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Cytogenetics

Compromised or Unacceptable

In Clinical Cytogenetics, the designation of a specimen as “unacceptable” is a critical decision that carries significant weight. Unlike routine blood draws, many cytogenetic specimens are obtained via invasive, painful, and expensive procedures (e.g., bone marrow aspiration, amniocentesis). Consequently, “rejection” is a last resort. The laboratory scientist must distinguish between specimens that are absolutely non-viable (Unacceptable) and those that are suboptimal but potentially salvageable (Compromised). Managing these specimens requires a strict protocol of documentation, physician communication, and specialized processing techniques

Unacceptable Specimens (Absolute Rejection)

Certain conditions render a biological specimen fundamentally incompatible with cell culture and cytogenetic analysis. Processing these specimens is futile, wastes reagents, and delays valid diagnostic testing. These specimens must be rejected, and the requesting clinician must be notified immediately to arrange for a recollection if possible

  • Fixation in Formalin: This is the most common and irreversible error, particularly with solid tissue biopsies and products of conception sent from surgery. Formaldehyde works by cross-linking proteins and DNA. This process kills the cells instantly and fixes the chromosomes in a rigid state, preventing the cell division required for karyotyping. While FISH or molecular extraction might be attempted (though often with poor results due to DNA fragmentation), cell culture is impossible
  • Frozen Specimens: Unless the specimen was cryopreserved in a specific medium containing a cryoprotectant (like DMSO), freezing a raw specimen is destructive. The formation of ice crystals within the cytoplasm punctures the cell membrane and organelle membranes, causing cell lysis upon thawing. Standard freezer or dry ice transport destroys the sample
  • Gross Labeling Errors: Any specimen that is unlabeled or mismatched (the name on the tube does not match the requisition) represents a catastrophic patient safety risk. In many institutions, these are “irretrievable,” though strict “exception” protocols involving physician signature and acceptance of liability may exist for precious specimens (e.g., amniotic fluid)
  • Wrong Anticoagulant (EDTA/Lavender Top): EDTA is a chelating agent that binds calcium and magnesium. Calcium is a critical cofactor for the polymerization of microtubules during spindle fiber formation. Without free calcium, cells cannot undergo mitosis. Therefore, EDTA blood is unacceptable for conventional chromosome analysis (karyotype), though it may still be viable for Microarray (SNP array) which does not require dividing cells

Compromised Specimens (Suboptimal but Processed)

A compromised specimen is one where viability or quality is questionable, but the “precious” nature of the sample dictates that an attempt at culture must be made. These samples are processed with a disclaimer, often requiring modified protocols to maximize the chance of success

Hemolysis

Hemolysis indicates the rupture of red blood cells (RBCs), releasing hemoglobin into the serum/media. This can occur due to traumatic venipuncture, forcing blood through a small needle, or exposure to extreme heat

  • The Problem: Free hemoglobin is toxic to living cells in culture. It can inhibit cell division (mitotic index) and reduce the quality of banding
  • The Solution: The laboratory scientist should wash the specimen immediately upon receipt. Centrifuging the sample and replacing the supernatant with fresh culture media or sterile saline removes the toxic hemoglobin before culture initiation

Clotted Specimens

Clotting occurs when the specimen is not mixed adequately with the heparin immediately after collection

  • The Problem: The fibrin clot traps the cells of interest (lymphocytes or leukemic blasts). If the cells are locked inside the clot, they cannot access the nutrients in the media or the mitogens required to stimulate division
  • The Solution
    • Mechanical Disaggregation: Mincing the clot with sterile scalpels or forcing it through a syringe
    • Enzymatic Digestion: Treating the clot with streptokinase or collagenase to dissolve the fibrin mesh and release the cells
    • Caveat: These salvage techniques increase the risk of contamination and cell damage, often resulting in lower yields

Delayed Transport / Old Specimens

Viability decreases exponentially over time

  • The Problem: As cells sit in the tube, they deplete nutrients, pH changes, and waste products accumulate. Granulocytes in the sample begin to die and release toxic enzymes
    • Newborn Blood: Viable for 3–4 days
    • Adult Blood: Viable for 48–72 hours
    • Bone Marrow: Viability drops significantly after 24 hours
  • The Solution: “Old” specimens are often set up with increased mitogen concentrations (e.g., higher PHA) or cell-growth supplements (e.g., L-Glutamine, Giant Cell Tumor Conditioned Media) to coax the remaining viable cells into division

Leaking or Broken Containers

A breach in the container compromise sterility

  • The Problem: The primary risk is microbial contamination (bacteria/fungi) from the outside environment. A secondary risk is the loss of volume
  • The Solution: If sufficient volume remains, the sample is processed using a “heavy antibiotic” regimen (e.g., Penicillin/Streptomycin plus Gentamicin or Nystatin) to suppress bacterial overgrowth. The culture is monitored daily for turbidity

Documentation & Reporting

The handling of compromised specimens requires rigorous documentation to protect the laboratory and inform the physician regarding potential limitations of the result

  • Receipt Verification: The condition of the specimen (e.g., “Received clotted,” “Received hemolyzed,” “Received >48 hours post-collection”) must be noted in the Laboratory Information System (LIS) at the time of accessioning
  • The Disclaimer: The final cytogenetic report must include a comment indicating the suboptimal nature of the sample. This manages clinical expectations regarding potential culture failure, low resolution of chromosomes, or limited number of cells analyzed
  • Culture Failure: If a compromised specimen fails to yield metaphases, the report is issued as “Unsuccessful Study.” The documentation of the specimen’s initial condition provides the explanation for the failure, distinguishing biological limitations from laboratory error