De-incredibles Group 9 (TG02)

Jean
Neela
Debbie
Xin Ni
Zhenling


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Sunday, November 9, 2008

SIP posting : week 20

YES! Finally it’s the end of SIP. But it also means submission for reports and all are coming. Hmm.. Anyway, I was posted to blood bank for the last week. But I am going to post on another test done in the Haematology lab, which I was in for 3 weeks before blood bank lab.

In Haematology lab, it is actually one of the busiest lab. There are constantly a lot of samples sent to the lab to be tested. There are only two types of specimen sent; blood and CSF samples. I am going to share on Erythrocyte sedimentation rate test. In the lab, they make use of the method Sedi-rate P4-Micr System to test for ESR.


Principle

Erythrocyte Sedimentation Rate is the rate of sedimentation of red cells in the first 50minutes. This will happen when anticoagulated whole blood is allowed to stand for a period of time, which the red cells will settle from the plasma. The increase in ESR shows indicates some abnormalities in the body.

Sedimentation of the red cells actually occurs in 3 stages.
1) Preliminary stage: In the first few minutes, rouleaux occurs and aggregates forms.
2) The sinking period: This is where the sinking takes place at a constant speed.
3) Last stage: This is where the rate of sedimentation slows which is due to the aggregates settling at the bottom of the tube.


Materials

Ø Aquisel tube with stopper filled with 0.8ml of Trisodic Citrate. (Available commercially)
Ø Pipette (for ESR)
Ø Rack/Aquisel tube styoform holder
Ø Mechanical pipette
Ø Pipette tips
Ø Timer



Methods

1. The EDTA blood samples sent to the lab should be mix well.
2. 320µl of blood were pipetted from the EDTA sample to an Aquisel tube containing Trisodic Citrate.
3. The Aquisel tube is mixed well.
4. The pipette is introduced into the Aquisel tube. The pipette is pushed downwards until the blood fills the whole pipette. The blood level should be at “0” on the markings of the pipette.
5. The timer is set to 50minutes and time starts.
6. A small LIS generated sticker is to be stick over the Aquisel tube.
7. Another small LIS generated sticker is to be pasted on the ESR record book. (For record purposes)
8. A “miscellaneous testing” is stamped on the request form and initiated.
9. After 50minutes exactly, the values on the pipette is read and results are taken down in the ESR record book and the request form, which is also to be signed for traceability.
10. The results are to be entered and verified manually in the LIS system.


Reference value

Neonates 1 to 2 days old : 0 to 4mm/50minutes
Neonates 3 days to 1 month old : 0 to 10mm/50minutes
Paediatrics 2months to 12years old :0 to 10mm/50minutes
Adults more than 12years old :0 to 10mm/50minutes


IF the ESR values are higher than the reference value, it can means:
Ø Increase in fibrinogen and/or globulin
Ø Decrease in albumin concentration
Ø Decease in cell pack volume
Ø Tuberculosis, Rheumatic fever, kidney disorder, systemic Lupus Erythematosus (SLE)
Ø Anemia


*Things to take note
The specimen tubes should be checked, there should not be any blood clots or platelet clumps, if there are clumps present, the results would not be vaild. The test should also be done in 4 hrs after the blood collection as the red cells would become spherical after that and it affects the results. The ESR pipette should be left standing on a rack upright, if there is any tilting, it accelerates the ESR. If the blood is lysed, the results will also have error.



That's all!

Zhenling
TG02


=) 11:46 AM

5 comments



Tuesday, November 4, 2008

Sip posting

Hey guys, this is my last posting for our SIP.

