Codec 5

20th week

2008-11-09T22:41:00.002+08:00

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

2008-11-01T15:29:00.006+08:00

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 =)

Week 18 of SIP

2008-10-27T22:22:00.003+08:00

Alright, its been a little bit of a delay trying to post my part up, due to the hectic and busy long weekend I had, thus leaving me with no time to do. So as time whizzes by, its week 18 of the internship programme, and 4 1/2 months have passed. 2 MORE WEEKS TO GO! And also, this would be the last posting by me. I think I am gonna miss SIP!!

Basically, I am starting in my company's Thomson Medical Centre outlet this week (after completing Paragon & Bukit Merah outlets), where patients do take a queue number and wait for their turn to be attended to us. I will thus share with you tests on Immunology - Dengue Duo Casette Test

WHAT IS DENGUE?

Dengue is a flagivirus found in the tropics, and is characterised by Aedes aegypti and Aedes albopictus. It is characterised by a spectrum of clinical manifestations, and is accompanied by dengue fever and shock syndrome.

Principle:

Dengue -specific IgM or IgG antibodies binding to anti-human IgM or Ig-G antibodies immobilized in two lines across the casette membrane. Collodial gold complexes contain recombinant dengue 1 - 4 antigens captured by the bound patient's IgM or IgG to give visible pink lines. A procedural control is done to indicate that the assay has been performed correctly.

In Dengue Duo Casette test, IgM and IgG are determined using a single addition of serum, plasma and whole blood. Differentiation between primary and secondary infection can be made through a single application of serum, plasma and whole blood. In primary infections, serum IgM antibodies can be detected from dengue patients as early as 3-5 days after the onset of fever, persisting for 30 - 90 days. Secondary infection can be characterised by high IgG levels that may be accompanied by elevated IgM levels.

The results can be intepreted by:

From left to right:

Primary Infection : Positive for IgM antibodies
Secondary Infection: Positive for IgM & IgG antibodies

Secondary Infection: Positive for IgG antibodies

Negative: No detectable IgG or IgM

That's all! Enjoy your final 2 weeks of SIP!

Lloyd Lam TG02 0607775D

WEEK XVII ATTACHMENT

2008-10-18T10:30:00.006+08:00

Hello all once again!

Time really does fly doesn’t it? And we’re suddenly finding ourselves staring at the final few numbered weeks of our attachments. Hope the projects are getting along fine there. Anyway, this would be my final blog entry regarding my attachment, but nonetheless, I hope you guys would enjoy it all the same.

For this entry, I’d be touching on G-6PD screening on neonatal blood. Basically, G-6PD is the abbreviation for the enzyme Glucoes-6-phosphate dehydrogenase. It is indirectly involved in the prevention of oxidant damage to the RBC, in that it is necessary for the maintaining of adequate quantities of Glutathione, an important buffer to oxidants within a RBC (see diagram below).

In the event that the production of NADPH is impaired, insuffiecient quantities of Glutathione would be regenerated, allowing cellular oxidants to accumulate, thereby resulting in erythrocyte injury and hemolysis.

This screening procedure makes use of a buffer(provided in the kit) which contains Glucose-6-P and NADP+, both of which would react with G6PD(if present in the erythrocyte) to give Gluconate-6-P, NADPH and H+. In turn, NADPH produced in the reaction would fluoresce under long-wave UV-light. However, in the event that there is a marked deficiency or complete absence of the enzyme, no fluoresce would occur. In short, the below equation describes the reaction taking place in the screening test.

The procedure for this screening is as follow:

1. Fill up well(s) with 50mL of buffer(provided in kit), one well per patient.

2. Using an applicator stick, add <1>

3. Allow the mixtures to incubate at r.t.p for 10mins. Meanwhile, label on the filter paper the necessary details such as that of date and time of experiment, and identity of each sample at the area where the sample would be placed on the filter paper.

4. Using a micropipette, place a drop(10-20mL) of each of the incubated sample into each respective the circles on the filter paper.

5. Incubate the filter paper containing the samples in the oven(42°C) for a further 15mins, or until the filter paper has dried.

6.View the filter paper under a long-wave UV-lamp in a dark room. Samples obtained from normal/ slightly reduced G-6PDH activity will show strong fluorescence. Failure to fluoresce, as mentioned above, suggests a total or marked deficiency of the enzyme G-6PD.

7. Mark out on the paper “present” for samples that fluoresce. Samples that do not fluoresce would be sent to a biochemist to obtain a titre count for any G-6PD present.

Alrights. That’d be all for this entry of mine. Thanks all for reading once again. All the best to your projects.

Alexander Soo TG02
0608122H

Replies to comments

2008-10-10T23:53:00.003+08:00

Hey guys. Sorry for the late replies to your comments. As the answers to your questions could be quite long, i decided to have it here instead.

To Ting Jie and Lyn

I have found out that the need for separation of the unbound ligand from the bound complexes indeed takes a longer time due to the steps involved.

I will illustrate using the dextran-charcoal method to remove the unbound radiolabelled ligand. After incubating the samples overnight, instead of counting the samples as for SPA, this method requires the addition of charcoal reagent and incubation for 30 min at 4oC. Afterwhich, the samples have to be centrifuged for at least 5 min. Supernatant has to be decant into vials and subsequently the addition of liquid scintillant. However, even after the addition of scintillant, there is still a need for 1h of incubation before counting.

Hence, as compared to the use of SPA, we can say that it actually takes about an addition of 2 more hours to complete the whole RIA process.

Furthermore, it has to be noted that while adding the charcoal reagent to the samples, the solution has to be in suspension. The time taken for the addition of the reagent also has to be rapid so that incubation time between the sample and the charcoal reagent does not differ too much across the assay. Ideally, the speed should be between 2 to 3 min. This however, takes practice and depends on the technican skills.

Hence, because of these factors it can increase the chances of human error.

While with the use of SPA, these steps are eliminated and thus decrease the human errors.

To Lloyd

The principle of scintillation counter is quite physics and lengthy. So not to bore you, I just give you the main principle.

Basically, the photons (electromagnetic waves) of light emitted from the radioisotopes are detected by a light-sensitive device called as the photomultuplier tube (PMT).

Firstly, interaction between the photons and the photocathode occur first, ejecting photoelectrons. The photoelectrons then accelerate to a series of dynodes (metallic element) where each dynode is at a higher position voltage than the previous one. The first strike of the photoelectrons to the first dynode will release secondary electrons which in turn will strike on the second dynode to release more electrons. This will continue for 12 dynode stages, each time the electrons increase at about 10to the power of 7.

The interaction between the photocathode and the photons also provide an electrical pulse where the amplitude of the electrical pulse will be proportionate to the amount of photons that interact with the photocathode and hence will be proportionate to the energy of the beta particle in the sample.

There is also a circuit incorporated into the counter where it can give a total pulse proportional to the total photon yield of the event, for example in 60 sec

Thanks for the comments.

Xin Yi
TG02

week 16 of SIP

2008-10-10T22:38:00.004+08:00

Hi everyone =) 16 weeks have passed and we are one month away from the end of attachment. Hope you guys are doing fine.

It's my turn to tell you guys more about what I'm doing in the lab I'm in and what my project is about.

As mentioned peviously, I am in a lab that mostly deals with pharmacogenetics. That is how genetic variation affects the drug metabolism. So, bascially everyday I'm doing PCR, gel electrohporesis, purification and sequencing. In school, we were only taught the basics but in my lab, I got all hands-on.

In my previous post, I discussed about how genptyping is done and it's significance. This week, my last post of the whole SIP, I will teach you guys an euqilibrium that most labs dealing with genetic population studies use. It is known as the Hardy-Weinberg Equilibrium, short-form HWE.

Hardy-Weinberg Equilibrium
In population genetics, the HWE principle states that genotypic/ allelic frequencies in a population remain constant from generation to generation unless specific factor are changed.

In HWE, there is 5 assumption.

1. Larger Population Size: The population must be large to minimise random sampling errors. With a large population, the number of changes that occur by chances alone becomes insignificant.

2. Random Mating: There musn't be any mating preference. For example, an AA male does not prefer an aa female.

3. No Mutation: There shouldn't be any mutationthat will result in a change in the alleles. This is because mutation is the source of genetic variation.

4. No Migration: This means that there shouldn't be any exchange of genes between a population and another population. A migration of gnees from a population to another will result in a change in the population's genetic frenquency.

5. No Natural Selection: No alleles are being selected over other alleles. If selection were to occur, the specific alleles selected will tend to be more common.

HWE equation
It is an equation used to estimate and determine the allelic and genotypic frequencies in different populations. This is done after all the genotypes of all subjects on all the SNPs are done. It can also be known as statistical analysis.

HWE equation:
p2 + 2pq + q2 = 1

where, p= frequency of the dominant allele (eg: A)
q= frequency of the recessive allele (eg: a)

For a population to be in genetic equilibrium, p+q must be equal to 1.0 (ie.: the sum of the frequencies of both alleles is 100%)

So, (p+q)2=1
=> p2 + 2pq + q2 = 1

p2 denotes the frequency of homozygous dominant (AA).
2pq denotes the frequency of heterozygous (Aa).
q2 denotes the frequency of homozygous recessive (aa).

