10th wk blog

Subject: Cytogenetics

Name of the Tests: Overnight Harvesting of Amniotic Fluid (AF) Sample using BRDU
HI people it’s me, TING-JIE again. This is the 10th week, and we are already half way of our SIP program, hope everyone having fun at their work place.

This week I am going to blog about what we will do after we set up the Amniotic Fluid (AF) sample in cytogenetics laboratory (which I blogged in the last blog entry). The next step after setting up is the harvesting of AF sample.

The purpose of harvest is to obtain sufficient cells at the metaphase stage with chromosomes of acceptable lengths. Remember my last blog I mentioned that for standard cytogenetic procedure, the cells being studied must be at late prophase or metaphase for analysis. That why the harvest step have to carry out to arrest cells in the metaphase.

Method:
1. Add 25 µl of 2.5mg/ml BrdU (Bromodeoxyuridine) and 25 µl of Colcemid® working
solution (1 in 12 dilution) to the culture dish

2. The next morning with a transfer pipet, remove the culture medium completely

3. Gently add 2 ml of warm 0.8% Na citrate hypotonic solution and let it stand at room temperature for 30 minutes

4. Prepare fresh cold 3 parts of methanol and 1 part of glacial acetic acid fixative, mix well

5. Gently add 1 ml of cold fixative to pre-fix the cells and leave for 2 minutes

6. Use a vacuum pump to discard the hypotonic-fix mix , and slowly add 2ml of cold fixative,leave to stand for 20 minutes

7. discard fixative and add another 2ml of cold fixative, leave to stand for 20 minutes

8. repeat the procedure with another 10 minutes cold fixative

9. remove the fix and blow dry with a fan until it is completely dry

10. check the chromosome spread quality with a phase contrast inverted microscope

11. gently remove the cover slip with a pair of splinter forceps

12. mount it with 1 drop of mounting medium onto a pre-labeled microscope slide with the cell side facing upwards

13. bake the slides for 1 hour at 90℃

Now the slides are ready for staining using routine G-banding method, which I will explain in the next blog.

Principle of the test:

Colcemid® is the mitotic arrestant. It prevent the formation of spindle fiber, which normally pull the sister chromatid to opposite poles for incorporation into the 2 daughter cells. Therefore it works by stopping cells in synthesis, and collect a large population of cells ready to head into division together, so that we can get more metaphases.

However, colcemid® will cause the shortening of chromosome or chromosome condensation, when the chromosome condense, the subbands merging into bands, to prevent the shortening of chromosomes a releasing agent has to be added.

Releasing agents such as thymidine, which will produce a greater number of bands as it inhibit chromosome condensation. In our lab, we use BrdU (Bromodeoxyuridine) which is an analog of thymidine as the realeasing agents.

Treatment with the warm 0.8% Na citrate hypotonic solution is to increase cell volume as it swell the cells and enable the cells to disperse wider, thus facilitate chromosome analysis. It works by creating concentration gradient across the cytoplasmic membrane so that the water will rush into the cells by active transport. To increase the effectiveness, pre-warming the hypotonic solution can speed up water transport across the cell membrane and also softening the cytoplasmic membrane.

Pre-fix the cells help to harden the cells and preserve the chromosomes which make cells more resistant to the shock of the pure fixative.

Fixation procedure removes the water from the cells, kills and preserves them, hardening the membranes and chromatin. It also prepares the chromosomes for the banding procedure later.

Yap that’s all for this week, Hope you all understand=)

TingJie
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