Laboratory Diagnostics, Specimen Collection, and Biosafety Issues

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Laboratory Diagnostics, Specimen Collection,and
Biosafety Issues
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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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The Specimen Collection Kit
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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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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 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
• Oropharyngeal swabs
• Lower respiratory tract specimens
• Bronchoalveolar lavage or tracheal aspirates
• Other specimens
• Nasal swab
• Nasopharyngeal swabs
• Collect the sample on several different days

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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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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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Field Data Collection Form

• Patient name
• Unique identification number
• Patient demographic information
• Patients 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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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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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

• Initial testing at State Public Health Laboratory
• Call CDC Hotline before sending specimens for
  reference testing
• 770-488-7100
• Send Priority Overnight
• Include inventory sheet and CDC case ID number
• Coordinate with the laboratory

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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 CDC

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Laboratory Tests
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Laboratory Diagnosis for Avian Influenza

• Tests for respiratory samples
• PCR-based techniques
• Virus isolation
• Immunofluorescence
• Rapid antigen detection
• Blood used for
• Measurement of specific antibodies (sera)
• Viral isolation (whole blood)
• PCR-based techniques (sera)

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Laboratory Tests

• Virus Isolation
• Gold standard for detection of avian influenza
• Technically difficult and requires BSL-3
  laboratory
• PCR Based Techniques
• Sensitivity depends on the particular test, the
  influenza strain, and the type of specimen used
• Sensitivity 80 - 100
• Specificity 90 - 100

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Serological Tests

• Paired serum samples are most useful
• Acute sample
• Within 7 days after symptom onset
• Convalescent sample
• More than 12 days after symptom onset

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Interpreting Serology Tests
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Summary

• Maintain adequately stocked specimen collection
  kits and store them properly when they are not in
  use.
• Oropharyngeal swabs and lower respiratory
  specimens are the best specimens to collect.
• Collect multiple specimens (respiratory and
  blood) on multiple days.

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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 states 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 CDC.

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Laboratory Practice Exercise
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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/gui
  delines/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/gui
  delines/humanspecimens/en/index.html