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Cytogenetics

Specimen Requirements

The foundation of any successful cytogenetic analysis lies in the quality of the specimen received. Unlike clinical chemistry or hematology, where analytes are often stable or cells are counted in a static state, cytogenetics requires viable, dividing cells. If the cells die during collection or transport, or if they are prevented from entering mitosis due to improper additives, the analysis will fail. Therefore, the laboratory scientist must strictly enforce specimen requirements regarding collection vessels, anticoagulants, sample volume, and temperature control

General Principles of Anticoagulants & Media

For almost all fluid specimens in cytogenetics (blood and bone marrow), the required anticoagulant is Sodium Heparin (green top tube). Heparin works by preventing the conversion of fibrinogen to fibrin, keeping the sample fluid without being toxic to the cells

  • Why Sodium Heparin?: It is the most non-toxic anticoagulant for lymphocyte and bone marrow cultures. It allows cells to remain viable and capable of division
  • Lithium Heparin: While often acceptable, some historical data suggests lithium ions can be slightly more toxic to cells than sodium ions; however, many modern labs accept lithium heparin if sodium heparin is unavailable
  • The Problem with EDTA (Lavender Top): EDTA (Ethylenediaminetetraacetic acid) is generally unacceptable for cytogenetics. EDTA works by chelating calcium and magnesium. Since calcium is a necessary cofactor for the enzymes involved in the mitotic spindle apparatus, EDTA effectively prevents cell division. While molecular testing (FISH or microarray) might sometimes be salvaged from EDTA, traditional karyotyping is usually impossible
  • The Problem with Formalin: Formalin fixes tissue by cross-linking proteins. This kills the cells instantly, rendering them incapable of growth in culture. Specimens received in formalin must be rejected for karyotyping

Peripheral Blood (Constitutional Studies)

Peripheral blood is collected for constitutional analysis (congenital anomalies, infertility, intellectual disabilities) to analyze T-lymphocytes. Since mature lymphocytes do not spontaneously divide, they must be stimulated with a mitogen (Phytohemagglutinin - PHA) in the lab

  • Container: Sodium Heparin (Green top) vacutainer
  • Volume
    • Adults: 3 to 5 mL is the standard requirement
    • Pediatric/Infants: 1 to 2 mL is usually sufficient
    • Neonates (hard stick): A minimum of 0.5 mL can sometimes be cultured successfully using micro-methods
  • Transport Conditions: Maintain at room temperature (20–24°C). Extreme heat (car dashboard in summer) will cook the cells; freezing will lyse them. Specimens should ideally reach the lab within 24 hours, though viable cultures can often be established up to 48–72 hours post-collection

Bone Marrow (Acquired/Oncologic Studies)

Bone marrow is collected to investigate hematologic malignancies (leukemia, lymphoma, multiple myeloma). In these cases, the lab is analyzing spontaneously dividing leukemic blasts. No mitogen is needed (usually), but the cells are fragile and already compromised by the disease process

  • Container: Sodium Heparin (Green top) vacutainer or a syringe rinsed with sodium heparin
  • Volume: 1 to 3 mL of marrow aspirate
    • Note: The “first pull” of the aspiration is best for morphology smears. Cytogenetics typically receives the second syringe. If the volume is too large, the sample may be hemodiluted with peripheral blood, potentially masking the leukemic clone
  • Transport Conditions: Strictly room temperature. These cells are highly sensitive. Transport should be immediate (stat). If transport time will exceed a few hours, the marrow should be placed in a transport medium (buffered culture media with antibiotics) to maintain viability

Amniotic Fluid (Prenatal Studies)

Amniocentesis is performed typically between 15 and 20 weeks of gestation. The fluid contains fetal cells (amniocytes) shed from the skin, respiratory tract, and urinary tract

  • Container: Sterile, conical plastic centrifuge tubes (e.g., Falcon tubes) or the syringe used for the draw. Glass tubes are avoided due to cell adherence issues
  • Volume: 15 to 20 mL
    • Note: The first 1–2 mL of fluid drawn should be discarded (or used for chemistry) to minimize the risk of maternal cell contamination (MCC) from the needle passing through the maternal skin and uterine wall
    • Early Amniocentesis (10-14 weeks): Requires smaller volumes (1 mL per week of gestation) to protect the fetus
  • Transport Conditions: Room temperature. Protect from light (though less critical for cytogenetics than for bilirubin analysis). The sample must be sterile; a contaminated fluid will result in bacterial overgrowth that destroys the culture

Chorionic Villus Sampling - CVS (Prenatal Studies)

CVS is performed earlier than amniocentesis (10–12 weeks). It involves biopsying the developing placenta (chorion frondosum)

  • Container: Sterile tube containing transport media (usually a balanced salt solution or culture media with antibiotics and Heparin). The media prevents the tissue from drying out
  • Sample Size: 5 to 20 mg of villi. The tissue should look like branching coral or fluffy white threads
  • Transport Conditions: Room temperature or cool (wet ice) if transport is delayed. Never frozen

Solid Tissues (Skin, Products of Conception, Tumors)

These specimens are cultured to detect mosaicism (skin biopsy), cause of spontaneous miscarriage (Products of Conception - POC), or solid tumor genetics

  • Container: A sterile screw-top cup or tube containing transport media (Hanks Balanced Salt Solution, RPMI-1640) or sterile saline
    • Critical: If media is unavailable, sterile saline is the backup. Never send tissue dry (it will desiccate and die) and never send in water (hypotonic shock will burst the cells)
  • Sample Size
    • Skin: A 3–4 mm punch biopsy or a small wedge. Full thickness is required to get the dividing fibroblasts from the basal layer
    • POC: 0.5 cm to 1 cm cubed of fetal tissue. Identification of fetal parts (villi, membranes) is crucial to avoid culturing maternal decidua
    • Solid Tumor: 0.5 to 1 cm cubed of viable tumor tissue (avoiding necrotic centers)
  • Transport Conditions: Room temperature is acceptable for short transit. If the delay is significant (overnight), the specimen should be kept cool (refrigerated or on wet ice) to slow metabolic decay and bacterial growth, but never frozen

Summary of Rejection Criteria

While laboratories strive to salvage every specimen, certain conditions render a sample unacceptable for cytogenetic analysis:

  • Frozen specimens: Ice crystal formation lyses cell membranes
  • Fixed specimens: Exposure to formalin or alcohol kills cells
  • Wrong Anticoagulant: EDTA or Citrate (unless only DNA microarray/FISH is ordered and validated)
  • Gross Contamination: Leaking containers or obvious bacterial smell/cloudiness
  • Severe Hemolysis: Released heme can be toxic to cell growth
  • Clotted Specimens: Clots trap the dividing cells, making them inaccessible for culture initiation