CGGE112 Final

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245 Terms

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Constitutional Genetics
heritable and in all body cells
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Suspension culture
A method of growing cells in a liquid growth medium
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Anchorage Dependent culture
cells grow adhered to a substrate
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primary culture
The initial cell culture established from a tissue.
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confluence
the percentage of culture vessel inhabited by cells
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Contact inhibition
arrested cell growth/division as cells come in contact with each other
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subculture
transferring some cells to a new vessel to reduce density
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Growth intervals
the time in culture it takes for cells to growth (human cells divide up to 100 times)
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Lag phase
calls are adapting to culture and dividing so slow
determined by
- number of cells
quality of conditions
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Log phase
cells are actively dividing and proliferate exponentially - optimal time to subculture
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Stationary phase
cells growth slows as cells approach 100% confluence
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Death phase
number of viable cells decreases
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accessioning
receiving a specimen and recording the acquisition in order of receipt
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Antibiotic
prevents bacterial growth Penicillin and streptomycin
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Antifungal
for long term culture to prevent fungal growth use Amphotericin B fungizone
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Centromere

primary constriction - where spindle fibers attach

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p arm
short arm
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q arm
long arm
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telomere
terminal end of chromosome
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metacentric
centromere in middle
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submetacentric
centromere slightly off center
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satellites
repetitive DNA in the short arm of acrocentric chromosomes
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centromeric index
P arm expressed as a % of the total chromosome length
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arm ratio

ratio of Q arm to P arm

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Value
comparing the proportions of arms to one another and the total length
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Analyzing
counting and checking all bands of chromosomes in a cell to look for abnormalities
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counting
counting and examining sex chromosomes in a cells when scanning large number of cells for an aneuploidy
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Scoring
examining a specific chromosomes or a region of a chromosomes in each cell to check for a specific abnormality in a large number of cells
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karyotyping
taking a metaphase spread, isolating chromosomes and lining them up by the homologs
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P-ter
distal p arm
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q-ter
distal q arm
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Ploidy
the number of complete sets of chromosomes in the cells of an organism
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haploid
normal sex cell/gametes with only one set of chromosomes
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diploid
a normal somatic cells that contains two complete sets of homologous chromosomes
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Euploid
containing chromosomes in complete sets - exact multiple of haploid number
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aneuploid
abnormal number of chromosomes in an incomplete set
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polyploidy
any multiple of 23 greater than diploid
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nondisjunction
failure of a pair of homologous chromosomes or sister chromatids to separate normally during division
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monosomy

loss of a chromosome - only viable with X

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trisomy

gain of a chromosome, viable for X, Y, 13,18,21

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Features of Down Syndrome
flat face, eyes up, cognitive delays
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features of edwards syndrome
overlapping digits 1 and 5, rocker bottom feet/heels, head shape issue
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Features of Patau syndrome
cleft lip/palate, extra digits small eyes
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features of Turner syndrome
fetal hydrops or cystic hygroma, kidney/heart problems
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Klinefelters syndrome
infertile small testicals, tall with decreased muscle
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Features of triple XXX
tall, learning delays
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Mosaicisim

one cell line has a mitosis error leading to two viable cell lines

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Chimerism
two cells line come together in one zygote
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5 reasons peripheral blood is preferred specimen

  • easy to obtain/minimally invasive

  • long nicely banded chromosomes

  • lots of cells

  • easy to culture

  • quick turn around

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explant method

Mince sample with a scalpel and stick pieces to the growing surface - cells will migrate from the adherent explants

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Enzyme method of culturing
Digest pieces of sample with a proteolytic enzymes into a near single cell suspension and then disperse cells throughout the growing surface
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7 Factors In cell culture environment
1. essential nutrient
2. suitable vessel
3. osmotic pressure
4. temperature
5. humidity
6. CO2
7. pH
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Advantages of Blood over other samples in culturing
- useful genetics information
- ease of collection
- rapid turnaround
- high quality results
- easy transportation
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Disadvantages of Blood over other samples in culturing

  • cell viability is variable

  • biohazardous

  • may not be representative of the full body

  • rare resistance to mitogen

  • discomfort with needles

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3 Categories of checks to make during sample recipeit
1. Requisition
2. sample label
3. sample quality
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What specimen container is used for blood in cytogenetics
Sodium heparin vacutainer coated in an anticoagulant prevents fibrinogen from converting to fibrin (prevents clotting)
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4 Reasons why we acession
1. acknowledge receipt for tracking
2. document anything that would compromise result
3. followup up on issues
4. assign internal lab number
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Laboratory information system
Software system where lab data is recorded managed and stored
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Examples of "2nd Identifiers"
Health care number, date of birth, medical record number
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Microculture