I was posted to Histopathology lab for 2 weeks and everyday was packed with things to be done due to low manpower. Anyway, in the lab everyone has to be well rounded in all aspects such as immunology, sectioning, trimming/passing and so on.
The day starts with blocking/embedding of fixed specimens into paraffin wax that were prepared overnight. Next, the lab technicians will shave the blocks and immersed them in softeners for around ten minutes to achieve a smoother and softer cut to get the ribbons for fixing onto the slides. (Note that we have learnt these during pathology so I won’t go in detail, but you guys can ask questions about it)

The most interesting task that I look forward to everyday would be the passing part in the afternoon when we will retrieve all the specimens for the day and sort them accordingly. Passing is done by lab technicians whereas trimming is done by pathologists and the dictations are assisted by the technician. The difference is that the pathologists trim/section the larger kinds of specimens such as colon, breast tissue, stomach, etc and frozen section specimens (specimens received from the operating theater) whereas the technicians will pass specimens such as breast lumps, appendix, prostatic cores, etc. In our lab, appendix, intestines and breast tissues are common and it is quite rare to receive specimens such as aborted fetus, nose (however we did received it for the first time 3 weeks ago), leg, etc. I helped out daily with the dictation part by writing the measurements of the specimens received, their description and comments given by the technicians.

The thing that I would like to further touch on would be the retrieval of prostatic cores.
It is done through needle biopsies for the diagnosis of prostate cancer by the pathologist. A minimum of 8 cores are required and it is made up of 4 from each side of the prostate gland and the 4 cores are from the apex, mid, periurethral and base. The whole procedure is done using a biopsy gun which is inserted through the wall of the rectum into the prostate gland area. I’m not clear of the whole procedure as I don’t get to see it.
Next, during passing of the prostate cores, the length are noted and inked blue before being wrapped in a filter paper and place into a cassette for fixing. Note that the cassettes are to be placed in formalin as soon as possible to prevent the cores from drying causing shrinkage leading to inaccurate diagnosis.

These are examples of stained prostate core slides:

Retrieved from http://www.hopkinsmedicine.org/hmn/F02/feature2.html

Yup, that is all, I’m not sure are the information sufficient cause in the histology lab, although I get to observe the procedure of the immunology staining process, but we are not allowed to do anything and there is not much learnt there. =)
Debbie
TG02


=) 3:31 PM

6 comments



Sunday, November 2, 2008

SIP sharing: LC-MS/MS

This will be my last post.1 more week and internship is completed. I am still in chemistry lab (or to be more specific is chromatography lab). Last few posts have shared thin layer chromatography and column chromatography. This post will be on liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS is the last step that will be carried out for my MP. It is used for chemical analysis of samples.

Chromatography means separation of components of sample based on chemical or physical properties of sorbent. (e.g. hydrophobic interaction, hydrogen bonding etc)

(Image of LC-MS/MS: taken from www.agilent.com)
What is LC-MS/MS?
LC-MS/MS instrument consisted of high performance liquid chromatography (HPLC) coupled to mass spectrometer (MS). HPLC will separate the sample and detect by UV and MS carry out mass analysis.

Since LC-MS/MS consists of 2 part, HPLC and MS, I shall explain them separately.

HPLC
HPLC has an injector that will inject the sample into the column.
A pump then force sample through column.
It is based on the same principle as column chromatography (mentioned in my second post). The difference is that in column chromatography, the sample flows through sorbent by gravity and is more manual while in HPLC, the sample is forced through the column by pressure and is more automated.

After separation of sample, the components are detected by UV detector of HPLC. Different components can be detected at different UV wavelength. Therefore, it is important to set the UV detection at a wavelength which the analyte of interest can be detected. Other than UV detection, retention time also differs for different compounds since different compounds have different affinity for the sorbent. So, different compounds remain in sorbent for different duration.

Sorbent (stationary phase) often used are reversed phase and normal phase. An e.g. of sorbent for reversed phase is C18 and e.g. of sorbent used for normal phase is silica gel. In normal phase, compounds are eluted in increasing polarity (i.e. least polar to most polar) while reversed phase is vise versa. Of course, elution of compounds depends a lot on mobile phase used. (e.g. methanol, acetonitrile water etc)

As mentioned in alex’s post (http://codec-5.blogspot.com/2008/07/attachment-experience-from-weeks-i-ii.html), parameters that affect results can include injection volume, stationary phase, mobile phase, UV wavelength, flow rate, column diameter etc.




