Hi, this week I shall give a brief overview on what Cytogenetics lab does. This is one of the few labs which still relies alot on manual work by medtechs.
Cytogenetics is the study if chromosome structures and their behaviour to discover any abnormalities in humans, and identify the medical condition that is caused by the abnormality in that particular chromosome. There are many different types of abnormalities and every type contributes to a different medical condition.
All cells undergo the cell cycle, a process by which the cell replicates through different phases. One of which is the M phase, where an adult cell splits into two daughter cells. The M phase is further categorized into two different processes: mitosis, where cell's chromosomes are divided between the two daughter cells, and cytokinesis, where the cell's cytoplasm divides and forms distinct cells. Prometaphase, metaphase, anaphase and telophase are stages that take place within mitosis.
All cells undergo the cell cycle, a process by which the cell replicates through different phases. One of which is the M phase, where an adult cell splits into two daughter cells. The M phase is further categorized into two different processes: mitosis, where cell's chromosomes are divided between the two daughter cells, and cytokinesis, where the cell's cytoplasm divides and forms distinct cells. Prometaphase, metaphase, anaphase and telophase are stages that take place within mitosis.
M Phase in Cell Cycle
(Source: http://www.infoplease.com/cig/biology/cell-cycle-interphase-mitosis-cytokinesis.html)
Metaphase is a stage in which condensed chromosomes align in the middle of the cell before being separated into each of the two daughter cells. Because the structure of chromosomes is the clearest at this stage, cells will be stimulated to stop growing at this stage for analysis. The metaphase chromosomes can be studied in spontaneously dividing cells or in cells that have been stimulated to divide in culture.
Metaphase
(Source: en.wikipedia.org/wiki/Metaphase)
3 general processes that must take place in order to study the chromosomes are:
1) Culturing – Supplying the cells with nutrients to allow to proliferation
Before obtaining the metaphases of cells, cultures may be needed. The decision of whether to culture the cells lies on the specimen type. Spontaneously dividing samples such as bone marrow and lymph node may be set up for a direct harvest. Other samples such as tissues may require culturing for several days to allow the cells to proliferate. Once the sample has been cultured, harvesting is performed.
2) Harvesting – Obtain metaphases of cells, the stage at which chromosomes can clearly be seen
When cells are growing spontaneously, they will be harvested to obtain metaphases for analysis. In harvesting, three standard protocols will be used: mitotic arrest with Colcemid, hypotonic treatment with KCl (potassium chloride) and fixation with 3:1 methanol: acetic acid.
a) Mitotic arrest with Colcemid – Cells are arrested at the metaphase stage to enable the capturing and analysis of the chromosomes. Colcemid is used to prevent spindle formation, a process by which sister chromatids are pulled to opposite poles for incorporation of into the 2 daughter cells. It also promotes chromosome condensation, a process that can be affected by increased exposure time and concentration.
Different exposure time to Colcemid can affect the quality and quantity of the chromosomes; the condensation effect is greater when exposed for a longer period of time (ie. chromosomes are smaller in size but more). However, different culture and harvest methods may react differently to the Colcemid reaction. Therefore, to obtain the desired result, cells will be exposed to Colcemid for:
2 hours (long exposure time: more but shorter chromosomes)
20 mins (short exposure time: lesser but longer chromosomes)
b) Hypotonic Treatment with KCl – After arresting the cells at metaphase stage, they need to be treated with a hypotonic saline solution to increase the cell volume so that chromosomes can spread out. However, prolonged exposure may cause weakening of the cytoplasmic membrane, thus increasing the risk of the cell bursting and chromosomes to escape.
c) Fixation (3:1 methanol: acetic acid) – The purpose of fixing the cells is to remove the water content as well as to preserve them be hardening the membranes and chromatin, and somehow prepares chromosomes for the bending procedure.
3) Karyotyping – Chromosomes will be pair up to check for abnormalities
After fixing the cells, the slide needs to be stained to be able to visualize the bands on the chromosomes. Stained slides can now be karyotyped.
Normal Karyotype
Down Syndrome (extra chromosome 21)
(Source: http://www3.geneticsolutions.com/?id=1530:1873)
Klinefelter Syndrome (extra sex chromosome X)
(Source: http://www3.geneticsolutions.com/?id=1530:1873)
Ka Hang
TG02
8 comments:
October 18, 2008 at 9:45 PM
Hi Ka Hang!
The topic of your sharing looks interesting, so I hope you would not mind me asking a few questions.
1) For the mitotic arrest, I am unclear on the distinction between shorter and longer chromosomes... What do you mean by shorter and longer?
2) Is there any example of how different culture and harvest methods affects the colcemid reaction?
3) Do you know the exact mechanism of colcemid reaction on the spindle formation prevention and chromosome condensation?
4) I am interested in the Klinefelter Syndrome... How will an extra sex chromosome X affect the patient?
That's all to my curiousity.
Thank you very much!
