20th week

Subject: Cytogenetics

Hello everyone, this is the end of SIP, week 20. Hope all of you learnt a lot of things from the SIP. =)

After 20 weeks of SIP, I do learn a lot from Cytogenetics laboratory. From receiving sample to cell culturing , harvesting, slide making, staining, mounting of slides and analysis of chromosome.
This week I am going to shared with you the chromosome banding and staining techniques- GTG banding.

Chromosome banding and staining techniques – GTG banding

Materials
· 4 coplin jar
· HBSS
· Trypsin- EDTA (10X, sterile)
· 7.5% v/v NaHCO3
· Gurr’s Buffer (PH 6.8)
· Wright’s stain
· Giemsa stain
· Clinical Lab Reagent Water (CLRW)


Methods
1. Prepare 4 clean and dry coplin jars and add working solution in the following orders:
· 1st jar: 47 ml HBSS + 3 ml 0.5 sterile T-EDTA (10X sterile). Adjust PH of HBSS using 7.5%v/v NaHCO3 to a darker pink color. Add the trypsin just before banding.
· 2nd jar: HBSS (1X) approximately 50 ml.
· 3rd jar: 50 ml Gurr’s Buffer (PH 6.8) + 7.5 ml Wright’s + 5 drops Giemsa stain.
· 4th jar: Clinical Lab Reagent Water (CLRW) approximately 50 ml.

2. Use one trial slide to determine the optimal trypsin time and staining time as the length of trypsin exposure and staining time may vary from batch to batch.

3. Normally trypsin time for blood is around 2.30-3 minutes.

4. Rinse in HBSS in 2nd jar.

5. Place slide to stain in the 3rd jar.

6. Staining time for blood, 3.30 minutes.

7. Rinse in CLRW and blow dry immediately.

8. Assess the quality of the banding under a brightfield microscope.

9. If the chromosomes are uniformly stained but no bands are seen, the trypsin time is too short.

10. If the chromosomes are swollen and bands are indistinct and the edges of the chromosomes are fuzzy, the trypsin time may be too long or the slide baking was inadequate.

11. In both case, repeat the trial procedure and determine the charges in trypsin and staining times until a sharp pattern of bands is achieved.

Note: slide aged longer on a hot plate or an oven give better contrast. Over-staining or under-staining with Giemsa/ Wright’s stain may also result in poor quality preparations.

12. Mount slide with mounting medium in fume hood.

13. Airs dry for 5-10 minutes.

Now the slides are ready for analysis.


Principle of chromosome banding and staining techniques- GTG banding

In our lab, we use Giemsa & Wright stain. Giemsa stain is the most popular stain for chromosome analysis, a dilute concentration of Giemsa or Wright stain will induce spontaneous chromosome banding. Wright stain is also becoming popular for G banding. It is extremely useful for high resolution chromosome analysis because it gives a sharper resolution and reveals fine bands.

We always use one slide from each patient or each cell type e.g. blood as trial slide. The trial slide will be band first to gauge the trypsinization time. This is done to prevent over-trypsinization and under-trypsinization of chromosomes.

Under-trypsinized chromosomes have indistinct bands and little contrast and usually appear fuzzy in appearance.

Whereas, over-trypsinized chromosomes have sharp bands but often appear frazzled at the end. This is because too much contrast between land mark bands and pale telomeres.

Extremely trypsinized chromosome are very pale after staining and very swollen, therefore to prevent undertrypsinized or overtrypsinized chromosomes, gauging of trypsinization time is very important.

After trypsinization step, it is important to stop its action to prevent over-trypsinization. The fecal calf rinse is used to stop the action of trypsin as serum contains α1-anti trypsin which inhibits the trypsin from further digestive action. In our lab, Hanks’s balanced salt solutions (HBSS) were used to stop trypsin action.

Hope you all understand my blog, and feel free to ask any question.
See you all in school =)

CHEN TING JIE
TG02
0608495H