(Example of HPLC chromatogram: Image taken fromhttp://www.ualberta.ca)
X-axis is the time and Y axis is absorbance (AU). Absorbance is proportional with the concentration. Each peak represents one fraction.

The above is just explanation on HPLC…..now is moving on to mass spectrometry instrument.

MS
Why need mass spectrometry?
UV detection can only detect compounds with double bonds or ring structure. Compounds without those properties will not be detected by UV. Thus, mass spectrometry is needed since all compounds have mass.

Mass spectrometry instrument consists of a ion source, mass analyzer and mass detector.

Ionisation is required to forms ions for detection by mass spectrometry. Two main ionization modes that are often used are the electrospray ionization (ESI) and the atmospheric pressure chemical ionization (APCI).

Mass analyzers will store the ions and eject them through mass filter to be detected by mass detector. The ions are ejected by varying voltage.
(will not be elaborating more on this part since lots of physics (voltage, direct current (DC), alternating current (AC) etc) is involved and the entry will become several times longer)

A mass spectrum shows the fragmentation pattern of the analyte.i.e it shows the quantity of ions at different mass to charge (m/z) ratio. By comparing mass spectrum of sample with a database containing mass spectrums of thouasands of compounds….the identity of the compound in a sample may be figured out.


Image of a mass spectrum. Taken from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htm
X-axis is the m/z value and y-axis is the abundance.

Lim Xin Ni
TG02
0607325H


=) 9:56 PM

2 comments



Sunday, October 26, 2008

SIP last entry-Heamatology

Hellllooo! This is the last blog entry for SIP! 2 more weeks for SIP to end!
I'm currently attached to heamatology lab now.
This blog entry would be on cytospin.
I'll try to keep it concise:D

Introduction

For oncology patients, CSF is usually taken for testing. This is so as infiltration of blast cells into the CSF can occur for oncology pateints. To confirm such findings, CSF smear is done to examine blast cells which is important.

To make a CSF smear, a cyto-centrifuge is used. This centrifuge is specially designed for CSF and fluid sample.
A normal healthy individual should not have any blast cells in the CSF. This is used as a reference for screening of blast cells in the CSF.

How is it done..

1. Firstly CSF is collected in the EDTA tube.
2. 300 microlitre of CSF sample is then pipetted into the sample chamber of the cytopad.
3. A glass slide is labeled with a small LIS label and initialed by the medical technologists.
4. It is then loaded right behind the chamber frame
5.It is balanced by placing a empty chamber on the opposite side.
6. The lid is then placed and the whole rotor is placed on the drive hub and spinned for 5 mins.

After centrifugation, cells and the sediments in the CSF is being deposited on the glass slide.The glass slide is taken out for staining and the cytopad is discarded into the biohazad bag.

Staining...

The glass side is air-dried and stained manually using Leishman's stain.

1. The glass side is flooded with Leishman's stain
2. Then, twice the amount of phosphate buffer is added on top of the stain
3.The glass slide is left to stain for 5 mins
4. The slides are them washed and left to air dry on the rack
5. Slides are then viewed under the microscope under high power (40X, 100X)

If cells appear to be disintegrated, then the whole process have to be repeated. But if the cell population is too high, then the sample may be eluted in saline.

This whole process only takes about 15 mins! That's it for this blog entry:D
Hope u guys have enjoyed ur SIP...cya back in sch!:D

Neela
TG02






=) 7:12 PM

9 comments



Sunday, October 12, 2008

Week 16: SIP sharing

Hi, this week i will be sharing on virus amplification.
Similar to what Shu Hui has previously shared in one of her entry on how to cultivate virus by infecting cells in a flask, my lab also uses this method for viral amplifcation.

Introduction
To amplify (or multiple or to increase in the number of) a specific virus, we need to have cultures of the host cells specific to the virus. The virus is used to infect the host cells, so that it is able to replicate and increase in number using the host cell machinery. We make use of this mechanism to amplify our virus stocks, once the volume is running low, when the virus titer (pfu/ml) is too low or when the virus stock is too old for usage.

