Analyze Number of Cultures
The cytogenetics laboratory creates multiple “cultures” for every patient sample. A culture is an independent flask, tube, or dish where cells are grown. Analyzing cells from only a single culture vessel is risky because it introduces Culture Bias and the potential for Pseudomosaicism (artifacts arising in vitro). To ensure the result represents the patient and not a lab accident, the analysis must be distributed across independent culture systems
The Two-Culture Rule
For almost every specimen type (Blood, Marrow, Amnio, Tissue), the standard guideline is to initiate and harvest at least two independent cultures
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Risk Mitigation
- Failure Protection: If one flask gets infected (bacteria/fungus) or fails to grow (no mitosis), the second flask serves as a backup to save the case
- Artifact Detection: If an abnormality (e.g., Trisomy 7) is seen in Flask A but not in Flask B, it raises the suspicion of a culture artifact (pseudomosaicism). If it is seen in both flasks, it is confirmed as a true biological clone
- Analysis Protocol: When selecting the 20 cells for analysis, they should be drawn roughly equally from both cultures (e.g., 10 cells from Flask A and 10 cells from Flask B)
Solid Tissue & Amniocytes (In Situ vs. Flask)
For prenatal (amniotic fluid/CVS) and solid tissue samples, the definition of “culture” is more specific because these cells grow adherently in colonies
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In Situ Method (Colony Counting): Cells are grown directly on coverslips
- Protocol: Analysis typically requires examining cells from at least 2 independent vessels (e.g., two different petri dishes)
- One-Colony Rule (Pseudomosaicism): If all abnormal cells come from a single colony within one dish, the abnormality is presumed to have arisen during culture division (a mitotic error in the lab). It is reported as pseudomosaicism (likely normal fetus)
- Multi-Colony Rule (True Mosaicism): If the abnormality is found in multiple colonies across different dishes, it is considered True Mosaicism (present in the fetus)
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Flask Method: Cells are grown in flasks and trypsinized (mixed up) before harvesting
- Limitation: Because the cells are mixed, you lose the “colony” context. A single artifactual mutant cell can divide into 100 cells, making it look like a huge clone (100% of the flask)
- Requirement: Therefore, using two separate flasks is absolutely mandatory. If Flask A is 100% Abnormal and Flask B is 100% Normal, the result is ambiguous (likely Level III Mosaicism or artifact), requiring further testing
Synchronized vs. Unsynchronized Cultures (Blood)
For constitutional blood cultures, labs often set up different types of cultures to maximize resolution
- 72-Hour Synchronized: Treated with chemicals (Methotrexate/Thymidine) to block cell division and then release it, producing long, high-resolution chromosomes. This is the primary culture for analysis
- 72-Hour Standard (Unsynchronized): Allowed to divide naturally. Produces shorter chromosomes but has a higher yield/success rate
- Strategy: The laboratory scientist prioritizes the Synchronized culture for the high-quality 5-cell analysis but may use the Standard culture to finish the 20-cell count or as a backup if the sync fails
Oncology Cultures (Mitogen Stimulation)
Leukemia and Lymphoma cells can be finicky. Tumor cells might not divide spontaneously, while normal cells do. To wake up the tumor cells, different “cocktails” (cultures) are set up
- B-Cell Mitogens (e.g., DSP-30 + IL-2): Essential for Chronic Lymphocytic Leukemia (CLL). Without this specific culture, you will only analyze normal T-cells and miss the cancer
- Standard Unstimulated: Used for myeloid leukemias (AML/CML)
- Analysis Strategy: The analyst must examine the specific culture designed to stimulate the suspected disease. Analyzing the wrong culture leads to a False Negative result