Formalin to preserve morphology of trophozoites
Specimen Storage
Different specimens have different storage requirements
• Specimens must be stored appropriately to preserve integrity
• Viruses and bacteria are temperature sensitive
• Environmental conditions can affect viability of microorganism (may cause
inaccurate lab result)
• In any outbreak investigation, it is essential to consult with the
receiving laboratory about how to handle the most likely specimen
types before sending a team into the field.
Storage requirements
• suitable biocontainers
• transport media, when necessary
• storage facility
• portable equipment with appropriate temperature control
• safety equipment and procedure
• rapid reliable transport.
Blood smears
• Timing of collection
• Anytime of the day
• Amount to be collected
• 5-10 drops, preferably thick and thin smear
• Method of collection
• capillary blood obtained thru finger prick, for infants prick the heel
• Initial processing, Handling and Storage
• Make smear in glass slides then fix in methanol or other fixative, store in room
temperature
• Transport / Shipment conditions
• Transport within 24hours, in room temperature
• Typical disease/syndrome: Malaria

Blood cultures
• Acute stage of illness or prior to antimicrobial therapy
• For 20ml blood broth
• Adults: 2-4ml (1:5 to 1:10)
• Children: 1-2ml (1:10 to 1:20)
• Infants: 0.5-2ml
• Venipuncture and aseptic technique, e.g. 70% alcohol THEN iodine; use of
infusion broth
• Avoid hemolysis
• Initial Processing, Handling and Storage
• Place in infusion broth
• Transport upright with cushion and at room temperature within 24 hrs
• Typical disease/syndrome: Neisseria, typhoid, meningococcemia
Serum
• Virus: within 4 days to 28 days from onset of illness
• Bacteria: depending on the bacteria under suspicion but must be
prior to antimicrobial therapy
• One red or yellow top tube
• Collect aseptically by venipuncture
• If with centrifuge:
• Allow to clot for 30 minutes at room temp, then centrifuge at 1500 RPM
for 5-10minutes;
• If with no centrifuge
• Place inside refrigerator and allow clot to retract for 1-2hours
Serum
• Storage
• If yellow top tube is used: store it at ref temp (2oCto 8oC)
• If red top tube: Pipet out serum and transfer to cryovial before
storing at ref temp (2oCto 8oC)
• Transport with icepacks (2oCto 8oC)
• Typical disease/syndrome: Acute Hemorrhagic
Fever, Acute Dermatological syndrome
Cerebrospinal fluid (CSF)
• Bacteria: acute stage of illness prior to antimicrobial therapy
• Virus: within 7 to 10 days from onset of illness
• Dedicated vials for bacteriology and virology (1 to 3 ml each vial)
• Must be performed only by trained medical practitioner inside a health
facility
• Collect aseptically by lumbar puncture and transfer in a sterile container
CSF
• Initial processing, Handling and Storage
• For bacterial testing:
• Can be kept at room temperature for <24 hours
• If >24 hours, transfer to:
• Trans-isolate medium
• Blood culture bottle or agar (if test requested is culture)
• For viral testing: Store in ref temp (2oCto 8oC)
• Transport within 24hours, in room temperature for bacteria
• Transport within 24hours, in with icepacks (2oCto 8oC) for virus
• Typical disease/syndrome: Acute Neurological Syndrome

Fresh stool sample and stool swab for
bacterial test
• during active phase, prior to antimicrobial therapy
• Thumb size of fresh sample
• Use of plastic in toilet bowl to catch stool or diaper, it should be
unmixed with urine in clean, dry and sterile container
• After collection, swab the fresh stool sample with 2 swabs and place
these into a Cary Blair transport medium
Fresh stool sample and stool swab for
bacterial test
• Swabs in Cary Blair can be kept at room temperature for <24 hours
• If >24 hours, store in ref temp (2oCto 8oC)