For the type of virus we are using, the virus has 3 phases in its infection cycle.
The 3 phases can be classified into the early infection, late infection and very late infection.
Also, this virus we are using has two unique morphology, one is the budded form and the other is a polyhedron form.

During each phase of the NATURAL infection cycle, certain event takes place in the host cell as follows:

Early infection (0-6 hours) : Virus enters the host cells and decodes itself
Late infection (6- 24 hours) : Replicates and buds out of host cells without lysing the cells; this give rise to the budded form of the virus
Very late infection (18-24 to 72 hours) : Single or a few budded virus comes together and gets encapsulated in a polyhedron coat; this give rise to the polyhedron form of the virus.
When the host cells eventually lyses during this phase, the polyhedron form of the virus will be released.

Note
*The virus we use in the lab is genetically modified for research purposes. The polyhedron gene is deleted from the virus, because only the budded form of the virus is useful to our research. The polyhedron form of this virus is not able to infect any cells and thus cannot be manipulated for research purposes such as protein expression or gene delivery
Therefore, even til the very late infection cycle, only budded virus will form and buds out of the host cells we infect, when this genetically modified virus is use.* However, the host cells will still lyse after >72 hours.


Method
We use tissue culture flask as shown in (picture 1)to seed the host cells. These tissue culture flask are the same as those used to culture mammalian cells which has a inner flat treated surface for easier cell attachment.

We use different sizes of the flask, depending on the scale of our amplification.

There are 3 types of flask:
T25 (Yellow) : Smallest flask (25cm square)
T75 (Green) : Medium flask (75cm square)
T175 (Red) : Largest flask (150cm square)

T25 flask requires 5 million host cells
T75 flask requires 10 million host cells
T175 flask requires 20 million host cells

Picture 1: T25, T75 and T175 Tissue culture flask, Taken from www.wwmponline.com/cart/images/T/flasks-01.jpg

Before the required number of host cells is seeded into the flask, medium has to be first added into the tissues culture flask.
This volume of medium, is just enough to cover the surface of each flask:
T25 flask requires 5ml
T75 flask requires 10ml
T175 flask requires 18ml


Then the required volume of cells (depending on the concentration no. of cells/ml) is added to the medium in the flask. The flask is held horizontally and rocked back and forth at a gentle angle of about 30 degrees. It is rock for a few times to evenly spread the cells so that the cells covers the whole surface of the flask as it begins to attach to the treated surface of the flask. These flask are incubated for 1 hour (at 27 degrees celcius, 5% carbon dioxide incubator) for the cells to attach.

The cells are incubated at 27 degrees celcius because it is a non-mammalian cell.
The flask cap has a unique 0.22micrometers membrane, which allows carbon dioxide to diffuse into flask.

After the cells attach it looks like this:
This is just one small part of a T175 flask under 10x magnification

Picture 2: Virus Host cell (Taken with permission)

After the cells has attached to the flask treated surface, a certain volume of the medium is pipetted out to concentrate the cells in a smaller volume. This would allow the virus to attach to the cells more easily later, when the virus is added.

T25 leave behind 1ml

T75 leave behind 3ml

T175 leave behind 5ml

Add ~100microliters of the virus stock (the current one that we have) to the concentrated cells. The volume to add can vary, depending on how old the virus stock is. A older virus stock may have a lower virus titer (pfu/ml), so we can add more volume such as ~200microliters .

These flask are then put on a rotator (for 2 hours) which continuously rock back and forth to allow the virus to find and attach to the cells.

After the virus attach to the cells (assuming, because we can't see any changes yet), we add more medium to each flask to maintain the infected cells in the flask.

T25 add 5ml
T75 add 10ml
T175 add 18ml

The infected cells are incubated for 72 hours (at 27 degrees celcius, 5% carbon dioxide incubator), so that the cells are now in the very late infection cycle.

These are how the cells look like during the very late infection cycle: under 10x magnification with 1.6x aperture

Signs to take note of: Cells look swollen, has black dots in the centre of the cells, uneven surfaces of the cells and some cells have even fuse together.

