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Molecular Biology

Assay, Method, & Instrument Selection

In Molecular Biology, the selection and design of assays and instrumentation are foundational Quality Management activities. Unlike the Core Laboratory, where “plug-and-play” analyzers are the norm, Molecular Pathology often requires the laboratory to choose between FDA-cleared kits and Laboratory Developed Tests (LDTs), or to design complex primer sets for novel targets. This decision-making process is a strategic balancing act involving clinical utility, operational throughput, financial feasibility, and regulatory compliance

Clinical Needs Assessment

Before a specific instrument or method is chosen, the laboratory administration must define the clinical question the test is intended to answer. This “Needs Assessment” dictates the required performance characteristics of the assay

  • Target Population and Prevalence
    • Screening Assays: Used for high-prevalence, asymptomatic populations (e.g., Chlamydia screening). Design priority: High Sensitivity (to avoid false negatives) and High Throughput (automation)
    • Diagnostic Assays: Used for symptomatic, complex cases (e.g., Leukemia sub-typing). Design priority: High Specificity (to avoid false positives) and Resolution (sequencing capability)
  • Turnaround Time (TAT) Requirements
    • Stat/Rapid: For time-critical conditions (e.g., Meningitis, Stroke/Flu differentiation), the lab must select “Sample-to-Answer” platforms (e.g., cartridge-based PCR) that offer 1-hour results but often have lower throughput
    • Routine/Batched: For chronic monitoring (e.g., HIV Viral Load), the lab can select high-throughput batch analyzers that run 96 samples simultaneously but take 4–8 hours to complete
  • Reimbursement Landscape
    • The selection process must verify that CPT codes exist for the proposed test and that the “Cost Per Test” (Reagents + Labor + Amortized Instrument Cost) is lower than the expected reimbursement. If a test uses a proprietary method with no billing code, the lab may not be paid

Method Selection: IVD vs. LDT

The most significant decision in molecular laboratory operations is whether to purchase a commercial kit or develop the test in-house. This choice defines the regulatory burden and the flexibility of the laboratory

  • In Vitro Diagnostics (IVD)
    • Definition: Commercially manufactured test kits that have been cleared or approved by the FDA (510(k) or PMA)
    • Pros: Lower regulatory burden (requires “Verification” only); standardized reagents; manufacturer support for troubleshooting; lower liability risk
    • Cons: Higher cost per test; “Black Box” technology (users cannot modify parameters); dependent on the vendor’s supply chain (backorder risk)
  • Laboratory Developed Tests (LDT)
    • Definition: An assay developed, optimized, and validated within a single laboratory. It utilizes “Analyte Specific Reagents” (ASR) or “Research Use Only” (RUO) components
    • Pros: Significantly lower reagent cost; adaptable (can rapidly add new targets, e.g., a new COVID variant); total control over assay parameters
    • Cons: Highest regulatory burden (requires full “Validation”); high liability (the Medical Director takes responsibility for safety); requires highly skilled staff to maintain and troubleshoot

Instrument Platform Selection

When selecting the physical hardware, the laboratory must evaluate how the instrument integrates with existing infrastructure and workflows. A key distinction is made between “Closed” and “Open” platforms

  • Closed Platforms (Sample-to-Answer)
    • Characteristics: The instrument, software, and reagents are a locked ecosystem. Users load a sample, and the machine performs extraction, amplification, and detection automatically
    • Advantages: “Walk-away” automation reduces labor costs; lowered risk of contamination (closed system); standardized protocols reduce operator error
    • Disadvantages: Users are locked into one vendor’s price and menu; if the vendor discontinues a kit, the instrument becomes a paperweight
  • Open Platforms
    • Characteristics: Separate instruments are used for extraction, liquid handling, and amplification (e.g., a generic 96-well thermal cycler)
    • Advantages: Flexibility to use reagents from any manufacturer; the same cycler can run an HIV test in the morning and a Factor V Leiden test in the afternoon
    • Disadvantages: High labor requirement; increased risk of pipetting error and contamination (open-tube handling); complex data integration

Assay Design Considerations (for LDTs)

If the laboratory chooses to design an LDT, the Quality Management focus shifts to the biochemical design of the primers, probes, and controls. Poor design leads to “Technical Failure” regardless of how expensive the instrument is

  • Target Selection (In Silico Analysis)
    • Conserved Regions: Primers must be designed to bind to genomic regions that are highly conserved (stable) across all strains of a pathogen. Designing primers against a hyper-variable region (like the HIV env gene or certain Spike protein domains) increases the risk of False Negatives due to mutations
    • Specific Regions: The target sequence must be unique to the organism. BLAST (Basic Local Alignment Search Tool) analysis is performed to ensure the primers do not accidentally bind to human DNA or normal flora (cross-reactivity)
  • Oligonucleotide Thermodynamics
    • Melting Temperature (\(T_m\)): Forward and Reverse primers must have matched \(T_m\) (typically within 2°C of each other) to ensure they anneal efficiently at the same step in the PCR cycle
    • GC Content: A GC content of 40–60% is ideal to prevent secondary structures (hairpins) that inhibit binding
    • Amplicon Length: In clinical PCR, shorter amplicons (<150 bp) are preferred because they are more efficient and tolerant of fragmented/degraded DNA often found in clinical specimens (e.g., FFPE tissue)
  • Multiplexing Design
    • When detecting multiple targets in one tube (e.g., Flu A + Flu B + COVID), the emission spectra of the fluorophores must be distinct (e.g., FAM, VIC, CY5, ROX) so the instrument can distinguish the signals (“Crosstalk” prevention)

Internal Control Strategies

A critical component of assay design is the Internal Control (IC). In molecular biology, a negative result is only valid if the Internal Control is positive. If both are negative, the result is “Invalid” (Inhibited)

  • Endogenous Controls (Housekeeping Genes)
    • Amplifying a gene naturally present in the patient sample (e.g., RNase P or Beta-globin)
    • Function: Verifies that the sample was collected correctly (human cells are present) and that extraction worked
    • Limitation: It does not prove the pathogen-specific primers are working
  • Exogenous Controls (Spiked)
    • Adding a synthetic DNA/RNA template (e.g., a Phage or Plasmid) to the sample before extraction
    • Function: This is the gold standard for monitoring the entire process: Extraction efficiency, Reverse Transcription efficiency, and PCR inhibition. If the Exogenous Control fails to amplify, it proves that “Inhibitors” (like Heme or Mucus) are present in the sample

Physical Laboratory Design for Selected Methods

The selection of a method dictates the physical requirements of the laboratory (Facilities Management)

  • Space and Infrastructure: High-throughput “Open” systems require strictly separated rooms (Pre-PCR, Extraction, Post-PCR) to prevent contamination. “Closed” cartridge systems can often be placed in a smaller, single-room footprint
  • Environmental Controls: If selecting Next-Generation Sequencing (NGS), the facility requires high-grade HVAC to control humidity (static electricity damages flow cells) and rigid temperature control (thermal fluctuations cause sequencing errors)