Week 17

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.

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

(Source: arnica.csustan.edu/.../cell_division.htm)

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

Answers to Week 16 posting (For Li Ping and Ernest)

I understand that the answers I provide is quite long, but it's going to give you a clearer picture in some of the applications of flow cytometer, especially regarding my MP. Please don't give up on the 'long' post and read it =)

Before I start answering questions, I would like to touch on a few terms first (you could skip this part if you wish):

1) Fluorescence-based techniques
2) What is autofluorescence?
3) What is ‘true negative’?

Answers:

1) You should know that fluorescence-based techniques use fluorochromes in many different ways; one of the ways is to utilize the ability of dye to influx in the cells and ability of cells to efflux out the dye. Examples of such dyes are: Hoechst 33342 and Rhodamine.
These fluorochromes emit a certain ‘light’ wavelength after excitation from a light source (eg. Laser beam) that is detected by a detector in the Flow cytometer. Please take note that flow cytometer is mostly used to analyze live cells.
2) Autofluorescence is ‘weak’ autofluorescence emitted by cells not stained with the fluorochromes. Therefore, they are considered to be ‘false positive’ fluorescence.
3) As you will know, before the immunofluoresence staining of a cell, the cell should not express any fluorescence, right? So, ‘true negative’ is the sum of ‘negative’ and ‘false positive’ fluorescence.

To Li Ping’s Questions

Answers:

I believe you are confuse over the word ‘true negative’, when I mention about ‘true negative’, I meant the portion of cells in a heterogeneous cell population that are not stained positive. Only 'unstained' tube consist of 'negative' results as it is not stained at all. Other 'stained' tubes consist of both stained and unstained cells, so the data from 'unstained' tube is used to identify the cells that were not stained, something like a negative control.

And yes, it is correct to say that the value obtained will be used as benchmark, however, this benchmark is to identify the amount of ‘true negative’ population in other tubes. You must know that the cell sample we used is a heterogeneous population of cells and so, not all cells will be stained with the dye we used.

Different gates are used for different experiments. For my experiment, I used that series of gating that I mentioned before:

a. Scatter gate:
used to gate cluster of cells which our cells of interest is located and exclude all other ‘rubbish’ cells.
b. Side Scatter (SSC) gate:
used together with FSC for doublets discrimination and exclude all but single cells.
c. Forward Scatter (FSC) gate:
used together with SSC for doublets discrimination and exclude all but single cells.
d. Viability gate:
identify viable, living, cells. Cells not stained by Propidium Iodide (PI), one of the viability dyes that could be used, are live cells.
e. Live gate:
exclude ‘rubbish’ cells, like red blood cells, and non-viable cells that are not successfully excluded by previous gates. Also exclude other cells that are not of interest.
f. SP gate:
compare “Hoechst 33342 only” against “Hoechst 33342 + Verapamil” tubes to identify and gate SP cells.

Note: Blockers, like verapamil, inhibit ABC transporters that give a ‘tail-like’ cell profile of SP cells that are low Hoechst blue and red, on the plot graph.
Hoechst emits a wavelength that can be detected at two different channels with two different detectors.

To Ernest Questions

1) Yes, you can interpret it as that way. ‘Unstained’ tube contains cells that are not stained. This is used to compare with other ‘stained’ tubes, which consist of both stained and unstained cells in its pool of heterogeneous cell population. It helps to identify cells that are not stained from the rest of the stained cell population. Something like a negative control.

2) ‘PI only’ stands for the tube that is only stained by propidium iodide, which is one of the viability dyes that could be used. Cells not stained by PI are live cells.

3) You can add as many dyes as you can, however, people usually 2 different dyes plus a viability dye at most. Also, it doesn’t make sense to add too many dyes as there will be complications like spectrum overlap of fluorescence that spills into other ‘light’ channels and gives false positive results.
(For example only: Imagine having PE dye fluorescent spills over into FITC dye channel and gives a false positive result in the FITC dye channel as the fluorescence detected in FITC channel is actually from the PE dye)

4) Please refer to above answers to Liping’s questions.

5) Please do not confuse my experiment to detect SP cells from an unrelated experiment of DNA analysis that also uses Hoechst 33342 dye. SP cells are known to actively efflux out drugs and dyes like Hoechst 33342, which is why Hoechst 33342 is used in our experiment design. And we compare Hoechst red to Hoechst blue channels to observe our results.

As I mentioned in answering Liping’s question:

“Blockers, like verapamil, inhibit ABC transporters that give a ‘tail-like’ cell profile of SP cells that are low Hoechst blue and red, on the plot graph.
Hoechst emits a wavelength that can be detected at two different channels with two different detectors.” (Quoted)

6) Refer to above, answer to your question 5.

I really hope both of you understand more about my SP experiment. If you have any more doubts, feel free to ask again.

Also, to understand more about my SP experiment, please read my 3rd posting of “Double posting! (2nd part: SP Profiling in Stem Cells)”, before asking me any related questions.

Many thanks
Quan Jun