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Cytogenetics

Reagent Performance & Sterility

In Clinical Cytogenetics, the quality of the final result - the karyotype or FISH analysis - is directly dependent on the quality of the reagents used to grow and process the living cells. Unlike clinical chemistry, where reagents are often stable chemicals, cytogenetics relies on biological matrices (Fetal Bovine Serum, culture media, enzymes) that are prone to contamination, degradation, and lot-to-lot biological variation. Therefore, a robust Quality Control (QC) program for reagents is mandatory to prevent culture failure and ensure accurate diagnosis

New Lot Verification (Parallel Testing)

Before a new shipment (lot number) of a critical reagent is put into routine use, it must be verified against the currently approved lot. This process, known as “parallel testing,” ensures that the new reagents do not negatively impact cell growth or morphology

  • Critical Reagents requiring testing
    • Culture Media: (e.g., RPMI 1640, AmnioMAX, MarrowMAX)
    • Serum: (e.g., Fetal Bovine Serum - FBS). This is the most variable reagent; different batches contain different levels of growth factors
    • Mitogens: (e.g., Phytohemagglutinin - PHA)
    • Harvest Chemicals: Colcemid, Hypotonic solution, Fixative components
  • The Testing Protocol
    • A control specimen (typically a surplus normal peripheral blood sample) is split into two cultures
    • Culture A: Set up using the Old/Current Lot
    • Culture B: Set up using the New Lot
    • Both are processed simultaneously under identical conditions
  • Performance Criteria
    • Mitotic Index (MI): The new lot must yield a comparable number of metaphases. A significant drop in MI indicates the new lot is inhibitory or lacks necessary nutrients
    • Chromosome Morphology: The chromosomes must be long, well-spread, and not “fuzzy” or “rotten.”
    • Banding Quality: The enzymatic digestion (Trypsin) using the new reagents must produce crisp G-bands

Sterility Monitoring

Bacterial, fungal, or mycoplasma contamination is the primary cause of culture failure. Reagents are the most common vector for introducing these contaminants

  • Manufacturer’s Certificate of Analysis (CoA)
    • For commercially purchased sterile reagents, the lab must obtain and file the CoA. This document certifies that the manufacturer tested the lot for sterility (bacteria/fungi/mycoplasma) and endotoxins
  • In-House Sterility Checks
    • When “Complete Media” is prepared in the lab (e.g., mixing Basal Media + Serum + L-Glutamine + Antibiotics), the final mixture must be checked for sterility before use on patient samples
    • Protocol: An aliquot of the newly mixed media is placed in a culture tube and incubated at \(37^{\circ}\text{C}\) for 48–72 hours
    • Pass/Fail: The tube is visually inspected for turbidity (cloudiness) or a pH shift (color change). If the media remains clear, the batch is released for use
  • Visual Inspection (Daily)
    • Laboratory scientistsmust inspect every bottle of media before use
    • Phenol Red Indicator: Most media contains this pH indicator
      • Red: Neutral (Good)
      • Yellow: Acidic (Indicates bacterial contamination)
      • Purple: Basic (Indicates fungal contamination or loss of \(CO_2\))

Probe Verification (FISH Reagents)

Fluorescence In Situ Hybridization (FISH) probes are expensive and sensitive to light and temperature. New lots must be verified to ensure they bind to the correct chromosomal target with sufficient intensity

  • Verification Steps
    • Hybridize the new probe lot to a known normal control (for enumeration probes) or a known positive control (for rearrangement probes, if available)
  • Evaluation Criteria
    • Signal Intensity: The signal must be bright and distinct
    • Specificity: The probe must bind only to the intended target (no cross-hybridization)
    • Background: The background nuclei must be dark, without “haze” or non-specific binding that interferes with scoring

Documentation & Traceability

Regulatory standards (CAP/CLIA) follow the rule: “If it is not documented, it was not done.” A strict paper trail connects the patient result back to the specific reagent lots used

  • Reagent Logbook: A central log must track:
    • Reagent Name and Manufacturer
    • Lot Number
    • Date Received
    • Expiration Date (Manufacturer’s)
    • Date Put Into Service (QC Pass Date)
    • QC Laboratory scientist Initials
  • Worksheets: Patient worksheets must record the Lot Numbers of critical reagents (Media, Probe) used for that specific case. This allows for retroactive investigation; if a cluster of culture failures occurs, the lab can quickly identify if they all shared a specific lot of media

Storage & Expiration Management

Proper storage preserves reagent performance. Documentation must reflect that storage conditions were maintained

  • Labeling
    • Upon receipt, reagents are labeled with the “Received Date.”
    • Upon opening, reagents are labeled with the “Open Date”: and the “New Expiration Date.”
    • Note: Many reagents have a stable shelf-life until opened, after which they degrade rapidly. For example, L-Glutamine degrades into ammonia at \(4^{\circ}\text{C}\) within a few weeks, which is toxic to cells
  • Aliquoting
    • To prevent repeated freeze-thaw cycles (which degrade enzymes and antibiotics) and to minimize contamination risk, bulk reagents are often aliquoted into single-use tubes upon arrival
    • These aliquots must be labeled with the full identity and expiration date of the parent lot
  • FIFO (First-In, First-Out): Inventory should be arranged so older lots are used first to minimize waste due to expiration