Here is one website that I kind that it's quite good at explaining HWE.
Website: http://www.phschool.com/science/biology_place/labbench/lab8/intro.html

That's all on HWE. Hope you guys understand my explaination. Take cares everyone. =)

Lyn
0611027D
TG02

week 15

2008-10-05T22:26:00.006+08:00

Subject : Cytogenetics

Hi friend, now already week 15, 5 more weeks of SIP. Hope you all doing well at your workplace.
In the previous blog I shared with you the harvest method and the next step which I am going to shared with you in this blog is the Slide Making method.

Slide making

The ultimate goal of Slide making

to obtain chromosome that have the following characteristics on the phase contrast microscope:

-Mostly medium gray to dark gray chromosome. Light gray chromosomes yield cells with poor contrast between bands and very black images may have increase cytoplasmic background.

-Little chromosome scattering.

-Minimal number of chromosome overlaps, for accurate counting and bnads analysis.

-Very thin cytoplasmic background so that trypsin banding will be optimal.

-Proper concentration of cell fixative suspension. Too dilute a suspension yields slides that are time consuming to scan for metaphase, and too concentrated a suspension may interfere with the banding.

-Chromatids that are together and not split apart.

Materials

-Clinical Lab Reagent Water (CLRW)
-Sterile 10 ml round-bottomed tube
-1ml and 10 ml serological pipet
-Glass Pasteur pipet
-Disposable plastic sterile and non-sterile transfer pipet (3ml)
-Microscope glass slide, frosted end

Methods

1. Remove from the fridge a beaker of cold, wet pre-cleaned slides kept in CLRW.

2. Take a slide and flick off excess water and wiped with toweling paper before dropping the cell suspension.

3. From a height of 10-20 cm from the slide surface, drop about 3-6 drops of the cell suspension from a glass pipet along the upper edge of the slide, while holding the slide horizontally to the bench at 45°angle.

4. Wipe the back of the slide with a towel and firmly bang the slide on the bench top several times (this may help in chromosome spreading).

5. Air dry before placing it on a 56℃ warming tray.

6. Check the spread under phase contrast microscopy, adjust cell density or height from which the suspension is dropped accordingly.

7. Baked slide at 90℃ oven for 2 hours.

Now the slides are ready for staining, which I will share with you all in my next blog entry.

In order to achieve the goal of the slide making, there are some variable that we have to take note of:

Wet VS dry slide
In our laboratory, wet slides are preferred, by dropping the fixed cells on wet slides, it facilitates spreading due to the immediate reaction of the water meniscus. The energy of dehydration from fixation is returned as a change in the free energy of mixing between fixative and water, which spreads the cells. Wet slides may further facilitate spreading, control by the use of different temperature of water coating to speed up or slow down drying time. Cold wet slides will slow drying, increase spreading. While warm slides will accelerate drying time.

Angle of the slides

Slides made with long edge down tend to be more uniform, especially if the angle is kept 20-30°, cells are placed in the upper one third of the slides and allowed to move downward. Greater tilt angles may speed up the drying process at the upper end of the slide compared to the lower end which gives uneven, inconsistent slides. Uniform drying can be achieved by tilting slides at one angle for part of the drying time and another for the final drying.

Hope my blog entry is clear for everyone, any question feel free to ask. =)

CHEN TING JIE
TG02
0608495H

Week 14

2008-09-26T13:42:00.009+08:00

Lab Technique: Radioimmunoassay

It’s the 14th week already. 6 more weeks to go. Hope you guys are doing fine. =)

Last week I mentioned about faecal extraction. So now, after the extraction has been done, we can now measure the glucocorticoid level.

To measure the glucocorticoid levels in the faecal sample, radioimmunoassay (RIA) is used. Just to refresh your memory. The principle of RIA is based on the competition between unlabelled ligand and a fixed amount of radiolabelled ligand for a limit amount of antibody. The proportion of the bound labelled ligand is inversely related to the concentration of the unlabelled ligand.

An essential step in RIA is the separation of the unbound radiolabelled ligand from the Ab-Ag complexes. This is done so that the unbound radiolabelled ligand will not interfere with the counting and hence cause inaccurate result. However, this step can make the whole RIA procedure to be time consuming and can be prone to human error due to additional steps needed as compared to using SPA.

What is SPA?

SPA refers to scintillation proximity assay that makes use of SPA reagent to eliminate the separation step. The SPA reagent contains either a secondary antibody or protein A that is bound to a fluomicrosphere. The fluomicrosphere will produce light if it is bound to a radiolabelled ligand. Light will not be produced if the SPA reagent is bound onto an unlabelled ligand. Unbound radiolabelled ligand will also not produce light.

F = Fluomicrosphere with protein A or secondary Ab attached

Ab = Primary Ab

L = Unlabelled Ligand

L* = Radiolabelled ligand

To calculate the concentration of the unlabelled ligand, a machine called the scintillation counter is used. Depending on what kind of isotope used, the scintillation counter can vary. If a beta emitting isotope is used, a beta-scintillation counter will be used and if a gamma emitting isotope is used, a gamma-scintillation counter will be used. The scintillation counter will calculate the amount of radiolabelled ligand by detecing the light and then using a software, the concentration of unlabelled ligand can be determine by interpolating from a standard curve. By detecting only the light produced by bound radiolabelled ligand, this eliminate the need for the separation step.

This is a beta-scintillation counter.

(Wallac 1414 Liquid Scintillation Counter)

Picture taken from: http://www.gmi-inc.com/Genlab/Wallac%201414%20liquid%20scintillation%20counter.html

In our lab, tritium (3H) is used. And since tritium is a beta emitting isotope, a beta-scintillation counter will be used. FYI, handling of any radioisotope compound has to be done in a radioactive room. The radioactive room looks very much like a normal lab. It is just that in front of each bench, there would be a shield and we are expected to work behind the shield for safety reason.

Again, depending on whether a beta emitting radioisotope is used or a gamma emitting isotope is used, the shield could vary. Beta emitting isotopes are weakly penetrative, hence, a plastic shield is sufficient enough to block the rays. In fact, a piece of paper is already enough to block off the beta-rays. Gamma emitting isotope such as iodine 125 is more penetrative. Hence, when using 125I, we are expected to work behind a metal shield. Also, as the rays emitted can be absorbed through skin, gloves as to be wore at all times!

And of course, we will have controls to ensure that our results are valid.

As the steps involved in carrying out the RIA is quite long, I will just give a brief outline on what is done. When measuring different ligands, different set of standard concentration, antibody and tracer will be used. Hence, for illustration, I will focus on cortisol.

First we have to prepare our standards so that a standard curve can be plotted.

6 standards are prepared with concentration consisting of 188, 375, 750, 1500, 3000 and 6000 fmol/0.1ml by serial dilution starting from the standard of the highest concentration. FYI, the standard with the highest concentration is already pre-prepared by the staff in the lab.

After which, we need to prepare the SPA reagent and antibody mixture and also the 3H cortisol tracer.

Because the controls used are in serum, there is also a need for the extraction of steroids as the steroids will be bound to its binding globulin. In this case, we don’t extract the steroids like what was done on the faeces. The controls are first diluted in distilled water and then heated at 60oC for 30 minutes. The heating is sufficient to dissociate the steroids from its binding globulin.

Next, we will dilute the faecal samples 50x. After which, 100 µl of standards, PBS, faecal samples and controls will be aliqouted into another set of test tube. PBS is used to measure the total binding of the radiolabelled ligand to the SPA reagent. 200 µl of SPA reagent and antibody mixture is then added to all tubes followed by 100 µl of 3H cortisol tracer.

The test tubes are then incubated overnight (18-24h), followed by counting it in the scintillation counter.

That’s all. Hope you guys understand. I know, the post is very long. Oops. x_x

Xin Yi
TG02

REPLIES TO COMMENTS(This is not a blog entry)

2008-09-25T20:10:00.003+08:00

Hi there Miss Chew and friends,
First, thank you Miss Chew for your kind comments and taking the time to go through even this section of the blog. Really appreciate it.(=

Up next is the answers to questions that have been posted. As you'd noticed, as the replies are rather long, a blog entry is more convenient.

To Sutiana:

Hi there Sutiana, your question is:
“is this the first test that will be conducted when the doctor request for fecal occult blood testing? bcoz here in my lab, wen such request is ordered, we carry out another test before the 0C-light test.we take 0C-light test as the confirmatory result for fecal occult blood testing.”

ANS:
Thanks for your question there, appreciate it. And yes, in the lab I’m in, this kit is just the primary screening, as in the first test that’d be done. The confirmatory test would be an endoscopic examination on the patient to observe for ulcers, lacerations etc in the lower GI of the patient. However, I’m surprised that your lab uses this as a confirmatory test as under the instruction manual for the kit, it’d said that the kit is not suitable as a substitute for confirmatory tests. So yeah, you might want to check that up.

To Xinni:

Hi there Xinni, your question is:
“since single blue line means negative and double blue line means positive…..so the antibodies on the single line that indicates negative (control) is different from that on the additional line to indicate positive? If so, what is the antigen which binds to the antibodies on the single line that indicate negative?”

