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Cytogenetics

Specimen Preparation

The pre-analytical phase of Clinical Cytogenetics is unique in the laboratory setting because the analyte is a living, dividing cell. Unlike chemical analytes that are stable in serum, or DNA that can be extracted from fixed tissue, cytogenetic analysis requires the successful culture of viable cells. Therefore, the specimen preparation phase - encompassing collection, transport, and accessioning - is the most critical control point. Errors occurring here, such as the use of improper fixatives or misidentification, are often irreversible and result in culture failure. The following overview synthesizes the requirements for collection, transport, and the administrative management of test requests

Specimen Collection & Transport

The integrity of the culture begins at the bedside. The laboratory scientist must ensure that specimens are collected in conditions that preserve sterility and mitotic capability

  • Specimen Requirements and Anticoagulants
    • Sodium Heparin (Green Top): This is the required anticoagulant for peripheral blood and bone marrow. It prevents clotting without inhibiting cell division
    • Prohibited Additives: EDTA (Lavender top) chelates calcium, preventing spindle fiber formation, and is generally unacceptable for karyotyping. Formalin is strictly prohibited as it cross-links proteins and kills cells instantly; formalin-fixed tissue must be rejected
    • Solid Tissues: Skin biopsies, products of conception (POC), and solid tumors must be transported in sterile media (e.g., RPMI/Hanks) or saline to prevent desiccation. They must never be sent dry or in water (hypotonic shock)
    • Temperature: Specimens should be maintained at ambient room temperature (20–24°C). Refrigeration is only acceptable for solid tissues during delays to slow autolysis; freezing destroys the specimen
  • Quality Factors (Viability, Cellularity, Contamination)
    • Viability: Assessed via transport time and media pH. A shift in the phenol red indicator to yellow suggests acidity (bacterial growth), while purple suggests alkalinity (loose cap)
    • Cellularity: Critical in Bone Marrow (risk of hemodilution reducing the yield of leukemic blasts) and Amniotic Fluid (bloody taps may obscure low-cellularity early gestation samples)
    • Contamination: Microbial overgrowth is the most common cause of failure. Maternal Cell Contamination (MCC) in prenatal samples (CVS/Amnio) poses a severe diagnostic risk, potentially leading to a misdiagnosis (e.g., analyzing maternal decidua instead of fetal villi)
  • Compromised vs. Unacceptable Specimens
    • Unacceptable (Reject): Specimens fixed in formalin, frozen without cryoprotectant, or unlabelled samples
    • Compromised (Process with Disclaimer): Hemolyzed samples (must be washed to remove toxic heme), clotted samples (treated with enzymes/mechanical disaggregation), or aged samples (>48 hours). These are processed to attempt salvage, but reported with caveats regarding potential limitations
  • Specimens for Multiple Tests
    • When one sample serves multiple departments (Microbiology, Flow Cytometry, Molecular), triage is based on sterility. Cytogenetics and Microbiology take priority because the sample must remain sterile for culture. Flow Cytometry (viability required) follows, with Molecular (DNA stability) often able to use leftover or fixed material

Specimen & Test Requests

The administrative intake of the specimen serves as the final safety barrier before testing begins. This process ensures the correct patient is tested for the correct clinical indication

  • Verification of Patient Information
    • The Two-Identifier Rule: Every tube and requisition must match exactly using two unique identifiers (Name/DOB or Name/MRN)
    • Clinical Correlation: The laboratory scientist must verify that the test fits the patient’s history. For example, a “Chromosome Analysis” on blood for a leukemia patient requires an unstimulated culture (to detect spontaneous blasts), while the same order for infertility requires a stimulated culture (PHA to grow T-cells). Mismatched sex (Phenotype vs. Indication) must also be resolved
    • Irretrievable Specimens: For unlabelled invasive samples (marrow/amnio) that cannot be redrawn, strict exception protocols involving physician liability waivers are employed
  • Test Prioritization (Triage)
    • STAT/Emergency: Reserved for cases requiring immediate medical/surgical intervention. Includes Newborns with anomalies (Trisomy 13/18 decisions), Acute Promyelocytic Leukemia (APL t(15;17) requires immediate ATRA therapy), and Late Gestational Age prenatal samples
    • Urgent: New leukemia diagnoses (AML/ALL) where chemotherapy planning awaits risk stratification
    • Routine: Infertility workups, constitutional developmental delay, and solid tumor prognostics
    • Workflow Adjustments: STAT cases may trigger “preliminary reporting” (e.g., reporting a positive FISH result immediately) or “short harvests” (harvesting cultures early to speed up TAT, even if cell yield is lower)