most common whole blood in media RBC do not interfere with set up quick and easy

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Macroculture
prepared by isolating WBC rich buffy coat - centrifuge
More labour/time intensive
More consistent if cells are counting before set up
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Purpose of Cell Culture
Increase the number of cells to look at
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2 types of culture media
1. Natural
2. Synthetic
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2 types of synthetic media
Basal Balanced Salt solution
Enriched
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Action of Basal Media in Blood Culture media
Essential nutritional requirements and adequate environment for culture
- inorganic ions
- glucose
- amino acids
-proteins
- vitamins
- carbohydrates
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Action of Serum (FBS) in Blood Culture media
Fetal bovine serum (derived from blood clotted) contains critical growth factors
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Action of Antibiotics in Blood Culture media
Penicillin/Streptomycin protects cells from microbial contamination
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Action of L-Glutamine in Blood Culture media
Essential AA for growth that is unstable and needs to be added to media older than 30 days
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Action of Anti-Coagulant in Blood Culture media
Sodium Heparin is inside the vacutainer and added to media to prevent blood from clotting so he WBC are free to grow and divide
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Action of Phytohemagglutinin in Blood Culture media

mitogen provokes immune response which stimulate the T lymphocytes to divide by transforming into lymphoblasts - changes the passage of molecules across the membrane to stimulate DNA and RNA synthesis

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Making media in the lab
Each element is aseptically poured into a common flask and then stored at 4 C until use
- always provide quality control by pouring a small amount into. tube and incubating to ensure there are no contaminants
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3 considerations of blood culture set up

  • set up aseptically by aliquoting media then blood

  • set up each patient in duplicate to account for contamination or ambiguous results

  • incubate duplicates to back up for failure or contamination

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Specimen retention
Do not discard vacutainer after use - store in fridge until suitable cells are confirmed
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4 reasons you may need to revisit a stored sample
1. unexpected findings
2. additional/followup testing
3. sample mix up
4. use it as a control
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Phases of the cell cycle
G1
Synthesis
G2 interphase
Mitosis
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Phases of Mitosis
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
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3 Pre-Analytical steps in Blood Culturing
1. Cell collection
2. Cell swelling
3. Cell fixation
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2 reagents used in Cell collection
Colcemid
Ethidium Bromide
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Outcome of too much time spent in Colcemid

Lots of cells are arresting in metaphase for too long so they continue to condense = TOO SHORT

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Outcome of too little time spent in Colcemid

Very few cells in metaphase with some in prophase and prometaphase = Long and squiggly

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Reagent for Cell swelling
Hypotonic solution of KCL

0.075M for blood harvest
0.03M for anchorage dependent harvest
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Outcome of too much time spent in Hypotonic KCL
Cells swell and membrane is stretched too much making it weak - chromosomes scatter is cells burst
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Outcome of too little time spent in Hypotonic KCL
Cells not swelling and chromosomes will not be spread enough for analysis - too much overlap
Possible RBC remain
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Outcome of too high concentration of Hypotonic KCL
Increase salt concentration moves towards isotonic so the cells are not swelling enough
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Outcome of too low concentration of Hypotonic KCL

Decreased concentration salt of makes a more hypotonic solution so much more water enters the cells - bursting

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Reagent for Cell Fixation
Fixative 3:1 Methanol: Glacial Acetic Acid
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Action of Methanol in Cell Fixation
dehydrates cells, precipitates DNA and hardens chromatin + membrane
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Action of Acetic Acid in Cell Fixation
Softening agent to increase spreading
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2 phases of cell fixation
1. Prefix
2. Full - fix
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Pre fix in cell fixation
A small amount of fixative added to hypotonic solution first to ease the cells into the process
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Full Fix in Cell fixation
Replacement of entire hypotonic solution with fixative resuspending the pellet each time to remove cellular debris
3x
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Outcome of not fully resuspending pellet between Fix changes
Poorly spread cells/clumping
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Disposal of Fix
In special waste container stored in flammable cabinet
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Contamination check in blood culturing
Prior to disturbing the sample asses the media clarity and colour
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Centrifuging in blood culturing
Used before changing solution, force a pellet of the cells we want to keep at the bottom leave the supernatant to discard at the top
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Suctioning in blood Harvest/Culture
Culture - sterile pipette
harvest - non sterile clean pipette

Always changes between patients
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The consequence of failure to resuspend at the hypotonic stages of harvest
Too much cytoplasm
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Harvest Documentation importance
Continuity and transparency - clear and appropriate documentation is completed
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2 Goals of Aseptic Technique
1. prevent spread of microorganisms from the specimen to the environment and us
2. Prevent spread of microorganisms from us and environment to the specimen
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Macroscopic evidence of contamination in media
Turbidity of the media
pH shift as the colour of the indicator