SIP sharing week 5

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

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

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

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.

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-