• Submit only the swab sample inside Cary Blair (not the fresh stool)
• Transport in room temperature transit time is ≤24hrs
• If transit time is ≥ 24hrs, transport with icepacks (2oCto 8oC)
• Typical disease/syndrome: Acute Diarrhea Syndrome
Stool samples for viral test
• Within 5 days of diarrhea: for Acute Diarrhea Syndrome
• within 14 days from onset: for AFP
• Thumb size or 5-10 ml fresh stool if diarrheic
• Collect two samples at least 24 hours apart for AFP cases
• Use of plastic in toilet bowl to catch stool or diaper, it should be unmixed with urine in clean, dry and
sterile container
• Store in ref temp (2oCto 8oC)
• Typical disease/syndrome: Acute Diarrhea Syndrome, Acute Flaccid Paralysis
Stool samples for parasite detection
• Anytime of the day
• 3 samples of around 5-10 ml or adult thumb size fresh stool
• Collect in a clean stool container
• Watery and soft specimen must reach the laboratory within 1 hr from time of passage
• mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative
• Transport in sealed plastic containers at room temp.
• Typical disease/syndrome: Acute Diarrhea Syndrome and Amoebiasis
Rectal swabs for viral test
• Within 14 days after onset of illness
• Virus: one rectal swab in VTM
• Use of Dacron tip swab
• Typical disease/syndrome: Only acceptable for Hand, Foot and Mouth Disease
Throat swab (Oropharyngeal swab) for
• Timing of collection bacterial test
• Acute stage of illness prior to antimicrobial therapy
• One oropharyngeal swab (OPS) in Amie’s medium
• Use of Dacron/Rayon tip swab

• OPS in Amie’s medium stored in room temperature
• Bacteria: transport at room temp within 24 hours
• Typical disease/syndrome: Diphtheria, Acute Respiratory Syndrome

Nasopharyngeal swab for bacterial test
• as soon as suspicion arises, before antimicrobial therapy
• one to two NPS in Amies media
• Use cotton swab with calcium alginate for bacteriology
• Typical disease/syndrome: Pertussis, Acute Respiratory Syndrome
Oropharyngeal and Nasopharyngeal
•
swab
Timing of collection
for viral test
• Within 7 days from onset of illness
• One Nasopharyngeal and one Oropharyngeal swab combined into one VTM (to increase viral yield)
• Use of Dacron/ Rayon tip swab
• Typical disease/syndrome: Acute Respiratory Syndrome, Measles
Sputum
• Within 7 days from onset of illness and prior to antimicrobial therapy
• Sputum: 1 tablespoon or approximately 5-15 ml
• Sputum: ask patient to take deep breath 3 times then cough out sputum into a wide-
mouth sterile container
• If to be transported immediately: Room temp
• If >24 hours, store in ref temp (2oC to 8oC).
• Virus: transport with icepacks (2oC to 8oC )
• Bacteria: transport at room temp within 24 hours
• Typical disease/ syndrome: MERS-CoV, Acute Respiratory Syndrome
Nasopharyngeal/ Endotracheal aspirate
• Within 7 days from onset of illness and prior to antimicrobial therapy
• Around 2mL
• Must be performed by trained medical practitioner inside health facility
• Use normal saline for irrigation and aspiration trap for collection
• Typical disease/ syndrome: Acute Respiratory Syndrome
Respiratory samples- Summary
Handling and Transport
All respiratory specimens except sputum are transported in appropriate media
• bacteria: Amie’s transport medium
• viruses: viral transport medium (VTM) or Universal TM (UTM). UTM commercially
available, longer expiry date, flexible storage temp, pre-positioned in the region
Transport as quickly as possible to the laboratory to reduce overgrowth by oral flora,
i.e. sputum samples
For transit periods up to 24 hours
• room temperature for bacteria
• With icepacks (2°Cto 8°C) for viruses
Urine
• From 2 weeks to one month from the onset of illness
• 5 to 10 ml
• If without catheter: Collect first morning midstream urine in sterile wide mouth container
• If with catheter: Open catheter bag valve and collect midstream urine in sterile wide mouth container
• Store in ref. temp (2oC to 8oC )
• Transport with icepacks (2oC to 8oC )
• Typical disease/ syndrome: Zika, Leptospirosis
Vomitus
• As soon as vomit is expelled
• Have patient vomit in a sterile collection pan
• Transfer sample to a sterile, wide mouth, leak proof, plastic container
• Store in ref. temp (2oC to 8oC )
• Typical disease/ syndrome: Food poisoning
Post-mortem samples
• Collection of tissues and/or bodily fluids:
• Must be done by pathologist/ trained medical professional
• Perform as soon as possible after obtaining consent
• For rabies:
• Must be done by trained medical professional (Kindly consult your local
rabies coordinator
Water sample
Water testing
Preparation
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 testing
• Collection
• Collect separate sample for bacterial and viral testing
• In sterile glass bottles or autoclavable plastic bottles, collect at least 250
ml of water sample from the source for each test type
• Clean, emptied, commercially available distilled bottled water can be used as
container
• Container must have:
• tight screw capped lid
• securely fitting stoppers/caps
• an overhanging rim
Water testing
Handling and transportation

