Basic Cytogenetics Flashcards
What is PHA and what does it do?
Phytohemagglutinin - an extract from the red kidney bean.
It primarily stimulates T-cells which respond to PHA as they would a foreign invader and differentiate into a immature lymphoblastic T-cell. This allows them to synthesise DNA and undergo division.
This response occurs during the first 24 hours and removal of the PHA does not stop the stimulation.
What is the metaphase stage of the cell cycle?
The point where a cell is going to divide.
Chromosomes are condensed and visible and lined up on the spindle ready to separate.
In order to obtain metaphases, what do we require?
Give some examples.
Either spontaneously dividing tissue:
- bone marrow and lymph node
- tumour tissue
- CVS tissue
- some leukaemia blood
- fetal or newborn blood.
Or tissue which can be stimulated:
- blood (mature lymphocytes)
- ?amniotic fluid?
What is Colcemid and what does it do?
What are the pros and cons?
A mitotic arrestant.
It inhibits the formation of the spindle. When the chromosomes line up at the metaphase plate, there is no spindle for them to attach to and therefore they cannot divide.
The chromosomes are then arrested in this position.
Pros:
Increased concentration dramatically increases mitotic index, straightens chromosomes, crisps chromatid edges and increases spreading as it releases them from the mitosis apparatus.
Cons:
Increased exposure time and concentration causes chromosomes to condense. Therefore concentration and exposure time kept to a minimum to achieve balance between mitotic index and quality of mets.
What is Thymidine and what does it do?
It is a cell cycle inhibitor/block. It can be used to synchronise the division of cells.
Excess Thymidine inhibits DNA synthesis and means cells collect at S-phase. The effect can be reversed by washing out the thymidine.
This means the cells in the culture are synchronised to the same point in the cycle. Washing out of the Thymidine allows to cells to continue their cycle and therefore they all continue from this point.
Why do we do a 72 hour culture in bloods?
These cultures have been treated with PHA. This stimulates them to undergo several rounds of division, the optimum time to harvest these samples is 60-70 hours and most cells will be on their second division by then.
What is BrdU used for?
It mitigates the condensation effect that colcemid causes.
What is the purpose of hypotonic solution? What happens if exposure is too long?
Creates a concentration gradient so that water moves into a cell by active transport. This increases the cell volume so that the chromosomes can spread out.
Hypotonic swells the lymphocyte cells and allows chromosomes to spread. Red blood cells burst/lyse because they can’t swell as much.
However! Likewise if a wbc is exposed to hypotonic for too long then it can burst and some or all of the chromosomes will escape!
What is used to ‘fix’ cells?
What does it do?
Is there a key step?
A mix of methanol and acetic acid, 3:1.
Fixation:
- kills and preserves cells
- removes water
- hardens membranes and chromatin
- prepares chromosomes for banding.
1st fix is most important - it creates turbulence so must be added slowly to prevent metaphase loss.
Subsequent fixes can be added quicker as cells are hardened.
Why should ‘fix’ be made fresh each time?
Methanol and acetic acid react together and absorb water from the air which deteriorates its fixing properties.
Are oncology samples stimulated to divide? Why?
No, acquired karyotype is obtained by looking at metaphases from spontaneously dividing cells.
Stimulation would mainly cause ‘normal’ cells to divide. There are ways stimulate abnormal cells but we don’t use them.
What are the pros and cons of using a blood sample to study a persons genetics?
Pros:
- relatively non-invasive/easy to obtain
- low cost
- short culture time
- widespread technique
- high quality chromosomes.
Cons:
- mosaicism! Abnormal cell line may get ‘cultured out’
- abnormal cell line may not be represented in blood tissue at all e.g. blood comes from mesoderm embryonic layer and won’t demonstrate genetic abnormalities confined to ectoderm or endoderm.
What are the main components of blood? Also name some of the other components.
- red blood cells (erythrocytes)
- white blood cells (leukocytes)
- platelets
Also:
Serum protein, lipids, fibrinogen, glucose, water, gases, salts, antibodies, excretory products (urea).
What are the different white blood cells present in peripheral blood? Which make up the highest %?
Lymphocytes 20-40% Neutrophils 40-60% Basophils 0.1% Eosinophils 1-3% Monocytes 4-8%
Why do we use less blood when setting-up a newborn sample?
They have a higher red blood cell count.
Red blood cells use up vital nutrients from the culture media.
Also, too many red blood cells may interfere with lymphocytes reaction to PHA (stimulation) which will ultimately effect division and growth of cells.
