CYTOGENETICS ( BONE MARROW)

Hi Guys!!! This is already the sixth week of our attachment and here i am to tell you and share with you ppl abt my experience in CYTOGENETICS lab. Til today, i am enjoying myself working in this lab. There are three different stations in cytogenetics lab. One is prenatal (lab) where you will handle amniotic fluid samples, bone marrow (lab) where you will get to know more about haematological diseases and finally FISH which is actually aneuploidy screening of prenatal specimen. Currently i am under BM in short for bone marrow lab. I have lots to say but I will be covering the basics of cytogenetics, procedures, etc.

What is Cytogenetics?

Cytogenetics is the study of chromosome strucutre, pathology, function and behaviour. Chromsomes are best studied at mitotic or meiotic metaphase. Studies such as FISH methods may utilize interphase cells.

The type of sample and culture techniques used in our lab:

Basically we receive bone marrow, bone core and sometimes perpheral blood for chromosome analysis. We receive both local and regional samples. For local cases (the day you collect the bBM from the patient and the day you receive the sample is the same date) we have to perfom two different cultures. One is Direct Harvest and the other one is either one of these, 24-, 48-, 72- hour culture with or without mitogen (growth factors) depending on the diagnosis of the disease. As for Regional cases (if the date of collection of specimen is different from the date we receive is different) we can only set up only one or two (24-, 48-, 72- hour) cultures but not Direct Harvest. The reason for this will be explained shortly!

What is Direct Harvest and 24-, 48-, 72-hour culture??

Direct harvest: This is done to capture only spontaneously dividing cells (both abnormal and normal ones) upon arrival of the sample.

Culture: Cells are cultured for the study of non-spontaneouls dividing components or cells. The type of culture (whether it is 24-, 48-or 72- hour) depends on the diagnosis of the disease. E.g in my lab we usually perform 24-hour culture (also known as unstimulated culture due to the absence of mitogen = growth factor) for myeloid disorders one such example would be acute myeloid leukaemia (AML). As for lymphoid disorders, we will usually do a 48-hour culture with Interleukin-2 (which is actually a growth factor) and this is known as stimulated culture due to the addition of interleukin. One e.g. of lymphoid disorder would be Acute lymphoblastic leukaemia( ALL). As for 72-hour culture, we will usually do this for Myeloma cases (disease) as the cells will grow very slowly. Interleukin-6 is added for Myeloma cases. As you guys know the interleukins are produced in the body and these are required for certain abnormal clones or cells to grow (in the body itself). This is the reason why we add interleukins (to provide in vivo environment).

What i am trying to say is some abnormal cells or clones will grow under different conditions. Certain cells gorw very rapidly and this can be captured only in Direct Harvest culture. However, there are also cells that grow very slowly (1 to 3 days) and in order to capture these cells after you cultured them ( providing the in vivo environment for the cells to grow).

The procedures and the purpose of these procedures:

1) Harvesting: The first step in most harvest procedure is to arrest cells in the stage in which they must be in for standard cytogenetic analysis: Metaphase or prometaphase. In order to arrest cells under metaphase stage we have to add Colcemide. Colcemide will stop all the cells at metaphase stage for analysis. The effect of colcemid is to prevent spindle fiber formation, which would normally pull the sister chromatids to opposite poles for incorporation into two daughter cells. It also causes the chromosomes to condense, a process which is accentuated by increasing the time of exposure and concentration.

2) Hypotonic treatment: The second major step in harvesting is treament with a hypotonic saline solution to increase the cell volume so that the chromosome can find or have sufficient space to spread out ( to get beautiful metaphase to analysis) *i will try to get photos to show you ppl wat i mean by "beautiful metaphase"*


*Note: the above two solutions are prepared in our lab and both solutions can be found in one tube *

3) Fixation: The third constant feature of chromosome harvesting is the fixation of the cells. This process removes water from the cells and preserves them, hardening membranes and chromatin and in some way, preaparing the chromosome for banding (staining) procedure.
Fixative used: 3:1 methanol acetic acid. Fixative will cause the cells to be stronger ( harden the cell membrane). Cells are then kept in the fridge for slide making. During the fixative process, red cells present in the sample are also removed thus giving us a clear suspension. Basically fixation strengthens the cells and also gives a clear suspension for slide making. ( Fixation = washing of cells)

4) Slide Making: Once cells have been well fixed, they are then dropped onto glass slides and dried using specific condotions for optimal chromosome spreading and morphology.
**Theory of air drying: As the fixative evaporates, the layer of fixative becomes thinner and the meniscus pushes down on the top of the cell, enlarging the area of the cell and pressing the metaphase between the upper and lower membranes, spreading them out. We want the chromsomes to spread so that suring analysis we wont get chromsomes that are clumped together thus not allowing us to analyse them clearly.

After slide making, the slides are then stained using the Wrights and Giemsa stain. This is known as GTG banding. the G bands by trypsin using Giemsa and WRight's stain. Giemsa/Trypsin banding invloves the treatment with a proteolyitc enzymes trpysin and subsequent staining with a combination of Romanowsjy dye (Giemsa and Wright's stain). Dark bands generally replicate their DNA in late S phsase, contain A + T rich DNA, appear to contain relatively few active genes and differ from light ands in terms of protein composition. THe G bands by this technique allows the identification of individual chromosomes and structural rearrangements.


After staining, the slided are then covered with the coverslip (using DPX) and dried for about 30 mins and the slides are ready for microscopic analysis.

Guys there is more to tell you but i dun want to bombard you guys with so much of info.. I try to summarize as much as i can. So if there is anything u are not sure abt please feel free to ask me.. As for the photos i will try to put them up asap so that u will noe wat am trying to say about metaphase chromosomes etc etc.. Alrite?? And sorry if this is too long!!! I try to shorten but i couldn. I think i have to do another posting to tok about LMQA. I will do it asap ok! SOrry for the late posting too!!

Have great week ahead ppl!

Vinodhini
TGO2
0503171A