ANS:
Boy thanks very much for this question. Am really glad to answer your question as I’d been thinking about how the control line functions too (as in the things involved in bringing about a positive reaction) I also did trying searching around for some information but could not find any, not about this kit directly at least. Fortunately, there are other kits in the lab too that includes a control line apart from the results. Some examples include that of the rotavirus and pregnancy screening. The same principle should probably apply to this kit as well. Thus that said, the following diagram should pretty much summarize what’s going on:

To Liyanah:

Hi there Liyanah, your questions are:
“you said that the kit is suitable for patients with hemorrhoids, diverticulitis, colorectal cancer, ulcerative colitis right, then what are the differences you would expect to see in the different diseases?and what to you mean by immunochromatographic reaction? do you mind explaining the term?”

ANS:
Well, regarding the first question, the terms “hemorrhoids”, “diverticulitis”, “colorectal cancer”, “ulcerative colitis” are just some examples on the long list of gastrointestinal disorders of which this screening is suitable(as in the side points). These are not diseases but symptoms (except for colorectal cancer). Nonetheless, I believe you might find the following descriptions to each of the terms helpful:
Hemorrhoroids: swelling/inflammation of veins in anus/lower rectum
Diverticulitis: rupture of lining in the colon, resulting in inflammation
colorectal cancer: colon/ rectal cancer
ulcerative colitis: ulcers/ sores in the colon

Also, the term “immunochromatographic” is from the two words “immunology” and “chromatography”. Thus that said, as “chromatography” implies, the reaction comprises of the migration of molecules (this would include antibodies, antigens etc as immunology implies) along the membrane on the test strip via capillary action.

To Dyana:

Hi there Dyana, your question is:
“You mentioned that the OC-light is a screening test rather than a confirmatory test rite? And presence of 2 blue lines would indicate that the result is positive rite? So it will indicate the patient is suffering from either of these;hemorrhoids, diverticulitis, colorectal cancer, ulcerative colitis, these of which give rise to abnormal bleeding thus found in the stool samples. Is there any follow up test that the doctor would request to ensure what disease the patient is suffering?”

ANS:
Well, thanks too for your question(s). As your question is slightly similar to Liyanh’s, I believe you would find my reply to her helpful too. Anyhow, just as I’d mentioned to Sutiana too, this kit is just a screening procedure and a confirmatory test, endoscopic examination, would have to be carried out. So for instance, if the cause of the intestinal bleeding is colorectal cancer, you’d probably expect to find some suspiciously abnormal lumps along the inner walls of the colon or rectum.

Alrights. Thanks all once again for your questions! Hope the answers satisfy once again! Outta here.

Alexander Soo TG02
0608122H

Week 13 is over! Like finally...

2008-09-21T20:12:00.001+08:00

Its the end of week 13 in case some of you aren't aware that our SIP has already been more than halfway through. Most of us cant wait for the 20 week attachment programme to be over, which is considered long for most courses. School life is seriously much better than working life, and all of us are seriously deprived for holidays to start once again.

So I am now currently based in the Paragon branch of my company. Yes, no more being based in the main office but at Orchard Road. What I am dealing now in Paragon clinics is doing on Immunology.

Like I mentioned in my previous post, automated machines are being utilized greatly, so I am gonna talk about automated machines, since manual work (what we learnt in practical labs) are no longer in use at all. The most common equipment used in Immunology would be

ADVIA Centuar

(Some of you would also be using this in your company)

There are many tests done for ADVIA Centuar, such as for Prostate Specific Antigen (PSA) - Tumour marker for prostate cancer or prostatic hypertrophy, prostatistis or even CA 125 (Cancer Antigen 125) - Glycoprotein produced by ovarian cancers. May signify cancers of gastrointestinal tract, breast etc. However, I shall concentrate on the test for HBsAg:

HBsAg (Hep B surface Antigen) - When a person is exposed to Hep B viral infection, the HBsAg will appear after three weeks. The antigen remains in the blood for 4 to 5 months, before dissapearing. In some cases, HBsAg will persist for long periods, thus people in this group suffers from chronic Hep B. However, some cases happen whereby there is no recovery at all, and these people are so-called carriers of Hep B. Carriers of Hep B have a high chance of getting liver cancer.

The detection of antibody to hep B signfies and determine immune status to HBV or disease progression in individuals infected with HBV. An increase in anti-HBs levels, together with loss of detectable circulating hep B surface antigen denotes convalscence in hep B.

PRINCIPLES:

The principle inolves a sandwich immunoassay using direct, chemiluminometric technology. HBsAg are covalently coupled to magnetic latex particles in the solid phase. In the Lite reagent, the HBsAg is labelled with acridinium ester. Non magnetic latex particles are added from the ancillary wall. The sample is incubated with lite reagent, solid phase, and ancillary reagent. Ag-ab complex will form if anti-HBs is present in the sample.

Reagents stored in the internal system

Steps - The system performs:

dispensing 100uL of sample into a cuvette
dispensing 50uL of Ancillary reagent and incubation for 2.75 mins at 37 degrees
dispensing 100uL of solid phase, 50uL of Lite reagent, incubation of mixture for 6.75 mins at 37 degrees
separating solid phase from mixture and aspirates the unbound reagent
washes cuvette and dispensing 300uL each of acid reagent & base reagent to initiate the chemiluminscent reaction

Results are then released by index values numbering system.

Any index value of less than 1.0 means negative for HBsAg.

Greater than 1 signifies positive for HBsAg. It is recommended to repeat the test to double-confirm. Confirmatory tests include ADVIA HBsAg confirmatory assay or additional HBV marker assays.

Careful quality control procedures include calibration done often & proper barcoding of samples.

Lloyd Lam 0607775D

ATTACHMENT WEEK XII

2008-09-13T18:58:00.013+08:00

Warm greetings to all once again!

It’s now the 12th week of attachment and time is really flying past isn’t it? Projects to rush, experiments to conduct, reports to start… Alright, shan’t dig into the wound already.

Anyhow, I’ve been posted to a hospital located somewhere in the central area just a week ago and have finally got to start my SIP proper. I must say that it’s a definite pleasure to be waking up everyday to head to work for the colleagues there are genuinely warm and nice, and patient and caring, and practically just like another family away from home. Great guidance has been provided by respective colleagues and supervisor in the various fields and areas of which I have been involved in thus far, and I have really benefited much from just these two weeks of experience. All things aside, much work still has to be completed, and somehow my colleagues have been able to blend in the aspect of being professional with their work pretty neatly with the social aspect. My respect is with these guys, period.

In the lab, there isn’t any clear boundary for the respective sections (for example: Micobiology, Haematology, Biochemistry etc). Instead, various areas with tables and respective machines atop would make up the respective “sections” (so you could imagine an office setting with groups of tables together except that these have machines and papers and equipment and on the tables as well). However, it is unbelievably well-organized and rather up-to-date with the current technology and methods. That said, in just a few walking paces, you could easily make it past a few “sections”. Thus, although I might have been officially posted to a specific section for a week, I could still bob around to the neighboring section (or table) to stick my hand around in my free time (for example, when there is no sample to process in the section I’ve been posted to). Hence, quite a few “sections” could be covered in just one day of work.

MICROFLUIDS

This week, I’ve been involved in a section called “Microfluids”. Back in TP, we’d probably be more familiar with it as Clinical Chemistry for it deals with the sampling of urine, stools, sputum, swab etc samples. That said, I shall be sharing on fecal occult blood testing.

OC-LIGHT©

The lab uses a kit called OC-LIGHT© to help as a screening process (screening meaning non-confirmatory/ non-conclusive, as in just an aid in the diagnosis) for asymptomatic gastrointestinal bleeding. Thus, other diagnostic procedures would have to be used (for example, proto sigmoidoscopy examination and barium enema) for conclusive results. However, this kit is used as it is useful for screening out negative samples and thus save crucial time and resources from the unnecessary screening of all samples with confirmatory methods (confirmatory methods are usually more tedious and expensive to carry out). The kit is suitable for patients with the following signs and symptoms - hemorrhoids, diverticulitis, colorectal cancer, ulcerative colitis, these of which give rise to abnormal bleeding thus found in the stool samples.

PRINCIPLE

The principle of the test is based on immunological detection of human Hemoglobin (Hb) A0 in feces. In other words, antibodies specific against human Hb play the main role in the immunochromatographic reaction. Thus, dietary restrictions are not a must before the test. This is a major advantage when compared to other similar fecal occult blood test such as. For instance, the stool guaiac test uses the peroxidase-like ability of hemoglobin to oxidize a chromagenic material in the presence of H2O 2. Thus, unlike that of OC-LIGHT, hemoglobin even from a patient’s diet would easily cause a false positive reaction to occur as it is not specific for human hemoglobin as the reaction is not specific to human hemaglonbin.

A positive result would see the buffered hemoglobin reacting with the antibodies on the test strip, thereby resulting two blue lines. However, a negative result would cause only a single blue line to appear(the control) as is seen to the left.

SPECIFICITY
This method has been tested on these types of hemoglobin - beef, pork, goat, sheep, chicken, pigeon and horse. All tests had turned up negative results. Thus, these foods in an individual’s diet would not interfere with the results.
PROCEDURE/ METHOD

1. Dilute the fecal sample with the buffer (provided in the kit) to stabilize the hemoglobin.

2. Remove the green cap with attached collection end.

3. Collect sample by stabbing into the feces at various places.

4. Return the cap and attached collection end to the bottle.

5. Tighten the cap securely and shake the vial vigorously.

6. Position sampling bottle with the green cap down.

7. Remove white cone nozzle.

8. Insert strip into the sampling bottle from the “dip” side.

9. Read the results in 5 minutes.

Alright, that’d be all for this entry. Thanks all for reading!

