Clinical Applications and Current Issues of Cytogenetics PDF

Clinical Applications and Current Issues of Cytogenetics Vienna Jamaica B. Cari-cari, RMT Lesson Objectives: 1. Characterize the different laboratory techniques in cytogenetics Examining and Analyzing Chromosomes - The study of chromosomes using traditional cytogenetic techniques requires...

Clinical Applications and Current Issues of Cytogenetics Vienna Jamaica B. Cari-cari, RMT Lesson Objectives: 1. Characterize the different laboratory techniques in cytogenetics Examining and Analyzing Chromosomes - The study of chromosomes using traditional cytogenetic techniques requires cells that are actively dividing. - Chromosomes are individually distinguishable under the light microscope ONLY during cell division and are best examined during metaphase. Metaphase chromosomes are obtained from: A. spontaneously dividing cells - bone marrow, lymph nodes, amniotic fluids, solid tumors tissue biopsies, chorionic villi - NO need of mitotic stimulants B. Cells that are cultured and chemically induced to divide in vitro - Prenatal diagnosis: Amniocytes - Postnatal diagnosis: Peripheral blood smears - Cancer studies: Bone marrow aspirate Cell Culture and Harvest Overview The MOST CRITICAL requirement: - Living cells capable of cell division should be received by the laboratory Specimen container: - Sterile - Properly labeled - patient’s name and a second identifier Overview of cell culture and harvest Cell Culture and Harvest Specimen Collection and Handling Sample Requirements PERIPHERAL BLOOD SPECIMENS - Sterile syringes, vacuum tubes - preservative-FREE Sodium heparin - Best result: Culture within 24 hours of collection - Room temp or Refrigerated above 4°C - Culture medium is sometimes added to small blood samples to avoid drying up - Repeat sample collection if: - Clotted, on ice, more than 24h old Sample Requirements BONE MARROW ASPIRATES - Same with peripheral blood - Processed without delay - Best sample for Cytogen lab: First few mL - Contain the highest proportion of cells - Later portion is diluted with blood, hence a reduced number of actively dividing cells in the sample Sample Requirements AMNIOTIC FLUID SPECIMENS - Amniocentesis (as early as 10 weeks of gestation) - Sterile container approved for cell culture - 15-30 mL - Earlier than 15 weeks = draw 1 mL for each week - Discard first few mL - may be contaminated with maternal cells SAMPLE REQUIREMENTS AMNIOTIC FLUID SPECIMENS Sample Requirements SOLID TISSUE BIOPSIES - Sources: tissue from stillbirths*, products of conception*, skin, chorionic villi**, lymph node, solid tumor - *One-of-a-kind specimens; can NOT be recollected - **Chorionic villi collection increases risk of miscarriage - common problem: Microbial contamination Sample Requirements SOLID TISSUE BIOPSIES - Small samples should be in sterile culture vessels containing growth medium (NOT formalin) - Larger samples are sent to the lab in toto for dissection - Solid tissue samples may be transported and stored on ice - slows enzymatic degradation of tissues - slows microbial growth Cell Culture and Harvest Culture Initiation THE BASICS OF A CELL CULTURE LABORATORY CULTURE INITIATION Growth Media - All specimens for chromosome preparation are grown and maintained in an aqueous growth medium A. Media for specific cell types B. Media for a broad spectrum of cell types - Commercial media are available either in A. powder forms that must be rehydrated B. ready-to-use aqueous solutions CULTURE INITIATION Growth Media - All culture media are balanced salt solutions with additives including salts, glucose, and a buffering system - Often used pH indicator: Phenol red - Acidic = yellow - Basic = pink or purple CULTURE INITIATION Growth Media - L-Glutamine - Serum - Antibiotics - Mitogens (Mitotic Stimulants) - Growth Factors CULTURE INITIATION Growth Media L-Glutamine - Added to culture medium just prior to use - Stored frozen - Unstable when stored (breaks down to D-glutamine) - D-glutamine can NOT be used by cells CULTURE INITIATION Growth Media Serum - Contains mitogen which helps in cell growth and division - Too little = does not allow maximum cell growth - Too much = detrimental effect on cell growth - Preferred: 10-30% Fetal Bovine Serum (FBS) CULTURE INITIATION Growth Media Antibiotics - Microbial inhibitors - Commonly used Bacterial inhibitors in tissue cultures: Penicillin/Streptomycin, Kanamycin, Gentamicin - Commonly used Fungicides: Nystatin, Amphotericin B CULTURE INITIATION