Picture 3: Infected virus host cells after 72 hours (Taken with permission)

When these signs are observed in the very late infection cycle, we assume that majority of the viruses has bud out of the infected host cells, (virus are too small to be seen under our phase contrast microscope. It can only be seen unless a high power electron microscope is used) and it is very likely that the cells are going to lyse soon. Virus has to be collected before the cells lysed. When the cells lysed, enzymes released can cause break down of proteins found on the surface of the virus and lowers the virus titer greatly.

Virus collection

Since the cells are attached to the flask surface, as the viruses buds out of the infected cells, it is released into the medium. Therefore, virus can be collected by pouring the medium from the flask into a 50ml tube. The infected cells remains attached to the flask surface and are discarded.

This tube is spin down at 1000rcf for 10minutes to remove any cells debris (from a small proportion of infected host cells that may have detached). The supernatant is collected in a new 50ml tube without disturbing the pellet of cells debris. The new tube is wrapped with non-shiny side of the aluminium foil (because viruses are light sensitive), and stored at 4 degree celcius for future use.


Jean Leong
TG02














=) 12:00 PM

7 comments



Sunday, October 5, 2008

SIP sharing - Week 15

Okie, time to blog again. I am attached to the biochemistry lab for this two weeks. During the campus discussion week, i've shared about G6PD deficiency testing. Now, i am going to blog on another test done in the biochem lab.


Except G6PD, most of the things done here make use of machines for testing. Neonatal Total Bilirubin is one of them that uses machine to analyse the results.


Background infor


We've learned how bilirubin is formed in school already. In case some of us forget, here is brief explanation of the process. Haemoglobin in the body is broken down into heme (iron and porphyrin) and globin (protein). Iron and protein are then re-utilized by the body while heme goes through a metabolic degradation where the porphyrin ring opened up to form biliverdin. Biliverdin is then further reduced to bilirubin, where it is transported to the liver via the plasma. It is in the liver where it conjugates with glucuronic acid to form bilirubin diglucuronide and excreted into the bile and then goes into the intestine. It will then reduced to form urobiliogen by the action of intestinal bacteria.

So, in this test, the sample that is collected is blood. The blood has to be spin down such that the plasma could be obtained to test for the bilirubin level.


Clinical Significance

When blood contains excessive bilirubin, it gets deposited in the tissues and cause the shade of yellow. It is also referred as jaundice or icterus. This condition may be due to 3 reason.
1) The bilirubin produced is more than the kidney can process and excrete. ( Hemolytic disease)
2) Having liver damage which resulted in inability to excrete normal amount of bilirubin produced.
3) The liver’s excretory ducts are obstructed and blocked.

Jaundice in the infants are quite common as their liver are immature or inefficient. Due to the deficient liver enzymes, the liver could not conjugate the bilirubin. The condition gets better when the liver is more matured and the enzymes are more functional. Possible treatment for the babies are phototherapy.


The bilirubinometer is used to measure the total bilirubin in the plasma of newborns. By measuring the absorbance of the plasma at 460nm, the concentration of bilirubin can be determined.

Methods

1) A heel prick is done and blood is collected via the capillary tube through capillary action.

2) A capillary tube is used to collect the blood of the newborn. When it is received in the lab, make sure that one end of the tube is properly sealed.

3) The capillary is placed in a plastic tube in the centrifuge in spin down at 3000rpm for 3 minutes. (make sure that one end of the tube is sealed and sealed end placed facing down)

4) Plasma should be visible at the top of the tube.

5) Spring-lock the capillary into a dispenser and slowly inject the plasma into a clean cuvette. ( Make sure there is no air-bubbles)

6) The capillary tube is then discarded and the cuvette is loaded into the bilirubinometer.

7) The measuring chamber will rotate downward to align the plasma of the patient to the optical path and results will be shown.

8) The results are to be recorded on the request form for entering into the system later on.


* If the reading exceeds the critical value of more than 300µMol/L, the test have to be repeated.


That's all folks.