Alexander Soo TG02
0608122H

Week 11 of SIP

2008-09-06T20:09:00.003+08:00

Hi everyone!! =) I'm back after 4 weeks. 11 weeks has passed and 9 more weeks attachment will come to an end.

I'm basically doing the same things everyday and week since I'm not attached to a routine lab. So it's just PCR, purification, sequencing, blasting, genotyping and data entering. And this repeats.

In my previous post, I discussed about what blasting is and what was done. This post, i will talk about genotyping.

Genotyping
This step is after blasting. After all the SNPs are found, you have to genotype all the samples for each and every SNP. You have to screen and look at all the samples and find the SNP. Before you can find the SNP, you have to copy a small fragment of the sequence either in front or behind that particular SNP from the reference sequence of a gene. Usually, the fragment is around 8-10 base pair long. Noone will copy a super long fragment to find in the DNA sequence of the samples. Similarly, too short the fragment won't be copied as well. This is because if the fragment is too short (around 4 or 5)- eg:AGTC, you may not be able to find that particular SNP. Possible reason why is 'cause the sequence 'AGTC' may be found at more than one postion on the DNA sequence. That is, for example at position 1103 there's 'AGTC' and at position 330, there's another 'ATGC'. Thus, it's important not to copy too short a fragment to find on you samples' DNA sequence.

After copying the suitable length of fragment in front or behind the SNP, you can start screening every samples. For example, the SNP is 334A>G. You have to screen every samples to see at the 334th position whether the subject has AA or AG or GG. In this case, AA is known as the wild type, AG is known as the heterozygous variant while GG is known as the homozygous variant.

Depending on the number of SNPs you have found during blasting and also the number of samples you have, you will have to screen that many times. For example, you have 100 samples and found 87 SNPs, you have to screen a total of 8700 times. It's a quite tedious process but it's necessary.

Genotyping is done using the programme Sequencing Analysis and similar to blasting, you have to look at the computer screen again.

After all the SNPs are genotyped for all the samples, you have to enter them into Microsoft Excel; data entering. In my company, it's programmed in such a way that we do not have to calculate the genotypic and allelic frequency.

Genotypic frequency is the frequency of a certain genotype in a population. For example, the frequency of Indians having AA for the SNP 334A>G. As for allelic frequency,it is the frequency of the population getting a paricular allele (eg: A) of a SNP (eg:334A>G) in their genes.

Allelic freqeuncy is expressed in percentage and it is calculated by taking the number of people in the population having a paricular genotype (AA) and multiply by 2 ('cause there's 2 A in the genotype AA). After that, plus the number of individuals having AG. For this value, you do not have to multiply by 2 'cause AG has only one A. After getting the value, divide it by the total number of alleles.

Example:
Number of people having AA= 5
Number of people having Aa= 4
So, take 5X2 and then add 4 to the vaule.
Total number of people= 10
So, total number of alleles= 10x2= 20
Thus, the allelic frequency for A in this paticulat population is 14/20= 0.7, which is also 70% of the population.

Genotypic frequency is easier to calculate. It is just taking the number of people having AA and divide with the total number of people in the population. Genotypic frequency is also expressed in percentage.

Example:
Genptypic freqency getting AA= 5/20= 0.25= 25%

After getting the allelic and genotypic frequencies, you will have to see if they are in Hardy-Weinberg equillibrium (HWE). I will touch on this in the next post; the principle of HWE, how to calculate and etc.

Hope my post is easy for you guys to understand. =)

Take cares everyone. See you guys at the next coming campus discussion.

Lyn
0611027D
TG02

10th wk blog

2008-08-31T23:28:00.004+08:00

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
Tg02
0608495h

Week 9

2008-08-24T23:57:00.005+08:00

Hi All,

How's everyone doing? Its Week 9 of SIP, and it seems that we are almost halfway through there..just 11 more weeks to go! Fast as it seems to be, its like we just started SIP and I believe all of us are cracking our heads over MP.

So I am currently in Specimen reception department, which is actually a admin dept. So I aint gonna talk about admin stuff. What I will share will be Serology dept, the dept that I was in 2 weeks ago.

Basically, almost all companies now uses automated machines to assist us in our work. Technology is really wonderful in such a way that it does things for us, leaving with much, much lesser things to do. Most of the practical work that we learnt in school are not applied in labs, due to the fact that manual work are no longer utilised in labs.

IMMULITE 1000

Immulite 1000 is used to measure two types of hormones levels in our body - DHEA & HGH. Therefore, it consists of two components:

Immulite DHEA-SO4

Immulite IGF-1

Immulite DHEA-SO4 measures dehyroepiandrosterone sulfate (DHEA-SO4), which is an adrenal sulfate in our body.

DHEA-SO4 is, in fact, a sulfated version of Dehydroepiandrosterone (DHEA), a steroid hormone that is thought to be the 'superstar' of all hormones.

Thus, the concentration of DHEA-SO4 relates to the amount of concentration of DHEA, as DHEA-SO4 is 500 times more than DHEA.

You just need the patient's blood serum to do this test.

DHEA:

DHEA is a natural-occurring hormone that decreases with age. It is produced in the adrenal glands and it may be also possible to be derived from the testes. It is said to be the precursor of testosterone and oestrogen, as they share similar chemical properties. DHEA levels increases & peaks when one reaches adulthood, but drop as one starts to age after his 40s. Thus, many diseases which correlate with age also correlate with low levels of DHEA production.

Immulite IGF-1 measures insulin-growth factor (IGF-1) levels, which is stimulated to be produced by Human Growth hormone (HGH) itself.

HGH:

HGH is a hormone that is produced in the pituitary gland & released only when the hypothalamus 'signals' for it. HGH levels decreases as one gets older. It promotes growth in the young by mainly promoting cell growth and height increases during youth. HGH is highly significant in the body as it is thought to be associated with 'youth'. As HGH decreases, the ageing process increases.

Thus, the immulite system involves determining the top two hormones that are involved in ageing. As such, the system is used to predict whether the patient is :

Suffering from growth disorder
Taking pills to supplement growth
Undergoing anti-ageing treatment

Principles:

The immulite 1000 system utilizes assay-specific, antibody, or antigen-coated plastic beads, as the solid phase, alkaline phosphtase-labeled reagent, and a chemiluminescent substrate. The coated bead, is found a proprietary plastic device, namely a test unit by Siemens.

Test unit

Antibody/antigen-coated Plastic bead

The test unit serves as a reaction vessel for the immune reaction, the incubation and even washing. The bead and sample's serum is first placed in a test cup, before being placed in the roller to enter the machine.

Sample Cups

Roller to place the sample cups, before entering the machine

After incubating the bead with the sample's serum and the alkaline phosphatase reagent, the reaction mixture is separate from the bead by spinning the Test unit at high speed on its vertical axis. The entire fluid contents (the sample, excess reagent and wash solution) is transferred to a coaxial waste chamber in the Test unit. The bead is left with no residual, unbound label. The amount of bound label is then quantiated with a dioxetane substrate that produces light. Light emission is measured by a Photomultiplier Tuve(PMT) and the results are calculated.

The results are given by values. There is a list of reference values for all age group by decades. However, there is no significant value for DHEA & HGH. Any value is acceptable, as deficiency or exceeding HGH & DHEA hormones does not cause any disease/harm to the body.

Any qns, feel free to ask me. I know its abit long, and lengthy. But I have no choice.

Lloyd Lam 0607775D

SIP Week 8 Sharing

2008-08-16T11:20:00.004+08:00

Subject: Lab Technique

Name of Test: Faecal Extraction

Hi people! Because I am in a research lab, I don’t get a chance of rotating around the different labs. And so, yes, I am still having my attachment at the endocrine lab.

Well, most of the time I am doing my MP. So, in this post, I am going to talk about a lab technique that is frequently being carried out in the lab for my MP.

Faecal Extraction. Yup. You guys didn’t read wrongly. Its faecal extraction. It is not really that smelly.

But before I go into that, let me discuss about my MP first. My MP has got to do with the clouded leopards. Clouded leopards are presently being classified as vulnerable and their actual numbers are unknown as there hasn’t been much research done on them. And because their numbers are declining, there are many conservation projects that are being undertaken by the zoos. However, the breeding process shows little success. This has been thought to be due to stress. Hence, for my MP, my main aim would be to establish their stress level and hope to be able to provide information to help reduce the stress brought about to the leopards.

To help analyse the stress levels, the hormones that we are measuring are glucocorticoids - cortisol and corticosterone and the samples used were faeces.

Why faeces?
This is because it can allow a more accurate result obtained. The collection of faeces for testing is a non-invasive method. Hence, it does not induce stress to the animal which could eventually lead to bias results. Urine could be another sample option but as we all know, it’s kind of hard to collect urine especially from such a fierce animal. Blood samples are not used as this is an invasive method in which it will tend to induce stress to the clouded leopards and thus could result in biasness in the results obtained. Furthermore, it is impractical to collect blood regularly.

Before we can measure the hormones, we need to extract steroids from the faeces first. This is done using the boiling method.

