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

WEEK 19

Hi guys. one more week and we will be ending our SIP.

Oh well, in this post, I am going to discuss about the development of a RIA which I had been doing to develop a corticosterone RIA. There are quite a number of assays that have to be done for example, like determining the optimum antibody concentration to be used, the right dilution for sample analysis, the optimum tracer concentration and the antibody specificity. Just to take note, determining the optimum antibody concentration and tracer concentration have to be determined first before any further tests (antibody specificity test, sample dilution) can be done.

However, in this post I m going to focus in determining the antibody specificity. In other words, determining the antibody specificity is just to see if the antibody will cross react with other compounds.

Just to recall, a normal RIA procedures will require standards, antibody, tracer and of course your samples. Usually, a RIA will require a separation step. However, because our lab uses SPA, the need for separation is eliminated (recall from last post).

Antibody Specificity

To determine how specific your antibody is, we do it through a cross reactivity assay. Like normal RIA procedures, we will incubate the antibody and tracer with the blank (assay buffer), standards and the compounds that are going to be used to determine the specificity of the antibody and then incubate overnight before counting them in the scintillation counter. Since the blank contain only the labeled tracer, it should have the highest count other than the total count (total radioactivity).

Compounds to be used for corticosterone cross reactivity assay are selected from the corticosterone synthesis pathway and the concentrations used for each compound are 10, 20, 50, 100 and 1000 ng/ml.

After counting in the scintillation counter, we will get the counts of all the compounds and then calculate the percentage of B/B0 for all compounds and plot in onto a graph of %B/B0 against log concentration.
B= Binding (counts of each compound)
B0 = Total Binding (Counts from the blank)

The antibody cross reactivity is then determined by having the standard concentration at 50% binding divided by the concentration of the competitive compounds at 50% binding, expressing as a percentage. If there are no binding of the competitive compounds to the antibody at 50% binding, the antibody will be seen as not having any cross reactivity to the compounds.

Below is a graph that i had plotted from the corticosterone cross reactivity assay that i had did.


ok. from the graph, we can only see that the red and green lines crosses the 50% B/B0 marking. So that means to say that the corticosterone antibody has cross reactivity to the compounds, cortisol and progesterone other than to its own antigen of interest ie corticosterone. From the graph, the corticosterone antibody is seen to have 100% reactivty to corticosterone, 3.8% reactivity to cortisol and 0.5% reactivity to progesterone. As the rest compounds do not cross that marking, the cross reactivity cannot be calucuated. So we take it as the antibody does not react to the compounds unless at very very very high concentration. In fact, the concentrations used for the assay are already very high. Hence, for the antibody to bind to the other compounds, the concentration has to be much higher than 1000ng/ml. Therefore, we can say that the antibody is specific for corticosterone.


Hope its understandable. Enjoy your last week at your work place.

Thanks.

Xin Yi
TG02 =)