CYTOGENETICS LAB PATHFLOW

• Cell culture


  The study of cells grown in vitro
• Cytogenetic procedures

  Techniques used to study chromosomes

  
• Cytogenetic procedures

  1. Cells actively dividing

  2. Chromosomes distinguishable during metaphase

  3. Metaphase chromosomes obtained from specimens with spontaneously dividing cells or chemically induced to divide in vitro
• Constitutional chromosome aberrations

  Studied in blood lymphocytes, skin, gonad, placenta to look for tissue mosaicism

  
• Blood may not always be available, so skin or other tissue may be used
• Tissues in suspension

  Easiest to culture, e.g. amniotic fluid, bone marrow, blood
• Solid tissues


  Require enzymatic dispersal before culturing, e.g. chorionic villi, skin, solid tumors, products of conception
• Cytogenetic request forms


  Include specimen type, test to perform, patient information, diagnosis, requesting physician

  
• Sample collection containers


  Sterile, standard stock or provided by cytogenetics lab
• Specimens to be cultured must never be frozen or exposed to excessive heat
• For cancer cytogenetics, only tumor tissue should be included, no normal tissue</b>
• Peripheral blood specimen


  Collected in sterile syringes or vacuum tubes with sodium heparin, set up within 24h, kept at room temperature or refrigerated above 4°C

  
• Bone marrow aspirate


  First few milliliters have highest proportion of cells, best for cytogenetics

  
• Amniotic fluid


  Collected from 10 weeks gestation, first few milliliters may be contaminated with maternal cells
• Skin biopsy


  Collected using dermal biopsy punch

  
• Chorionic villi sample


  Delicate collection to avoid confined placental mosaicism and microbial contamination, repeat collection increases miscarriage risk
• Products of conception, stillbirths, tumor biopsies


  One-of-a-kind specimens that cannot be recollected

  
• Microbial contamination is a common problem for many solid tissue samples
• Solid tissue sample transport

  Small samples in sterile culture vessels with growth/tissue culture medium, or sterile saline if no other option, transported on ice
• 1. Glutamine


  Amino acid essential for cell growth, must be stored frozen and added just before use
• Serum

  Essential for cell growth, 10-30% fetal bovine serum (FBS) preferred but has lot-to-lot variation
• Antibiotics


  Added to retard microbial growth, e.g. penicillin/streptomycin, kanamycin, gentamicin, nystatin, amphotericin B
• Mitotic stimulants (mitogens)


  Added to stimulate cell division, e.g. phytohemagglutinin (PHA) for T-lymphocytes
• Growth factors

  Additional factors used to optimize cell growth, e.g. giant cell tumor extract for bone marrow
• Buffers


  Maintain pH between 7.2-7.4, e.g. sodium bicarbonate with 5-10% CO2
• Culture vessels

  Suspension cultures in centrifuge tubes or T-flasks, adherent cultures in T-flasks or in situ on coverslips/plates
• Flask vs in situ culture methods

  Flask allows subculturing but mixes cell colonies, in situ preserves colony origin information

  
• Hanging drop cell culture


  Cells form spheroids at tip of suspended droplet
• Culture maintenance

  Optimal growth at 37°C, lower for cold-blooded animals, peripheral blood requires little maintenance
• True mosaicism (constitutional mosaicism) or an artifact of tissue culture (pseudomosaicism)
• Flask cultures

  1. Cells in the culture medium suspension are placed on the underside of petri dish lids

  2. The cells accumulate at the tip of the drop, spontaneously aggregate, and form spheroids

  
• Hanging drop cell culture


  Cells in the culture medium suspension are placed on the underside of petri dish lids. The cells accumulate at the tip of the drop, spontaneously aggregate, and form spheroids

  
• Culture maintenance

  1. Allowed to grow under specific conditions of temperature, humidity, and pH

  2. Until adequate numbers of dividing cells are present
• Culture maintenance for cold-blooded animals

  Lower temperature required
• Peripheral blood culture maintenance

  Culture vessels are placed in an incubator for a specified period of time, usually 72hr
• Amniotic fluid culture maintenance

  1. Harvested at 6–10 days, sometimes earlier

  2. Flask method, culture interval may be 2 weeks or more
• Biosafety cabinets are used in cell cultures to avoid microbial contamination
• Short-Tandem Repeat (STR) profiling is used to ensure the proper cell line is being cultured and not a different cell
• During sample collection, different cells may be collected instead of the intended cell, often not a problem for blood samples but can be seen in prenatal testing where fetal cells can be contaminated with maternal cells

  
• STR profiling can identify that a specific cell line is being cultured rather than another