Method:
1. Dry the faeces using a lyophilizer for about 2 days.
2. Pound the faeces into powdered form using a pestle.
3. Weigh out 0.200 ± 0.002g of faeces into the test tubes.
4. Add 4.5 ml of ethanol and 0.5 ml of distilled water to the test tubes.
5. Mark the levels of the solution in the test tubes.
6. Vortex briefly.
7. Boiled the test tubes in the water bath at 95˚C for 20 minutes.
8. Add 100% ethanol as needed to prevent the test tube from boiling dry.
9. Bring the volume up to pre-boiled level using 100% ethanol.
10. Centrifuge the samples at 2500 rpm for 20 minutes.
11. Pour off the supernatant into another set of clean test tubes.
12. Add 4.5 ml of ethanol and 0.5 ml of distilled water to the test tubes.
13. Centrifuge the samples at 2500 rpm for 20 minutes.
14. Pour off the supernatant into the test tube containing the first set of supernatant.
15. Dry down the second set of test tube using a water bath and purified compressed air.
16. Add 1 ml of PBS and vortex briefly.
17. Sonicate for 15 minutes.
18. Pour the extract into 12 x 75 mm plastic tubes and store them at -80˚C.

Hope you guys understand. =) How to go about analysing the hormones level will be explained in the next post.

Enjoy.

Xin Yi
TG02

the 7th week of SIP =)

2008-08-08T22:51:00.002+08:00

Hi people! Hope you guys are doing fine for SIP. =)

I'm Lyn and yes, I am back once again after 7 weeks of attachment. I know almost everyone is saying this but still, time really flies.

In my very first post, I had already mentioned briefly on what I do in the lab that I am in. This time round, I shall share with you guys on blasting. As I am in a research lab, I won't be changing departments now and then. Thus, the things that I do are more or less the same.

Blasting
Ok. Blasting is a step that the lab I'm in does after the sequencing step. Sequencing is done by a DNA analyser. After the whole sequencing procedure is done, the trace files for a particular primer is retrived by injecting your thumbdrive into the desktop that is linked to the DNA analyser to save them. Usually, you have to will retrieve 2 trace files in total for a primer. This is because we do sequencing for both forward and reverse.

Trace files are the end result that you get after sequencing is done. By transferring the trace files and opening them in sequencing programmes, you can get the electropheragram of each DNA sequence. In the lab I am in, the sequencing programmes they use are Sequencing Analysis version 5.2 and SeqScape version 2.5 (Applied Biosystems, USA).

As these programmes enables one to view the electropheragram, we are able to bast. Blasting means detecting SNPs in the different DNA sequence. By doing blasting, we are able to find out any known or new SNPs in the gene. Blasting is actually quite an eye-straining step to do. However, there are no alternatives to it. One has to look through all the DNA sequences to detect the SNPs.

How one detect SNP(s) is bascically through scanning the whole DNA sequence of each different DNA. A SNP is shown on the electropheragram as two peaks at the same base nucleotide position. Usually, only one peak is seen.

Sometimes, blasting is made tougher when there are a lot of 'noises' in the DNA sequence. 'Noise' in this case is peaks formed by excess reagents that interfere with sequencing. Usually these 'noises' will affect the jugdement of whether the peak is a SNP. This is because, some of the 'noises' are of such high peaks that one may think or assume that they are SNPs. According to what I'm taught, one can confirm a peak is a SNP when the peak is almost the same height as the peak of the correct base, or at least 2/3 the height of the peak of the correct base.

By comparing with the reference sequence of the gene, one can detect and confirm the polymorphisms in a gene. Reference sequence of a cetain gene most of the time shows the correct DNA sequence that is found in the majority of the healthy population.

When a SNP is found, one will record down or comment on the position of the SNP and also the change in base mucleotides, in the obtained reference sequence of the gene. Reference sequences of different genes can be found at the UCSC genome browser webiste (http://genome.ucsc.edu/).

That's all. I shall share with you guys more on what I am doing in my lab the next time i post again. Hope what i explained was clear.

Take care everyone and have fun! =)

Lyn
0611027D
TG02

Weeks V and VI Attachment

2008-08-01T23:37:00.023+08:00

Hello again Y’all!

Indeed six good weeks have flown by and it’s my blogging once again, just right before I knew it. Hope all is fine there and that all your lil’ test tubes are still returning every cheeky grin of yours.

Alright, all things proper, in the previous entry, a brief overview of the major project was posted. So you’d probably know by now what metformin is and some theory about HPLC. With that, in this entry, I‘d be putting up some stuffs involved in the method development stage of the project.

Preparation of an Extemporaneous Metformin Sample

These procedures here are practically the same as most other usual practices in the lab. The only difference is that the sample arrives in a tablet form instead of the usual powder form; thus an additional step is required here, followed by the usual.

Materials:

500mg metformin tablet
DI water
sonicator
Mortar and pestle
100ml volumetric flask
glass funnel
glass stirring rod
5ml dropper
filter paper (optional)***

Methods:

1. Thoroughly pulverize the metformin tablet into powder with the mortar and pestle.*

2. Rinse the drug particles off the pestle into the mortar with DI water.

3. Dissolve the powder using the stirring rod.

4. Transfer the drug solution into the volumetric flask using the glass funnel.

5. Rinse any remaining drug particles from the mortar into the volumetric flask.

6. Top up the rest of the volume with DI water till the 100ml mark, using the dropper when nearing the end.

You might be able to see a faint white line on the bottle immediately to the left.

A filled volumetric flask.

7. Shake the volumetric flask vigorously to dissolve any remaining powder in the solution.

8. Place the volumetric flask onto a sonicator** to aid in the dissolving process and removal of bubbles in the solution sample.

9. Filter the solution (optional) ***.

* You might notice that there is no weighing of any samples here to be dissolved, unlike that in the preparation for the standard sample. Instead, the entire tablet is used for the making of the sample.

** A sonicator is a machine that causes sonication, whereby sound (ultrasound) energy is used to agitate a sample for various samples such as that of speeding up the dissolving of a solute and the removal of gases (bubbles) from the sample.

*** Filtering is necessary for the sample in this experiment it would be used for HPLC. Thus, it would be removal of any undissolved particles would be necessary to prevent interference with the outcome of the results.

In this experiment, this sample is usually used as an unknown sample, whereby it is used to compare against a calibration plot from the standard and determine if it is accurate. In later phases of the project, it might also be used to facilitate a typical sample that would be made by patients themselves in stability tests.

Preparation of a Metformin Standard Sample

This procedure here uses a commercially available metformin standard, for the preparation of a solution with a concentration of 500ppm*. The procedures as you might probably notice are slightly different from that for the preparation of an extemporaneous metformin sample in that weighing is involved.

Materials:

Meformin standard

This is the one we have in the lab.

DI water
Electronic pan balance
50ml** volumetric flask
watch glass
glass funnel
glass stirring rod
5ml dropper
spatula
filter paper (optional)

Method:

1. Weigh out 25mg of metformin standard on the watch glass using the electronic pan balance.
2. Transfer the sample into the 50ml volumetric flask.
3. Rinse off any remaining metformin standard particles on the watch glass into the volumetric flask with DI water using the glass funnel to help collect the solution.
4. Top up the sample in the volumetric flask to 50ml, using the dropper when nearing the end.
5. Shake the sample vigorously to dissolve to metformin standard.
6. Sonicate the newly formed solution sample.
7. Filter the sample (optional).

* ppm denotes “parts per million”. The actual definition of it is quite complex and thus a simpler way of understanding it in this case is by looking at some examples such as 1mg/ 1000ml = 1ppm, 500mg/1000ml = 500ppm, 250mg/500ml = 500ppm etc. Essentially, the number of milligrams over a thousand milliliters is the number of parts per million.

** A 50ml volumetric flask has been selected here as it provides a sufficient amount for the carrying out of various tests within a suitable period of time while reducing unnecessary wastage of the standard (the standard is very expensive!).

In this experiment, the prepared metformin standard sample is usually used to chart a calibration plot for the determining of any samples of unknown concentrations and also to aid in determining the suitability a particular set of conditions for the quantification of metformin. Its concentration would usually be diluted to lower concentrations. Some commonly used concentrations are 50ppm and 100ppm.

An example of how dilutions are done is in the case of 50ppm for example, whereby 1ml of the 500ppm standard sample is transferred into a 10ml volumetric flask, with the rest of the volume being topped up with the mobile phase or DI water. Thus, simple calculations would confirm that 500ppm/10(DF) = 50ppm.

Alright, that’s all for this entry. Thanks for taking the time to read it and hope you’ve benefited somewhat. All the best to the rest of your attachments once again!

Alexander Soo TG02
0608122H

SIP sharing week 5

2008-07-22T13:34:00.012+08:00

Subject: Cytogenetics laboratory technique

Hi Friends!!

I am TING-JIE here! 5 weeks had already passed, time flies right? I do have fun at my attachment, all of the staffs are very nice to me, and I learnt a lot of new things from them, by the way, I am attached to Cytogenetics laboratory.

Basically there are 3 different sections in cytogenetics lab which include prenatal lab, bone marrow lab and FISH (fluorescence in-situ hybridization) lab. Currently I am under prenatal lab for the past 4 weeks.

And now I will be sharing with you all my experience in cytogenetics lab-prenatal section!
Firstly, what is cytogenetics?

Cytogenetics actually is the study of chromosome number and morphology, particularly as it relates to a normal or pathological state, the cells being studied are best at late prophase or metaphase of mitosis for analysis.