Zhenling
TG02


=) 12:00 AM

8 comments



Friday, September 26, 2008

SIP sharing---week 14

It is the 14th week!!!…..left 6 more weeks to go. Jiayou pple. As I am still in a chemistry lab for the last 14weeks and for the future 6 weeks, this week post will continue from my first post. (week 4) which is on my major project which is on phytoplankton (diatom and dinoflagellate are some e.g. of phytoplankton.)

Here is a recap:
1) Seed phytoplankton cells
2) Harvest (centrifuge) when culture is ready
(Centrifuging takes 1 whole day. Why? Cause got about 20 litre of culture to spin down and there is only 1 rotar available----1 rotar can only carry 6 bottles, each bottle has a volume of 250ml, so after calculating…figure out !?!)

3) On the supernatant, carry out SPE
a. Methanol loaded first to condition the cartridges and wash away any impurities
b. Then load DI water to remove methanol
c. Next, load the supernatant (this take many days)
d. Then, load DI water again to remove salts (cause seawater contains salts)
e. Spin down the cartridges to ensure all DI water is removed from cartridges
f. Lastly, load methanol to elute out the extract.

So, now I have the eluent which contains the solvent (methanol) with the extract I want.
To get the extract and remove the methanol, evaporation is carried out.

Technique: evaporation
Used for removing solvent
Equipment used: rotary evaporator



(Image taken from http://www.eyelausa.com/category.php?cID=1)
It is a very easy process but take very very long for large quantity of eluent.
The bottle on the right is the evaporating flask and on the left is the receiving flask.
Water aspirator is connected to the hook-like thing on the condenser. Condenser pump is placed in a beaker with water covering the pump and the remaining space in the beaker is filled with ice.
So first switch on the condenser (the tall tube with coil inside) and water bath (it take time for the water bath to reach the temp set).

What temp to set for the water bath?
Temp set is around 40Cto 50C as long as it is below methanol boiling point.
Depending on the compound want to obtain, if it is stable and does not react easily (meaning it has few hydroxyl(OH) groups), then it is alright to set at higher temperature but the temp must be below the boiling point.

Eluent is poured into the evaporating flask, filling around 1/3 of the flask. If the flask is filled too full, eluent might spill out while evaporating and the whole process need to be repeated again.

After everything is set, the water aspirator is on. Water aspirator provides the vacuum required. Next, the pressure is gradually reduced. The eluent in the evaporating flask had to be monitored constantly in case there is lots of bubbles (‘boiling”). When this occurs, the pressure is released a while then reduced again. If violent boiling occurs, the desired compound may spill over to the receiving flask.
When evaporating, methanol from the evaporator become vapours and travels up to the condenser. The vapours then condense on the cool surface of the condenser. The methanol in liquid form then dripped into the receiving flask.

This whole process continue until the eluent in the receiving flask left a very small quantity, then I top it up with more eluent. When all eluent has been poured into the evaporating flask, then I evaporate the remaining eluent till dryness.
And the residue in the evaporating flask is the extract that I want to get. The residue is of a very very small quantity. To get sufficient amount for LCMS (discuss about it in next post) …. I need to accumulate about 3 times of SPE eluent.
After accumulating, I use methanol to dissolve and suspend the accumulated dry extract. Then I transfer the extract into a small tube using a dropper. As there may be some extract then cannot be removed from the walls of the flask, sonication using a sonicator is carried out to detach the extract from the walls.
Next I dry the extract in the tube using stream of nitrogen gas. (Why nitrogen? Actually nitrogen is a common gas used for drying.)

For the cells,
Methanol is used to remove the cells from the centrifuge bottles.
The methanol containing the cells is transferred into a conical flask.
The cells is left in the flask for about 1 week. Meanwhile, the flask is sonicated.
Methanol can lyse the cells to get the extract from the cells.
Sonication helps to enhance the process of cell lysis because high frequency sound waves can shear cells.
After that, the mixture (cells in MeOH) is filtered to remove the cells.
Methanol extract is evaporated as mentioned above.

LCMS on the extract will be discussed in the next post.

6 more weeks to go.
Hope my explanation is clear.
Any doubts, questions or uncertainty, juz ASK. =D
Lim Xin Ni
TG02
0607325H


=) 2:16 PM

4 comments