• If to be tested within 3 hours: keep at room temp
• If delayed is expected: refrigerated sample (2oC to 8oC )
Food samples
Food samples
• Identify type of food product to be tested
• For Processed food: Coordinate with FDA for testing
• For uncooked meat: Coordinate with NMIS for testing
• For fish/shellfish: Coordinate with BFAR for testing
• Collect suspect food as soon as possible and submit to corresponding agency
• Collect aseptically – use sterile tools and 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 and pour about 250ml of the sample into a sterile container
Food samples
• Handling and transportation
• Transport as soon as possible
• Keep perishable food at 2 to 8oC
• Cool hot food rapidly:
• put containers under cold running water
• Pack samples properly to prevent spillage
• Contact testing lab regarding method of transport and anticipated time of arrival
Other samples
Contact surfaces
Examples:
• Hands
• Utensils/kitchen tools for food processing
• Hospital surfaces (for nosocomial infections investigation)
• Collection:
• moisten swab with sterile distilled water or sterile NSS
• Put the swab in an enrichment broth
• Can be kept at room temperature for <24 hours
• If >24 hours, store in ref temp (2oCto 8oC)
• Transport in room temperature transit time is ≤24hrs
• If transit time is ≥ 24hrs, transport with icepacks (2oCto 8oC)
Animal samples
Coordinate with appropriate Dept. of Agriculture office (BAI, RADDL, NMIS, etc.)
SESSION 4: SPECIMEN LABELLING
Labelling and specimen identification
• Correct labeling to identify specimens will determine how the
specimens will be stored and transported, as well as which tests a
laboratory will conduct on the specimens.
Clarity is key
Labeling clinical specimens
In a masking tape, write the following minimum requirements:
• Patient’s Complete name (Including Jr, Sr, III, etc.)
• Age/Sex
• Date of collection
Juan dela Cruz

40/M
01/01/2018
Glass slides for microscopy
Label slides individually
• If slide has frosted edge: Write here using pencil
• If without frosted edge: Write details in a masking tape
• ensure markings don’t interfere with staining process
Each slide should bear:
• Patient’s Complete name (Including Jr, Sr, III, etc.)
• Age/Sex
Labelling water sample
In a masking tape, write the following minimum requirements:
• Source of water sample
• If treatment plant, be specific if inlet or outlet sample
• Time collected
Pasig River
01/01/2018
7am
Labelling Considerations
• Should be written in block letters
• Use permanent markers when labelling
• If sample container has designated area for labelling: use this area
• If without:
• Do not write directly into the sample container
• Write information into masking tape and securely stick this into the
container
• Writing directly into sample container may cause label to be erased during
transport
• Information written in the sample MUST match information in the
accompanying Case Investigation Form/ Linelist of samples
SESSION 5: LABORATORY RELATED FORMS
Types of Forms
• Case Investigation Form: to be used for Vaccine Preventable
Diseases outbreaks only (AFP, Measles, Pertussis and Diphtheria)
• One sample must have one individual form
• Outbreak Notification Form with Linelist of samples: to

