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Cytogenetics

Multiple Tests

In the clinical laboratory, the most valuable resource is the patient specimen. This is particularly true in Cytogenetics, where samples are frequently obtained via invasive, painful, and risky procedures such as bone marrow aspiration, amniocentesis, or surgical biopsy. Often, a clinician will collect a limited volume of specimen but order a battery of tests across multiple laboratory disciplines (e.g., Microbiology, Hematopathology, Flow Cytometry, Molecular Genetics, and Cytogenetics). The laboratory scientist acts as the steward of these “precious specimens,” responsible for triage, prioritization, and proper aliquoting to ensure that all critical diagnostic information can be obtained from a single collection event

Principles of Triage & Prioritization

When a single specimen tube arrives for multiple tests, a strict hierarchy of processing must be established based on specimen stability, sterility requirements, and the methodology of the testing platforms

  • Sterility First (Microbiology & Cytogenetics): Tests that require the growth of living organisms (bacterial culture) or living cells (cytogenetic culture) require the specimen to remain sterile. Once a tube is opened in a non-sterile environment (such as Hematology for a slide smear), it is compromised for culture. Therefore, Cytogenetics and Microbiology typically take priority or must be aliquoted first inside a biological safety cabinet
  • Viability Second (Flow Cytometry & Cytogenetics): Both Flow Cytometry and Cytogenetics require viable cells. However, Flow Cytometry analyzes cells in their current state, whereas Cytogenetics requires cells to divide. If the sample volume is extremely low, the Pathologist may prioritize Flow Cytometry for a rapid diagnosis of leukemia lineage, or Cytogenetics for prognostic stratification, depending on the clinical suspicion
  • DNA/RNA Stability Third (Molecular): Molecular tests (PCR, Next-Gen Sequencing) are generally more robust. DNA can be extracted from dying or even dead cells. Therefore, Molecular departments can often utilize “leftover” material or the cell pellet remaining after Cytogenetics has harvested the culture

Bone Marrow Aspirate Triage

Bone marrow is the most common specimen type requiring multi-departmental coordination. A standard “leukemia workup” involves morphology, flow cytometry, cytogenetics (karyotype/FISH), and molecular genetics

  • The “First Pull”: The first 1-2 mL of marrow aspirated is the most cellular and undiluted. This is prioritized for Hematopathology to make direct smears for morphological examination. It is generally not suitable for Cytogenetics because it is often not in a heparinized tube
  • The “Second Pull” (Green Top): This heparinized sample is shared between Flow Cytometry and Cytogenetics
    • Splitting the Sample: If 3-4 mL are received, the sample is taken into a sterile laminar flow hood. 1-2 mL are removed for Cytogenetics culture setup. The remaining fluid is forwarded to Flow Cytometry
    • Low Volume: If only 0.5 mL is received, the lab must consult the ordering physician or pathologist. Often, the sample is diluted with media, a small amount is used for a “micro-culture” in Cytogenetics, and the rest is sent to Flow
  • Shared Pellets: If no specific tube was drawn for Molecular Genetics, the Cytogenetics lab can often provide the fixed cell pellet remaining after the harvest procedure. Although the DNA is fixed in methanol/acetic acid, modern extraction kits can isolate high-quality DNA from these pellets for PCR testing

Solid Tissue (Biopsy) Workflow

When a surgeon removes a tumor or a lymph node, the tissue must be divided between Surgical Pathology (Histology) and specialized testing (Genetics/Flow)

  • The Fresh vs. Fixed Conflict: Histology requires the tissue to be fixed in formalin. Cytogenetics requires fresh, unfixed tissue. Once tissue touches formalin, it is useless for karyotyping
  • The Workflow: The fresh tissue should be sent from the OR to Pathology immediately. The Pathologist or Pathologist’s Assistant (PA) examines the fresh tissue, sections a portion for Cytogenetics (placing it in sterile media/saline), and then places the remainder in formalin for standard histology
  • Touch Preps: If the tissue is too small to divide, the PA may perform “touch preps” (touching the fresh tissue to a glass slide) for FISH analysis before fixing the tissue. This allows for some genetic data (e.g., MYC amplification) even if the tissue is entirely formalin-fixed

Amniotic Fluid Co-Testing

Amniotic fluid is frequently tested for Alpha-Fetoprotein (AFP) and Acetylcholinesterase (AChE) in the Chemistry department, in addition to Chromosome Analysis in Cytogenetics

  • Centrifugation Strategy: The fluid is centrifuged upon receipt in Cytogenetics
    • The Pellet: Contains the amniocytes (fetal cells). This is resuspended in culture media and plated for chromosome analysis
    • The Supernatant: Contains the dissolved proteins (AFP). This is decanted into a separate tube and forwarded to the Chemistry department. This workflow allows one tube to serve both departments without compromising sterility or volume

Anticoagulant Compatibility Issues

A major challenge arises when the wrong tube type is shared

  • Sodium Heparin (Green): Acceptable for Cytogenetics, Flow Cytometry, and most Molecular tests (though heparin can inhibit PCR enzymes, requiring additional cleaning steps during extraction). It is generally unacceptable for Hematology smears (causes blue background staining)
  • EDTA (Lavender): Preferred for Hematology and Molecular. Unacceptable for Cytogenetics (karyotype) because it chelates calcium and prevents cell division. If only an EDTA tube is received, Cytogenetics may have to reject the karyotype request or attempt a “wash” protocol (rinsing cells with saline to remove EDTA) if the sample is irreplaceable, though success rates are low. Alternatively, the sample may be redirected for Chromosomal Microarray (CMA), which does not require dividing cells

Communication & Chain of Custody

Handling shared specimens requires rigorous documentation:

  • Aliquoting Log: If a primary tube is split, a log must be kept detailing which department took which volume, the time of the split, and the laboratory scientist responsible to ensure traceability
  • Physician Consultation: In cases of insufficient volume for all ordered tests, the laboratory scientist must never guess which test is most important. A pathologist or the ordering clinician must define the priority (e.g., “Prioritize the ruling out of Acute Promyelocytic Leukemia via FISH over the standard Karyotype”)