CGGE112 Final

Constitutional Genetics

heritable and in all body cells

Suspension culture

A method of growing cells in a liquid growth medium

Anchorage Dependent culture

cells grow adhered to a substrate

primary culture

The initial cell culture established from a tissue.

confluence

the percentage of culture vessel inhabited by cells

Contact inhibition

arrested cell growth/division as cells come in contact with each other

subculture

transferring some cells to a new vessel to reduce density

Growth intervals

the time in culture it takes for cells to growth (human cells divide up to 100 times)

Lag phase

calls are adapting to culture and dividing so slow
determined by
- number of cells
quality of conditions

Log phase

cells are actively dividing and proliferate exponentially - optimal time to subculture

Stationary phase

cells growth slows as cells approach 100% confluence

Death phase

number of viable cells decreases

accessioning

receiving a specimen and recording the acquisition in order of receipt

Antibiotic

prevents bacterial growth Penicillin and streptomycin

Antifungal

for long term culture to prevent fungal growth use Amphotericin B fungizone

Centromere

primary constriction - where spindle fibers attach

p arm

short arm

q arm

long arm

telomere

terminal end of chromosome

metacentric

centromere in middle

submetacentric

centromere slightly off center

satellites

repetitive DNA in the short arm of acrocentric chromosomes

centromeric index

P arm expressed as a % of the total chromosome length

arm ratio

ratio of Q arm to P arm

Value

comparing the proportions of arms to one another and the total length

Analyzing

counting and checking all bands of chromosomes in a cell to look for abnormalities

counting

counting and examining sex chromosomes in a cells when scanning large number of cells for an aneuploidy

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

karyotyping

taking a metaphase spread, isolating chromosomes and lining them up by the homologs

P-ter

distal p arm

q-ter

distal q arm

Ploidy

the number of complete sets of chromosomes in the cells of an organism

haploid

normal sex cell/gametes with only one set of chromosomes

diploid

a normal somatic cells that contains two complete sets of homologous chromosomes

Euploid

containing chromosomes in complete sets - exact multiple of haploid number

aneuploid

abnormal number of chromosomes in an incomplete set

polyploidy

any multiple of 23 greater than diploid

nondisjunction

failure of a pair of homologous chromosomes or sister chromatids to separate normally during division

monosomy

loss of a chromosome - only viable with X

trisomy

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

Features of Down Syndrome

flat face, eyes up, cognitive delays

features of edwards syndrome

overlapping digits 1 and 5, rocker bottom feet/heels, head shape issue

Features of Patau syndrome

cleft lip/palate, extra digits small eyes

features of Turner syndrome

fetal hydrops or cystic hygroma, kidney/heart problems

Klinefelters syndrome

infertile small testicals, tall with decreased muscle

Features of triple XXX

tall, learning delays

Mosaicisim

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

Chimerism

two cells line come together in one zygote

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

explant method

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

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

7 Factors In cell culture environment

1. essential nutrient

2. suitable vessel

3. osmotic pressure

4. temperature

5. humidity

6. CO2

7. pH

Advantages of Blood over other samples in culturing

• useful genetics information

• ease of collection

• rapid turnaround

• high quality results

• easy transportation

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

3 Categories of checks to make during sample recipeit

1. Requisition

2. sample label

3. sample quality

What specimen container is used for blood in cytogenetics

Sodium heparin vacutainer coated in an anticoagulant prevents fibrinogen from converting to fibrin (prevents clotting)

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

Laboratory information system

Software system where lab data is recorded managed and stored

Examples of "2nd Identifiers"

Health care number, date of birth, medical record number

Microculture

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

Macroculture

prepared by isolating WBC rich buffy coat - centrifuge
More labour/time intensive
More consistent if cells are counting before set up

Purpose of Cell Culture

Increase the number of cells to look at

2 types of culture media

1. Natural

2. Synthetic

2 types of synthetic media

Basal Balanced Salt solution
Enriched

Action of Basal Media in Blood Culture media

Essential nutritional requirements and adequate environment for culture

• inorganic ions

• glucose

• amino acids -proteins

• vitamins

• carbohydrates

Action of Serum (FBS) in Blood Culture media

Fetal bovine serum (derived from blood clotted) contains critical growth factors

Action of Antibiotics in Blood Culture media

Penicillin/Streptomycin protects cells from microbial contamination

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

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

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

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

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

Specimen retention

Do not discard vacutainer after use - store in fridge until suitable cells are confirmed

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

Phases of the cell cycle

G1
Synthesis
G2 interphase
Mitosis

Phases of Mitosis

Prophase
Prometaphase
Metaphase
Anaphase
Telophase

3 Pre-Analytical steps in Blood Culturing

1. Cell collection

2. Cell swelling

3. Cell fixation

2 reagents used in Cell collection

Colcemid
Ethidium Bromide

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

Outcome of too little time spent in Colcemid

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

Reagent for Cell swelling

Hypotonic solution of KCL

0.075M for blood harvest
0.03M for anchorage dependent harvest

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

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

Outcome of too high concentration of Hypotonic KCL

Increase salt concentration moves towards isotonic so the cells are not swelling enough

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

Reagent for Cell Fixation

Fixative 3:1 Methanol: Glacial Acetic Acid

Action of Methanol in Cell Fixation

dehydrates cells, precipitates DNA and hardens chromatin + membrane

Action of Acetic Acid in Cell Fixation

Softening agent to increase spreading

2 phases of cell fixation

1. Prefix

2. Full - fix

Pre fix in cell fixation

A small amount of fixative added to hypotonic solution first to ease the cells into the process

Full Fix in Cell fixation

Replacement of entire hypotonic solution with fixative resuspending the pellet each time to remove cellular debris
3x

Outcome of not fully resuspending pellet between Fix changes

Poorly spread cells/clumping

Disposal of Fix

In special waste container stored in flammable cabinet

Contamination check in blood culturing

Prior to disturbing the sample asses the media clarity and colour

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

Suctioning in blood Harvest/Culture

Culture - sterile pipette
harvest - non sterile clean pipette

Always changes between patients

The consequence of failure to resuspend at the hypotonic stages of harvest

Too much cytoplasm

Harvest Documentation importance

Continuity and transparency - clear and appropriate documentation is completed

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

Macroscopic evidence of contamination in media

Turbidity of the media
pH shift as the colour of the indicator