be used for all outbreaks
• Multiple samples can be included
CASE INVESTIGATION FORM
Case investigation form
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, travel history, anti-microbial drugs
taken prior to specimen collection
Laboratory information
• acute or convalescent specimen
• other specimens from the same patient
Outbreak Notification Form
Linelist
Reminders
• Use ballpoint/ fine pen (blue or black ink)
• Write legibly (Use block letters)
• Review data prior to submission (Sample data must match)
• Submit a copy of these forms to the testing lab together with
your samples
• Keep a copy of forms submitted
• Create a masterlist of all sample collected for each patient
(Useful for tracking of samples and during analysis of
outbreak)
Summary
Identify appropriate samples to collect
• Think critically while working with laboratory counterpart
investigator
• Identify epidemiologic, clinical, laboratory needs
• Estimate the number of samples needed to confirm the cause of
the outbreak as appropriate
• Define sampling strategy - mode of transmission, syndrome
• Seek external advice for atypical scenarios
• Collect samples ethically
Ethical Consideration in sample
collection
• Explain the procedure and the purpose of the test to the patient
and/or to parents/legal guardians
• Inform possible discomfort that may be experienced during the
procedure
• Inform them that they will be informed of the test result
• Maintain confidentiality by not divulging personal information
when reporting the results to persons not involved in the
care/treatment/management of the patient
Appropriately collect, label, package
and store samples
For each type of sample, know:
• Collection protocol
• Documentation and labeling requirements
• include patient data, outbreak information
• Tracking system
• Correct storage condition
• Appropriate biosafety measures including packaging and
transportation requirements (UN)
PRACTICUM- NPS/OPS
UPPER RESPIRATORY TRACT SPECIMEN
Method of Collection of Oro-pharyngeal swab (OPS)
1. Hold the tongue down with depressor
2. Use a strong light source to locate areas of inflammation and exudate in the posterior
pharynx and tonsillar region of the throat behind the uvula
3. Rub the area back and forth with a swab
4. Withdraw the swab without touching cheeks, teethy or gums
5. Immediately place the swabs in the container with transport medium
6. Break off the top part of the applicator sticks without touching the tube and tighten the
screw cap firmly
7. Label the specimen containers
8. Complete the laboratory request form
Picture: http://www.nytimes.com/imagepages/2007/08/01/health/adam/9550Throatswabs.html
Method of collection of NPS
1. Seat the patient comfortably and tilt the head
2. Insert a flexible swab through the nares parallel to the palate (not upwards)
until resistance is encountered or the distance is equivalent to that from ear
to the nostril
3. Of the patient indicating contact with the nasopharynx. Gently rub and roll
the swab. Leave the swab in place for several seconds to absorb secretions
4. Carefully remove the swab and insert it into the tube containing the
transport medium without antibiotics. Break off the top part of the
applicator sticks without touching the tube and tighten the screw cap firmly
5. Label the specimen tube, indicating left or right side
6. Complete the lab request form
7. Repeat on the other side
Get picture:COPAN flocked swab product brochure
Specimen Collection Supplies Multi-tool knife (Swiss knife or
Leatherman)
OUTBREAK FIELD KIT Alkaline peptone water
Personal protective equipment
Blood culture bottles
Power banks
Cary Blair medium/Stuarts transport
medium Rechargeable lamps
Centrifuge tubes Rubber bands
Collection vials for molecular diagnostics Solar panel kit
Disposable sample collection vials
Spirit lamp/ gas lighter
Disposable storage vials (5ml)
Trunk and smaller tool box
Disposable syringes (5ml)
Vaccine carrier with ice-packs
Glass slides and cover slips
Water Purification Tablets (eg. Aquatab)
Gloves (triple layer surgical)
Ziploc plastic bags
Labels-specimen / address
Field Test Kits / RDTs (as necessary)
Lancets
OT test kit and H2S water and needles
Masks testing kit
Pasteur pipettes/ pipettes and pipette RDTs - Malaria
aids
- Cholera
Scissors/Scalpel/blade - Dengue
- Viral hepatitis
Spatula, Forceps, Loop holder Water testing kits
Sodium hypochlorite concentrates (4%)
Forms & Reference Documents
Stationery (writing pads, pens, pencils,
Action Plan for preparedness, control,
erasers, sharpeners, glass marking pen
and containment of Outbreak
etc)
Epidemiological investigation report
Stool culture bottles
forms
Stuart’s transport medium
Epidemiological survey forms
Test tube racks
Incident form for biosafety purposes
Throat swabs
Laboratory request forms
Tourniquet
Outbreak investigation guide
Viral transport medium

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RITM Training Manual for

MODULE 3. SPECIMEN COLLECTION, HANDLING AND STORAGE

1. An improperly collected, handled or stored sample may lead to false

2. Unsafe specimen collection practices may expose the health worker

- characterized by safe and standard collection processes, methods and

Health Worker Biosafety Considerations

• specimen should be representative of the infectious process

• specimens should be of adequate quantity for the desired tests

• Documentation consists of labels and forms which ensure that a

• Five samples per week during the outbreak

• Typical disease/syndrome: Malaria

• Typical disease/syndrome: Acute Neurological Syndrome

• Transport / Shipment conditions

• Initial Processing, Handling and Storage

• Typical disease/syndrome: Diphtheria, Acute Respiratory Syndrome

Handling and transportation

Juan dela Cruz

• Outbreak Notification Form with Linelist of samples: to

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