What is chromosome? It is a long, stringy aggregate of genes that carry heredity information and are formed from condensed chromatin.

Why late prophase or metaphase? For this we will have to refer back to Molecular Genetics (MGEN) and Molecular Biology (MBIO).

In the late prophase, the chromatin condenses into discrete chromosomes. The nuclear envelope breaks down and spindles form at opposite "poles" of the cell. The chromosomes begin to migrate toward the cell center.

picture taken from http://iweb.tntech.edu/mcaprio/late_prophase.jpg

In metaphase, the spindle fully develops and the chromosomes align at the metaphase plate (a plane that is equally distant from the two spindle poles). At this stage the nuclear membrane disappears completely and chromosomes are held at the metaphase plate by the equal forces of the polar fibers pushing on the centromeres of the chromosomes. This is also the stage when the chromosomes are at their most contracted state that is why chromosomes are best study at metaphase.

picture taken from http://iweb.tntech.edu/mcaprio/metaphase1.jpg

Only in those 2 phase, chromosomes are ready to be study under light microscope, whereas in anaphase, the paired chromosomes (sister chromatids) separate and begin moving to opposite ends (poles) of the cell. Spindle fibers not connected to chromatids lengthen and elongate the cell. And in telophase, which is the final stage of mitosis, the chromosomes uncoil, the nucleolus reappears the nuclear envelope reappears, the spindle fibers disappear, and results in two daughter cells. Therefore there are no complete chromosomes for us to study at anaphase and telophase due to the progression into daughter cells.

picture taken from http://iweb.tntech.edu/mcaprio/anaphase2.jpg

http://iweb.tntech.edu/mcaprio/Telophase1.jpg

Prenatal diagnosis is requested by obstetricians and gynaecologists, through chromosome analysis, it helped in the clinical management of pregnancies by detecting possible constitutional abnormalities of the fetus. The specimens used for prenatal cytogenetic diagnosis include amniotic fluid (AF), chorionic villi (CV) and fetal cord blood (FB).

Amniotic Fluid (AF) contains cells derive from the amnion, from the gastrointestinal tract and from the skin of the fetus. Some of these cells are viable and are capable of undergoing cell division in vitro. Through amniocentesis, a sample is collected for the purpose of culturing the cells to obtain chromosomes that reflect the fetal karyotype.

Amniocentesis is a diagnostic procedure performed by inserting a hollow needle through the abdominal wall into the uterus and withdrawing a small amount of fluid from the sac surrounding the fetus.

illustration of the amniocentesis procedure

picture taken from http://atlasgeneticsoncology.org/Educ/PrenatID30055ES.html

Chorionic Villi (CV) sample is collected through the removal of a small amount of chorion of placental tissue. Chorionic villi are usually collected by aspiration biopsy with a thin plastic catheter inserted trans-cervically or trans-abdominally into the uterus. Chorionic villi consists of coral like projections that surround the embryonic sac in early pregnancy.

illustration of the chorionic villus sampling procedure

picture taken from http://atlasgeneticsoncology.org/Educ/PrenatID30055ES.html

Fetal cord blood (FB) is obtained from the umbilical cord, it is constitutional and is able to reflect the chromosomal constitution of the fetal.

The prenatal chromosome analysis is usually done in:
-Advanced maternal age, 35 years or older
-Recurrent miscarriages
-Abnormal ultrasound findings
-Previous child with a chromosome disorder such as Down syndrome
-One member of the couple is known to carry a chromosomal rearrangement or relatives of a child with a chromosome disorder
-Parental anxiety
-And other indications

Many women who have any above indications choose to undergo prenatal test, to reassure the normality of the foetus.

The whole flow of cytogenetics procedure is as followed:
Cell culture set up --> Harvest --> Slide making --> chromosome Staining
-->Analysis -->Karyotype

And now I will be sharing with you the procedure of amniotic fluid (AF) culture set up. For harvest, slide making, chromosome staining, analysis and karyotype I will post in another blog.

Amniotic Fluid (AF) culture set up

1.Usually the specimen will arrives in a 25 ml syringe, and we have to aliquot the specimen equally into 2x15ml pre-labeled V-shaped tubes(labelled with the lab number and patient’s name)

2.Spin the 2 tubes at 1200 rpm for 10 minutes in centrifuge.

3.After centrifugation, record the pellet size (small, medium, large) and appearance (clear or blood-stained) for future reference.

4.Use a sterile pipet to aspirate the supernatant to just above the pellet and discard it.

5.Break the pellet up by gently flicking the bottom of the tube.

6.Resuspend one of the tube with 1.0ml alpha-AM medium. Mix well and pipet 0.5ml of the suspension onto the sterile 22mm X 22mm coverslips which is pre-prepared in the 35mm petri dish. Labelled the cultures as culture “A” and culture “B”.

7.Resuspend 2nd tube with 1.0ml of alpha-BIO medium. Set up culture “C” and “D” with 0.5ml suspension each. It is important to ensure that the suspensions are confined to the coverslips. This is because only the coverslips will be mounted onto the glass slides for analysis and not the petri dish.

8.Place culture “A” and “C” in one incubator , and place culture “B” and “D” in another incubator.

That is basically what we will do when we receive AF sample.

For the alpha-AM media and Alpha-bio media I mention above in the steps are actually used to grow the AF cells. They contain the essential nutrients for the AF cells to grow. Actually there many different types of media used to culture AF cells, but in our lab we use Alpha-AM media and Alpha-BIo. The difference between Alpha-AM and Alpha-BIO is that Alpha-AM is commercially made while Alpha-BIO is homemade which contains extra nutrients that are not found in the Alpha-AM media. Furthermore Alpha-AM is commercially prepared thus they contain fixed amounts of the essential nutrient whereas for Alpha Bio, we have added extra ingredients to promote the growth of the AF cells.

Hope my blog is clear, if there is any doubt, feel free to ask me, I will try my best to answer your queries. =)

Ting-Jie
(0608495h)
TG02

SIP Sharing

2008-07-19T00:50:00.009+08:00

Subject: Endocrine System

Name of test: SHBG Test

Hi people!

Hope you guys had fun so far in your attachment. Well, I am posted to an endocrine research lab that also do some routine lab work. and so yup, I am dealing with hormones. Anyway, there are so many hormones being secreted by our body so it is kind of impossible to focus on all of them.

In this post, I will be focusing on the Sex Hormones-Binding Globulin (SHBG). SHBG is not a hormone but is actually a glycoprotein that is synthesized by the liver. But before I go into SHBG, I will talk about testosterone first.

Testosterone can be present in both males and females and are mainly bound tightly to SHBG. However, they can also be present in the circulation as either the unbound form or weakly bound to albumin and to cortisol-binding globulin. The unbound testosterone and those weakly bound are considered as bioavailable (non-SHBG bound).

Ok. Back to SHBG.SHBG binds to testosterone and 5α-dihydrotesterone with high affinity but to estradiol at a lower affinity. (Estradiol is an estrogen). So, naturally, as females have a higher concentration of estrogen (female sex hormones) than androgen (male sex hormones), SHBG will have a higher concentration in female than in male. And since SHBG mainly binds to testosterone, any changes in the concentration of SHBG can affect the testosterone concentration.

SHBG is usually ordered along with total testosterone in my lab to help measure the bioavailable testosterone in which bioavailable testosterone is the measure of the free circulating testosterone and those that are weakly bound to albumin and cortisol-binding globulin. This is to test for testosterone deficiency in men and in women for excess testosterone production. Measuring only the total testosterone alone does not show testosterone deficiency as you can have high amount of testosterone and high amount of SHBG which actually may results in testosterone deficiency.

Immulite 1000

Picture taken from: http://diagnostics.siemens.com/webapp/wcs/stores/servlet/ProductDisplay?catTree=100001,1015815,1015817&catalogId=-111&langId=-111&productId=172962&storeId=10001

Both the concentration of SHBG and total testosterone can be determined using a machine called Immulite 1000. Basically, everything is automated. So, I just have to load the sample in. =)However, before I can start using the machine, I have to purge the system to remove all bubbles that could be present in the tubing which in turn can affect the exact volume of reagent added. The probe also has to be clean to prevent any contamination. After which, I have to pipette about 120µl of the sample (plasma) into the sample cup. I then load the materials needed in this order: sample cup, dilution cup, leave a space and then the test unit. The machine will then do all the work and I would just have to wait for the results. But, before I can leave the machine alone, I have to make sure that all the samples are "accepted" by the machine. If there are errors detected by the machine, I will then have to troubleshoot.

Anyway, this machine uses assay-specific antibody or antigen-coated plastic beads, alkaline-phosphatase-labelled reagent and a chemiluminescent substrate. The coated bead is contained in an apparatus called the test unit in which it allows all reactions to occur.

The machine will add the sample and the alkaline phosphatise-labelled reagent into the test unit and then incubated. After which, the bead in the test unit are spun at high speed about the vertical axis to remove all unbound antibodies. Using the chemiluminescent substrate, dioxetane substrate, the amount of SHBG can then be quantified by a Photomultiplier Tube.

Picture taken from: http://diagnostics.siemens.com/webapp/wcs/stores/servlet/PSGenericDisplay~q_catalogId~e_-111~a_langId~e_-111~a_pageId~e_79518~a_storeId~e_10001.htm

The reference range is about 13-71 nmol/L for male and 18-114nmol/L for non-pregnant women.