Best wishes
Quan Jun
TG02
October 20, 2008 at 5:20 PM
hey kahang,
may i know the nutrients you mentioned for culturing right, what nutrients to do you used for culturing and is it different nutrients for different cells.
also, for the hypotonic treatment with KCl, you mentioned that long exposure can cause cell bursting and chromosomes to escape, thus how do i know how long should i do it in order for it to be okay?
Thanks!
Liyanah Zaffre
0607718D
TG02
October 22, 2008 at 10:21 AM
Hi Ka Hang,
May i ask what type of stain do you used to karyotype and what is the mechanism of aciton behind it?
Thanks!
From: Benjamin Ma
Class;tg01
0606181F
October 22, 2008 at 11:12 PM
Hi Ka Hang,
may I know what is 'bending procedure'?
also, I'd like to add on to QJ's question about the long/short chromosome - you also mentioned 'more/less chromosomes'. so,
for example, if I treat the cell with Colcemid for 20 minutes, then stain, I will not get to see all 23 pairs of chromosome? (and if 1 hour treatment, I will see complete set after the staining?)
Thanks =)
Nor Liyana
0607927A
TG02 - Group 8
October 25, 2008 at 9:49 PM
Hi QJ,
1) There are as much as 20 bands in each chromosome. Most of the chromosomes you see in karyotype diagrams do not have that many bands; this is because they are compact (short chromosomes). So if we were to get longer chromosomes, more bands can be seen and this may help in the identification of deletions or translocations of bands. In summary, shorter and longer is referring to the numbers of bands seen.
2) This depends on the nature of specimens. For example, for what I know, bone marrow specimens will produce shorter chromosomes.
3) Clocemid depolymerises microtubules (refer to metaphase diagram on blog post), thus limiting microtubule formation and inactivates spindle fibre formation in metaphase.
4) Ok first of all, people with Klinefelter Syndrome all have a male phenotype. These males can be affected in several ways:
Physical development: As babies, they have weak muscles and reduced strength. Their physical development may be slower than other infants. After about age four, they tend to be taller and may have less muscle control and coordination than other boys their age.
As these males enter puberty, they wont make as much testosterone as other boys. This leads to a taller and less muscular body, less facial and body hair, as well as broader hips. As teens, they may also have larger breasts, weaker bones, and a lower energy level than other boys.
Although affected males can have normal sex lives, they usually make little or no sperm. 95 to 99% of them are infertile because of their inability to produce sperms.
Besides all these, they may also experience delay in language and social development, such as learning to talk late, trouble using language to express thoughts and needs, problems reading, and trouble processing what they hear. Socially, they tend to be quieter and less demanding and shy.
Ka Hang
TG02
October 25, 2008 at 9:57 PM
Hi Liyanah,
Yes different types of mediums are used for different cell types. In culturing of amniotic fluid cells, complete chang basal and BIO-AMF-2 are used. These 2 mediums are designed for the culturing of human amniotic cells. Antibiotics and L-glutamine are present in both mediums to prevent the growth of bacteria that may cause contamination and to provide an essential amino acid respectively.
Two types of mediums are used so in case if the cells do not grow well in any one of the medium, the other can act as a back-up. All mediums contain a pH indicator to indicate pH change in the culture. For blood specimens, RPMI and M199 mediums are used as mediums.
As for the hypotonic treatment, there is a standard protocol stating the recommended KCl exposure time. To prevent cells from bursting, medtechs must strictly adhere to the recommended time. To facilitate that, a timer is always used to keep tract of the exposure time.
Ka Hang
TG02
October 25, 2008 at 10:05 PM
Hi Benjamin,
In chromosomes study, are different staining techniques to demonstrate different bandings. In my lab, the technique used is GTG, which demonstrates G-bandings by the use of trypsin and Giemsa + Wright’s stain.
Trypsin activity greatly determines the quality of the chromosome bands. The exposure time can be adjusted if the bands appear too dark or light after checking the first slide stained. The activity can also be altered by factors such as pH, temperature and concentration. At higher pH, the activity of trypsin increases and at lower pH, the activity decreases. Therefore for optimum demonstration of bands, the pH of trypsin must be controlled using the 7.5% sodium bicarbonate and exposure time must be adjusted accordingly.
Giemsa + Wright’s stain is used because it is able to show the fine bands of the chromosomes, which will be useful for chromosome analysis and in-situ coverslip cultures where more cytoplasmic background may be seen.
Ka Hang
TG02
October 25, 2008 at 10:09 PM
Hi Liyana,
Sorry typo error, I actually meant ‘banding’, not ‘bending’. Banding is the range of grey shadings with a fair amount if contrast shown along the entire chromosome after staining.
For the long/short chromosome, you can refer to my reply to qj. For more/less, you’ve got the wrong idea. The more/less is referring to the number of individual cells that are arrested at metaphase stage and not the number of chromosomes. Within every normal cell, there are 23 pairs of chromosomes.
For example, if treated for 20 mins, there may be 50 metaphases arrested (50 cells). With 23 pairs of chromosomes present in one metaphase, there are 50 of such metaphases that can be analysed. If treatment were to be 2 hours, 150 metaphases (150 cells) may be arrested. In this case, you would have more metaphases to can choose to analyse.
Ka Hang
TG02
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