Growth Media Antibiotics Signs of contamination: - Bacterial contamination: - Naked eye: Cloudy - Microscope: Presence (rods, cocci, and other morphologies) - Fungal contamination: - Naked eye: Woolly masses - Microscope: Branching hyphae CULTURE INITIATION Growth Media CULTURE INITIATION Growth Media Mitogens (Mitotic Stimulants) - For cells (esp. Lymphocytes) that do not spontaneously divide - T-cell mitogen: - Phytohemagglutinin (PHA) – Red kidney bean extract - B-cell mitogens: - EBV, E. coli Lipopolysaccharide, Protein A, Pokeweed CULTURE INITIATION Growth Media Growth Factors - Giant Cell Tumor extract (GCT) for bone marrow culture - Specially formulated amniotic fluid culture media CULTURE INITIATION Culture Vessels Samples: Culture Vessels or Methods: Blood and Bone Marrow - Sterile centrifuge tubes - Consist of single free-floating cells - Tissue Culture Flasks (T-flasks) Amniotic fluid, Chorionic villi, - T-flasks Skin biopsies, Solid tissues - In situ - Need to attach to a surface CULTURE INITIATION Culture Vessels Flask Method - Cells are grown on the inner surface of T-flasks - Cultures are treated with an enzyme such as trypsin - To remove the cells from the surface of the flask - To allow collection and harvest of individual cells CULTURE INITIATION Culture Vessels Flask Method CULTURE INITIATION Culture Vessels In Situ Method - Cells are grown directly on coverslips in small petri dishes, or in slide chambers - Harvested as primary cultures - NO enzymatic treatment prior to harvest - Subculture is NOT necessary CULTURE INITIATION Culture Vessels In Situ Method CULTURE INITIATION Culture Vessels Advantages of In Situ Method over the Flask Method - Main advantage: Provides information about colony of origin of a cell - Helps determine true mosaicism vs pseudomosaicism True Mosaicism Pseudomosaicism multiple colonies from more a single colony with all or some than one culture with the same cells exhibiting a chromosomal chromosomal abnormality abnormality CULTURE INITIATION Culture Vessels Advantages of In Situ Method over the Flask Method - Other advantage: Shorter turnaround time (TAT) since only primary cultures are harvested - Flask cultures are often sub-cultured, thus requiring additional days of culture time CULTURE INITIATION Specimen Preparation Samples: Preparation: Whole blood, Can be added directly to the culture medium Bone marrow aspirate White blood cells Can be separated (buffy coat) from other blood elements prior to inoculation to culture medium Amniotic fluid Centrifuged (800-1000 rpm) to retrieve viable cells. (amniocytes) - cell pellet - inoculated to culture media - supernatant - used in biochem tests for fetal defects Solid Tissue Must be disintegrated or finely minced Cell Culture and Harvest Culture Maintenance CULTURE MAINTENANCE Culture Maintenance Incubators maintained at 37 °C (optimal temp) Open System Closed System - Culture media are loosely capped - Culture media are tightly capped - Humidity = 97% - Humidification is self-maintained - CO2 = 5% - CO2 incubators are NOT required - pH = 7.2–7.4 - Risk of microbial contamination - Required for samples grown on coverslips using the in situ method CULTURE MAINTENANCE Growth Intervals Samples: Growth interval: Peripheral blood 72 hours Bone marrow Harvested directly, or additional 24-48 hours Amniotic fluid (in situ) 6-10 days Amniotic fluid (flask) 2 weeks or more Solid tissue (flask) 2 weeks or more CULTURE MAINTENANCE Growth Intervals Lag phase - No increase in number of living cells; Cells do not divide Number of cells Log/Exponential phase - Exponential increase in number; Cells actively proliferate Plateau/Stationary phase - Growth rate and death rate are equal; Growth limiting factors, depletion of nutrients CULTURE MAINTENANCE Growth Intervals Cell Culture and Harvest Cell Harvest CELL HARVEST Cell Harvest Cell Harvest - the procedure of collecting the dividing cells at metaphase, their subsequent hypotonic treatment and fixation, and the placement of the chromosomes on glass slides so they may be stained and microscopically examined. CELL HARVEST Mitotic Inhibitor - Arrest cell division - Colcemid - analog of Colchicine - binds to tubulin, thus obstructing the formation of spindle fibers or destroying formed spindle fibers - longer exposure = shorter chromosomes – preferred: longer chromosomes CELL HARVEST Hypotonic Solutions - Added to cells after colcemid - Water moves into the cell (osmosis) → cell swells → chromosomes spread out - Exposure too long = cells