Some indications for abnormal values of SHBG are
Decrease SHBG levels can indicate:
1. Hirsutism - “male-pattern” hair growth in women (found in decrease SHBG in women)
2. Hypothyroidism
3. Androgen administration

Increase SHBG levels can indicate:
1. Hyperthyroidism
2. Hepatic Cirrhosis
3. Pregnancy

Ok. That’s all. Enjoy.

Xin Yi
TG02

SIP 1st 3 weeks - MMIC Department

2008-07-13T00:41:00.008+08:00

Hey peeps! Its been 3 whole weeks since the start of SIP, and time seems to pass really fast! 17 more weeks and we are done with it. School life is undoubtedly better than working life I suppose. Wearing formal daily proves to be a torture to me!

So anyway, I am currently attached to Microbiology department for the 1st 3 weeks. I have 9 departments to clear, and 3 different locations to work. The reason is because my company has 3 branches (Bukit merah, Orchard & Thomson), so I will not be stuck in one place!

This week, being my final week in Microbiology (before moving on to Cytology), has been really great. What we deal in includes urine, stool and semen. There was a practical test for me to go through which involves the entire ID process for microrganisms, and I am glad that I managed to pass.

STOOL OCCULT BLOOD TEST

Intended use : Rapid, convenient and non offensive quantitiative method for detecting occult blood in the stool, and is mainly for professional use as an aid in diagnosis in gastrointestinal conditions.

TA DA..My hard work..you have 2 sections to dab two stool specimens in each test paper,if it turns blue (after adding the reagent), it is POSITIVE.

Principles: When stool specimens containing occult blood are applied to HEMA SCREEN test paper, the hemoglobin portion of the occult blood comes in contact with the guiaic. When the HEMA SCREEN peroxide developing solution is added, a guaiac-peroxidase like reaction occurs, and thus the results can be seen by:

BLUE-GREEN - POSITIVE (PRESENCE OF BLOOD IN STOOL)

BROWN/NO CHANGE - NEGATIVE

Thus, any trace of blue within 30 secs, signifies a positive test result. However, it must be noted that the results should be read quickily, as the colour may fade after 4 mins.

This test is easy and cheap. However, the setback is that it is not extremely accurate (could be a result due to pre-anayaltical error/human error).

ALTERNATIVE

The alternative is a procedure involoving stool specimen that is incubated and mixed in some sort of culture (cant rmb), instead of just using the pure stool specimen.

Next, a strip test paper is inserted, and allowed to incubate for 5 mins. Just like a HCG preganacy test, two lines shown = POSITIVE

one line shown = NEGATIVE

Here is the pic..for the alternative test (cant rmb the name).

Oh, and it must be noted too that all procedures have to be done in a fume hood! And of course, one must not puke and run away from the sight of stool specimens. I have dealt with VERY hard stool to VERY liquid stool specimens (diarrhea).

Alright, thats the main highlight of the microbiology work I have done. Till then, all the best for ur SIP & MP!

Lloyd Lam 0607775D

Attachment Experience From Weeks I & II

2008-07-06T10:45:00.013+08:00

Greetings to all! Hope all is good there!

My attachment had started off with quite a bang since day 1 last week, the very day I was given the objectives and scope of my major project(MP). You'd find out why as you go along.

Basically, I've been posted to nowhere unfamiliar - back to TP - for my first ten weeks of attachment. But during the entire duration there, I'd be focusing on just my MP only. In other words, you shouldn't be expecting much of any routine lab work but more of some jucier bits (I hope). Also, my MP's been snatched out from an area pretty much alien to our BMT option; it'd probably would fit more nicely under the subject title of Pharmaceutical Analysis (In other words, that's pretty good for me, as in think resume and you'd know why, hahs). And as with every new experience, I'm really hoping to grab more than just a thing or two out of it into my bag of knowledge.

Well, to start off proper, the MP's got to do with an antihyperglycemic drug called metformin, alongside an impressive masterpiece - High Performance Liquid Chromatography (HPLC). The gist of it is to come up with a method development and method validation to quantitate an extemporaneous form of metformin and test for its stability with the help of the trusty HPLC.

In case you might be scratching your head about metformin, again, it is an antihyperglycemic agent commonly used to treat patients with type II diabetes. It is believed to bring about its mode of action through three ways:
i. Decreasing gluconeogenesis in the liver
ii. Enhancing insulin senstitivity by increasing peripheral glucose uptake and utilization
iii. Decreasing intestinal absorption of glucose

Also, metformin has the chemical formula of C4H11N5. HCL when in generally all dosage forms, and would be described as strongly basic and polar, freely soluble in water, soluble in methanol, slightly soluble in ethanol, and practically insoluble in methlyene chloride, acetone, chloroform and ether. Unlike many other antiglycemic drugs such as that of sulfonylureas however, metformin generally does not cause hypoglycemia or hyperinsulinaemia. Nonetheless, as is inevitable with all drugs, metformin can bring about certain unwanted side effects such as nausea, stomach upset, diarrhea and even lactic acidosis in serious cases.

At present, metformin is only available in two types of dosage forms – the first being a more commonly prescribed tablet while the other is a relatively new liquid solution. However, despite the breakthrough of the relatively new liquid metformin solution, the problem regarding difficulty in swallowing of the drug in its tablet dosage form, often due to dysphagia, by a significant fraction of patients with type II diabetes - the elderly, still persists worldwide. This is so as the liquid solution of the drug, Riomet® produced by Ranbaxy Laboratories, is only available only in the U.S and not other countries, such as that of Singapore. Moreover, the disease is increasing in prevalence among the children population too, another group of patients who would have to share in the agonising task of swallowing a tablet due to their relatively poor coordination of muscles required in swallowing. On top of these difficulties, it should be noted that the treatment lasts for the rest of an individual's lifetime - an arduous burden to carry especially with the abovementioned difficulties.

This project, in relation to the developement of a new dosage form of metformin, hopes to develop a simple and efficient method for the quantitation and stability testing on an extemporaneously prepared metformin solution formulation with the help of HPLC. This in turn would allow for the determination of metformin concentration and stability in future newly-developed metformin solution formulations.

With some light shed onto the scope of the project, let's move on to some technical aspects shall we, whereby with technical aspect, I mean nothing else other than HPLC.

As the name suggests, HPLC is a type of chromatography, or a technique for the separation of mixtures of compounds in a sample. This separation takes place between a mobile phase -otherwise known as the solvent, and a stationary phase - otherwise known as the material in the column. Separation is dependent on different mechanisms such as that of adsorption, partition, ion-exchange and size-exclusion. In this project, the two common types of HPLC encountered are normal-phase HPLC and reverse phase HPLC. The difference between these two types is that in normal-phase HPLC, the mobile phase is of a low to medium polarity while the stationary phase is of a high polarity, whereas in reverse-phase HPLC, the situation is reversed in that the the mobile phase is of a medium to high polarity while the stationary phase is of a low polarity. In turn, the result is that the least polar particles are eluted first in normal phase HPLC and the most polar particles are eluted first in reverse-phase HPLC.

The following picture gives a really rough sketch on the basic parts of a common HPLC machine:

There are variable conditions/parameters in the machine that can affect the result for a tested substance/sample. For instance, the column type, type of mobile phase, flow rate, type of detector (UV or Diode Array Detector), temperature, wavelength, elution time and injection volume. The result in turn, would be translated into a graph, whereby the peak would be directly proportional to the concentration/ quantity of a particular substance present. The concentration is determined in a way that is pretty much similar to that of a spectrophotometer, whereby a UV light would emit rays through the stream of liquid coming out of the column while a detector on the opposite side of the stream would give a direct reading of the amount of light absorbed by the liquid. This amount of light absorbed would nevertheless be directly proportional to the concentration/ amount of a particular substance flowing through the beam.

The following is an example of how a typical result might look like, whereby the X-axis represents the duration of the run, and Y-axis represents the concentration of a substance that was run at that time during one of my lab sessions in the second week.

With all of that covered, perhaps the only thing that I've left out thus far is the progress of the project to date.

As of presently, under the guidance from my patient supervisors of the project, a simple formulation for the extemporaneous product of metformin has been achieved, and the first few runs of the sample on a HPLC machine carried out. The results in turn had provided better perspective into the responsiveness of the HPLC machine being used to metformin, thus providing a stpping stone to other subsequent objectives yet to come.

Alright, that'd be all for now. Thanks for going through the thick and densely forested entry of words and hope that it'd benefitted you somewhat there. Of course, all the best to the rest of the duration of your respective attachments.

Here's signing off,

Alexander Soo, TG02.

References:

1. RxList. Glucophage Clinical Pharmacology. Retrieved 23 June, 2008 from http://www.rxlist.com/cgi/generic/metformi_cp.htm

2. WebMD. Drugs and Treatments - Metformin Oral. Retrieved 23 June, 2008 from http://www.webmd.com/drugs/mono-7061-METFORMIN+-+ORAL.aspx?drugid=11285&drugname=Metformin+Oral

3. Riomet. What is Riomet. Retrieved 23 June, 2008 from http://www.riomet.com/about.asp

4.The World Intellectual Property Organization. (2007). Effervescent Metformin Composition and Tablets and Granules Made Therefrom. Retrieved 23 June, 2008 from http://www.wipo.int/pctdb/fr/ia.jsp?ia=EP2005%2F054757&IA=EP2005%2F054757&DISPLAY=DESC

5. (2006).Metformin Hydrochloride. British Pharmacopoeia 2007 Volume II. London: The stationery Office.

