Collection, Transport, Processing, and Storage of Samples for Viral Diagnosis

Mr. Parag Chauhan
  1. Accurate and timely viral diagnosis relies heavily on properly handling clinical specimens from collection to laboratory processing.
  2. The integrity of a sample is crucial as poor handling can lead to degradation of viral particles, contamination, and ultimately false or inconclusive results.
  3. This article explains the principles, methods, and considerations in collecting, transporting, processing, and storing samples for viral diagnostics.

 

Sample Collection

1.1 Purpose of Collection

The primary goal of sample collection is to obtain a specimen representative of the disease process without contamination or degradation of the viral content. It is essential to use appropriate methods and tools to ensure the sample is viable for subsequent analysis.

1.2 Principles of Sample Collection

    • Timeliness: Samples should be collected as early as possible during the acute phase of infection when the viral load is highest.
    • Aseptic Technique: Use sterile equipment and maintain a contamination-free environment to prevent the introduction of extraneous organisms.
    • Correct Site: Samples should be collected from anatomical sites most likely to harbor the virus based on clinical suspicion (e.g., nasopharyngeal swabs for respiratory viruses).

1.3 Types of Specimens Collected

Depending on the clinical presentation and suspected virus, various types of specimens may be required:

    1. Respiratory Specimens: Nasopharyngeal swabs, oropharyngeal swabs, nasal aspirates, or bronchoalveolar lavage for respiratory viruses like influenza, RSV, or SARS-CoV-2.
    2. Blood Samples: For viruses such as HIV, hepatitis B/C, or arboviruses (e.g., dengue).
    3. Urine Samples: For viruses like cytomegalovirus (CMV).
    4. Fecal Samples: For enteric viruses like rotavirus, norovirus, or enteroviruses.
    5. Skin Lesion Swabs: For herpes simplex virus (HSV) or varicella-zoster virus (VZV).
    6. Cerebrospinal Fluid (CSF): For central nervous system infections caused by viruses like enteroviruses or arboviruses.

1.4 Equipment and Supplies

    • Sterile swabs (preferably with synthetic tips like Dacron or nylon) and viral transport media (VTM).
    • Syringes, needles, and vacutainer tubes for blood collection.
    • Containers with tight seals for urine, stool, or tissue specimens.

1.5 Collection Guidelines

    • Clearly label specimens with patient identifiers, date, time of collection, and sample type.
    • Use personal protective equipment (PPE) to safeguard the patient and the collector.
    • Follow institutional guidelines for sample collection to ensure compliance with biosafety protocols.

 

Transport of Specimens

2.1 Importance of Transport Conditions

Viruses are often unstable outside the host and can lose infectivity if not transported under the right conditions. Optimal transport methods maintain sample integrity, prevent viral degradation, and minimize contamination.

2.2 Viral Transport Media (VTM)

    • Composition: VTM typically contains a balanced salt solution, protein stabilizers (e.g., bovine serum albumin or gelatin), antibiotics to inhibit bacterial overgrowth, and a buffer to maintain pH.
    • Purpose: Protects the virus during transit to the laboratory, especially if processing is delayed.

2.3 Temperature Requirements

    • Cold Chain Maintenance: To prevent viral degradation, most viral samples should be transported at 2–8°C.
    • Dry Ice or Liquid Nitrogen: For long-distance transport or specimens requiring ultra-cold temperatures (e.g., -70°C).
    • Room Temperature: Some viruses, like adenoviruses, are temporarily stable at room temperature.

2.4 Packaging Standards

    • Triple-layer packaging:
      1. Primary Container: Leak-proof container holding the specimen.
      2. Secondary Container: Watertight, labeled container with absorbent material to prevent leaks.
      3. Outer Container: Rigid outer packaging to protect against physical damage.
    • Label with “Biological Substance, Category B” if applicable.

2.5 Timeliness of Transport

    • Transport samples to the laboratory as soon as possible, ideally within 24 hours of collection. Delays can result in reduced viral viability and affect diagnostic accuracy.

 

Processing of Specimens

3.1 Receiving and Logging Samples

    • Verify sample details (e.g., patient name, specimen type) with accompanying requisition forms.
    • Assign a unique laboratory identification number for tracking.

3.2 Biosafety Measures

    • Process samples in a biosafety cabinet (BSC) to protect personnel from exposure to infectious agents.
    • Follow biosafety level (BSL) guidelines appropriate to the suspected virus (e.g., BSL-2 for most viruses, BSL-3 for SARS-CoV or hantaviruses).

3.3 Pre-Analytical Processing

    1. Swabs: Elute swabs into VTM by vigorous agitation or vortexing.
    2. Blood: Centrifuge blood samples to separate serum or plasma for testing.
    3. CSF: Aliquot directly for molecular or culture-based testing.
    4. Stool: Prepare a 10% phosphate-buffered saline (PBS) suspension or another suitable medium.

3.4 Diagnostic Methods

Specimens may be subjected to one or more diagnostic methods based on clinical suspicion:

    • Nucleic Acid Amplification Tests (NAATs): PCR, RT-PCR, or isothermal amplification for detecting viral RNA/DNA.
    • Culture: Isolation of live virus in cell culture systems (used less frequently but still important for certain viruses).
    • Serology: Detection of virus-specific antibodies (e.g., ELISA for HIV or hepatitis).
    • Antigen Detection: Rapid antigen tests for respiratory viruses like influenza or RSV.
    • Electron Microscopy: Rarely used but valuable for identifying novel or uncharacterized viruses.

Storage of Samples

4.1 Short-Term Storage

    • If immediate processing is impossible, samples can be stored at 2–8°C for up to 48–72 hours.
    • Avoid multiple freeze-thaw cycles as they degrade nucleic acids and proteins.

4.2 Long-Term Storage

    • Ultra-Low Freezers (-70°C or -80°C): Ideal for preserving viral RNA/DNA and infectious viruses.
    • Liquid Nitrogen (-196°C): Used to preserve highly sensitive or rare viral samples.

4.3 Stabilizers and Preservatives

    • For nucleic acid preservation, use commercially available stabilizers such as RNAlater.
    • For live viruses, include cryoprotectants like dimethyl sulfoxide (DMSO) or glycerol.

Challenges and Best Practices

5.1 Challenges

    • Degradation: Viral RNA/DNA is prone to enzymatic degradation, especially if samples are mishandled.
    • Contamination: The introduction of extraneous nucleic acids can lead to false positives.
    • Transport Delays: Affect sample viability and reduce diagnostic accuracy.

5.2 Best Practices

    1. Training: Ensure that personnel involved in sample handling are adequately trained in collection, transport, and biosafety practices.
    2. Standard Operating Procedures (SOPs): Follow standardized protocols for all aspects of sample handling.
    3. Quality Control: Use controls at every step to ensure the reliability and reproducibility of results.
    4. Communication: Maintain clear communication between clinicians and laboratory personnel regarding sample requirements and clinical suspicion.

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