burst - Exposure too short = poor spreading of chromosomes - 0.075 M potassium chloride (KCl) - 0.8% sodium citrate - dilute balanced salt solutions - dilute serum CELL HARVEST Fixative - 3 parts absolute methanol + 1 part glacial acetic acid - Stops the action of the hypotonic solution - Fixes the cells in the swollen state - Lyses RBCs (if present) CELL HARVEST Well-prepared slide: Slide Preparation - sufficient numbers of metaphases - Final step before staining/banding - well spread - not crowded - After fixation, cells are dropped - with minimal onto the glass slide overlapping only - In situ cultures are not dropped - no visible cytoplasm because they are already attached to a coverslip or other solid surface - Aged (after preparation) - Phase contrast microscope A. Overnight at 60 C B. 1 hour at 90 C C. Briefly using UV light CELL HARVEST CELL HARVEST Chromosome Staining and Banding Chromosome Banding and Staining - Stain bands along the entire chromosome: G-, Q-, R-banding - Stain specific chromosome areas: C-, T-, NOR-banding Chromosome Banding and Staining Technique: Treatment/Stain: Banding Pattern: G-banding Treatment: Trypsin Dark bands: A–T rich Stain: Giemsa Light bands: G–C rich R-banding Heated with Phosphate buffer Dark bands: G–C rich Stain: Giemsa Light bands: A–T rich Q-banding Stain: Quinacrine dihydrochloride Bright fluorescence : A–T rich (a fluorochrome) Dark/Dull bands: G–C rich Chromosome Banding and Staining Technique: Stained: Treatment/Stain: Banding Pattern: C-banding Constitutive Treatment: Barium Dark bands = Constitutive heterochromatin hydroxide Heterochromatin Stain: Giemsa * q arm of Y chromosome * Around centromere T-banding Telomeres Stain: Giemsa Dark bands = Telomere NOR-banding Ribosomal RNA Stain: Silver nitrate Dark bands = rRNA genes * NORs of Acrocentrics Chromosome Banding and Staining Additional notes - G-light bands are biologically more significant because they represent the active regions of the chromosomes, while the G-dark bands contain relatively few active genes - Q-banding – first banding method developed for human chromosomes - Distal long arm of the Y chromosome is the most fluorescent site in the human genome - C-banding is useful for determining the presence of dicentric and pseudodicentric chromosomes Chromosome Banding and Staining G-banding Chromosome Banding and Staining R-banding Chromosome Banding and Staining Q-banding Chromosome Banding and Staining C-banding Chromosome Banding and Staining T-banding Chromosome Banding and Staining NOR-banding Karyotyping Karyotyping Information obtained from a karyotype: - Number of chromosomes - Sex chromosome content - Presence or absence of individual chromosomes - Nature and extent of structural abnormalities Karyotyping Fluorescent in-situ Hybridization (FISH) FISH - “Marriage of classical cytogenetics & molecular technologies” - largely replaced special stains in many laboratories - Major advantages: - Ability to detect and characterize chromosomal abnormalities that are not routinely delineated with standard banding studies - Ability to study uncultured material (rapid results) FISH Principle - Based on fluorescently-labeled probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity - When two complementary sequences find each other, they will bind together or “Hybridize”  FISH Basic Procedure 1. The Probe and the Target DNAs (on fixed slides) are denatured using high temperature incubation in a formamide/salt solution. 2. The probe is applied in great excess, ensuring that the probe anneals to the specific target DNA. 3. Probe detection is accomplished by: a. ultraviolet (UV)-light excitement of a fluorochrome, such as fluorescein-5-thiocyanate (FITC) or rhodamine, that is directly attached to the probe DNA; or b. incubation of a hapten (biotin or digoxigenin)-labeled probe with a fluorescent conjugate FISH Basic Procedure FISH Results - Deletion = probe will NOT bind/hybridize - Duplication = more of the probes will bind/hybridize FISH Results FISH Results FISH Sample Results - specimen: amniocytes - probes for chromosomes 18 (2 aqua signals), X (green signal), and Y (orange signal). - Nuclei are counterstained blue with DAPI FISH Sample Results - specimen: amniocytes - probes for chromosomes 13 (2 green signals) and 21 (3 orange signals) = Trisomy 21 - Nuclei are counterstained blue with DAPI  Any Questions? (You, after every discussion) Reference book: Gersen, S. and Keagle, M. (2013) The Principles of Clinical Cytogenetics. 3rd Edition.