6. (2000). Metformin Hydrochloride. Pharmacopoeia of the People’s Republic of China Volume II. Beijing, China: Chemical Industry Press.

2008-07-05T14:27:00.002+08:00

Hi guys!

It seems that some of you are quite interested in knowing how does the Gel Doc works and how to operate it. I found this website whereby they teach you how to operate it. You guys can visit this website if you want to know more about Gel Doc.

Website: http://www.calpoly.edu/~bio/ubl/protocols_files/geldocdet.htm

Thanks and i apologise for the poor explanation on the Gel Doc.

-Lyn-

2008-06-27T23:02:00.003+08:00

Subject: PCR Techiques

Hi all! =) This is Lyn here. I will be the first in my group to share with you guys (and my group mates) about the things that i have done this first week of my attachment.

I'm attached to the research lab doing research relating to pharmacogenetics. My MP will be on that as well. Pharmacogenetics is basically the study of how the variability of the genetics of individuals affect the reponse to drug dosage. This is because, if an individual has a variant in his/ her gene, he/ she might need a lowerd drug dose as compared to an healthy individual with no variants. Thus, pharmacogenetics enables the prescription of the correct drug dose to the patient so as not to result in drug toxicity.

Bascially, what I do in the lab for this one week was preparing the PCR products using different primers, cast 1.5% and 2% agarose gel to carry out gel electrophoresis and the PCR which includes primer optimisation. Doing primer optimisation is actually to find out the optimum temperature whereby primers anneal to the DNA sample and result in bright sharp thick bands. I also get to do gel check for the PCR products that are prepared. It is similar to what we were shown during Mgen or Mbio practical sessions. However, in the lab, they use this machine known as the Gel-Doc to visualise the bands. In this machine, you place your completed agarose gel from electrophoresis process and turn on the UV so as to 'bring out' the bands. This machine is connected to a computer whereby there's a programme where you can print the video print of your agarose gel out. It comes out as a photo.

I also learnt about purification and sequencing. Purification is done actually to remove the excess primers and dNTPs that would interfere with the sequencing procedures. In purification, Exo-SAP is added to the PCR products. Exo = exonuclease 1: it cleaves and remove the excess primers. SAP = shrimp alkaline phosphatase: it cleaves and dephosphorylates the dNTPs so that they are unable to bind and carry out elongation.

Sequencing is done to determine the actual sequence of the DNA that is prodcued during the PCR that is complementary to the template DNA strand. This is done by adding the primers and the dye that contains dNTPs, ddNTPs and Taq polymerase. These ddNTPs are fluorescently labelled so that they can be detected and the sequence of the DNA strand complementary to the temlate DNA strand can be known. After the addition of the reagents needed, the samples are sent to the analyser, whereby the analyser will analyse the different ddNTPs that binds and also produce the peaks.

These are bascially what I have been doing for the first week of attachement.

Have fun at work for SIP. Take care everyone. =)

-Lyn-

What is CAP accreditation and CAP Lab Accreditation Programme?

2008-06-16T11:30:00.016+08:00

CAP is an authorized accrediting organization1 that promotes excellent pathology and laboratory medicine practices to ensure welfare of its customers are taken care of.1, 2

Upon completing and passed the inspection programme done by CAP, a laboratory will be granted CAP accreditation. The laboratory is now proven to produce the best results.3

Laboratories can undergo CAP Lab Accreditation Programme to acquire CAP accreditation. The programme is authorised by CMS and recognised by JCAHO. It satisfies the requirements of different laboratory settings, comprises of a variety of disciplines and testing procedures, and enables high level of service to be obtained.3

(96 words)

CMS: Centers for Medicare and Medicaid Services

JCAHO: Joint Commission on Accreditation of Healthcare OrganizationsReferences

1. METROPOLIS Health Services. (2005). College of American Pathologists [CAP]. Retrieved June 13, 2008, from http://www.metropolisindia.com/cap.asp

2. DNA Junction. College of American Pathologists (CAP) Laboratory Accreditation Program. Retrieved June 13, 2008, from http://www.dnajunction.com/accreditation/dna-cap.php

3. College of American Pathologists. (2008). About the Laboratory Accreditation Program. Retrieved June 13, 2008 from http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=/portlets/contentViewer/show&_windowLabel=cntvwrPtlt&cntvwrPtlt{actionForm.contentReference}=laboratory_accreditation/aboutlap.html&_state=maximized&_pageLabel=cntvwr

Done by Lyn

Why is there a need for CAP accreditation?

2008-06-16T11:29:00.002+08:00

By having a CAP accreditation, it can show that the laboratory has fulfilled the regulatory requirements, skilled in carrying out its specific tasks and that patient care and safety are always well taken care of to the highest possible quality, minimizing errors in laboratory results which can lead to wrong diagnosis and treatment.1,2 This thus, allows the existence of trust between the accredited laboratories and the third parties in the information generated during the testing procedures, preventing unnecessary verification that could save time and money.2 Furthermore, acknowledgement of test results generated in other countries can also be facilitated.2

(95 words)

1. CLSI eNews. (2007). Excellence in Laboratory Performance: CLSI and CAP Aligned for a Common Goal. Retrieved June 14, 2008, from http://enews.nccls.org/clsi/textonly/2007-11-01/4.html

2. Singapore Accreditation Council. (2005). SAC E-ALERT-SAC-CAP Joint Programme. Retrieved June 14, 2008, from http://www.sac-accreditation.gov.sg/news.asp?month=7&year=2005

Done by Xin Yi

Benefits of CAP accreditation

2008-06-16T11:24:00.002+08:00

CAP Laboratory Accreditation Program helps laboratories to meet and even exceed the requirements of the Centers for Medicare and Medicaid Services (CMS) and other Federal requirements as only CAP uses multi-disciplinary teams of qualified professionals to be inspectors. Under this program, CAP is able to attend the widest patient population and ensuring accurate and quality test results of patients. The laboratory standards are maintain by laboratories professionals, and from the self-inspection and participating in the inspection of other laboratories, the laboratory can learn the latest laboratory process and techniques.1,2

(89 words)

1. College of American Pathologists. (2005). Benefits of CAP Accreditation. Retrieved June 13, 2008 from http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=laboratory_accreditation%2Flap_info%2Fbenefits.html&_state=maximized&_pageLabel=cntvwr

2. METROPOLIS Health Services. (2005). College of American Pathologists [CAP]. Retrieved June 13, 2008, from http://www.metropolisindia.com/cap.asp

Done by Ting Jie

Features of CAP System Inspection

2008-06-16T11:17:00.011+08:00

A CAP System Inspection1 comprises of:

-utilization of standardized checklist and inspection dates for all sites

-integration criteria2 to ascertain that a common system is used to carry out inspections

-a pre-inspection meeting with a CAP inspection specialist to obtain relevant details and knowledge, and to assist the team leader in forming a team that is suitable in size and goal

-a CAP inspection specialist being present at the respective venues during the inspection to allow for a systematic management and communication

-allocation of one inspector to one inspection team of similar disciplines

-a post-inspection international consolidation meeting and a final report to determine system-level feedback

-a CAP system inspection document to indicate the laboratory’s accomplished performance

(124 words)

1. College of American Pathologists. (2008). Features of a CAP System Inspection. Retrieved June 14, 2008, from http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=/portlets/contentViewer/show&_windowLabel=cntvwrPtlt&cntvwrPtlt{actionForm.contentReference}=laboratory_accreditation/system_inspections_features.html&_state=maximized&_pageLabel=cntvwr
2. College of American Pathologists. (2006). Qualifying for a System Inspection. Retrieved June 14, 2008, from http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=laboratory_accreditation%2Fsystem_inspections_qualify.html&_state=maximized&_pageLabel=cntvwr

Done by Alexander

How is CAP Accreditation awarded?

2008-06-16T11:16:00.010+08:00

CAP Accreditation is awarded when a laboratory has proven that it meets the criteria and standards for laboratory accreditation.1 A qualified inspector will inspect every 2 years, and both inspector and laboratory will evaluate its own standards by comparing against the standard CAP checklist.1 The assessment of areas includes laboratory equipment, computer services and the handling of specimens.2 Laboratories have to correct and improve on any errors detected, as unsatisfactory results will cause the laboratory to lose its CAP accreditation.1 Thus, CAP ensures that laboratories are of high standards, safe and have excellent procedures to produce sustainable and up-to standard results.2

(100 words)

1. Hon Fon L. Mark. Setting the Standards for Cytogentics Laboratories. Medical Cytology. (2000). Retrieved June 15, 2008, from http://books.google.com.sg/books?id=VVn_Othsf0UC&pg=PA615&lpg=PA615&dq=how+is+CAP+accreditation+awarded&source=web&ots=cbXJDyjAaQ&sig=s6TJyLZ9klvl0ihqqS_aPGw1Kqo&hl=en&sa=X&oi=book_result&resnum=1&ct=result

2. DNA Junction. College of American Pathologists (CAP) Laboratory Accreditation Program. Retrieved June 15, 2008, from http://www.dnajunction.com/accreditation/dna-cap.php

Done by Lloyd