Urine Processing

Hello People.

This is Jordan Wong Wei Jie reporting for his entry for SIP Post.

First thing when we arrived into the clinical laboratory, we were greeted by our lab manager and deputy lab manager. Our deputy lab manager then brought us around the clinical laboratory and introduces us to several things we need to take note in the lab of the Hospital, such as the Mission and Vision of the Hospital as well as the 4 codes of emergency. Of course the most important of all, our deputy lab manager also emphasized was the hand wash and hand rub in the clinical lab, especially H1N1 is still around. He taught us the procedures of hand wash and hand rub with the specific type of reagent (Desmanol & MicroShield respectively).



This is a picture of Desmanol that we used for Handrub.

Desmanol® is a rapid acting alcohol based handrub for hygienic and surgical hand disinfection. It helps prevent the skin from drying out.

"Desmanol® has a broad spectrum of activity and is effective against MRSA and VRE, bacteria ( inlc. TB bacilli), fungi, viruses (polio, vaccina, adeno, herpes, herpes simplex viruses, HAV, HBV, HCV, HIV)"

This picture and information is taken from http://www.demkasakti.com/products/medical/schuelke/desmanol.html on 22 June 2009

After which we were separated into 4 different departments. I chose to be in haematology department currently. I was introduced to my supervisor. My supervisor then allocate me into a section dealing with urine samples, doing tests like Dip stick test, Drug test and Urine culture etc. Of course, there were several senior colleagues who have guided me through out, one of which was our senior in TP BMT.

Laboratory Information System

First of all, I was taught how to access to the Laboratory Information System (LIS). This lesson is very important because we will have to retrieve information on which patient wants to carry out a certain tests and what type of tests a particular patient wants. All of these data were actually being registered into the LIS by our admin staffs. After which our individual department can check which patient wants to do urine FEME test or urine culture test by typing in a code number that represents a certain test. For example, urine FEME test has a code number of 472100 and urine culture’s code number will be 457262.

Then, we will print a worksheet out with the information of the patients such as their name, their ID number and the type of test we are going to performed. ID number will be something like 090622-00XX. 090622 will refer to the date/month/year the test is performed and 00XX refers to the number of this patient doing the test. There are other functions of the LIS too such as registering patient samples into the LIS and approving the results of a certain patient so it can be sent to the doctor for diagnosis. LIS is definitely a very convenient system for all of us, it’s so accessible!

Urine FEME

I was then taught on how to process dip stick strips with urine samples using machines. Usually in school, after dipping the dip stick strip into the urine, we check by comparing with the colour chart manually. However, with the use of machines, there is not a need to perform this manual checking of one square at a time on the strip. The machine can help us to check all the squares in the strip and tell us the results for each component (Specific Gravity, pH, Ketone, Glucose, Nitrites, Blood, Bilirubin, Urobilinogen, Protein and Leucocytes) The old machine used was called Miditron Junior II and now there is a newer machine used called Cobas u 411 (Roche).

The process goes like this:

Firstly, we will get the list of patients who wants to have Urine FEME test from the LIS.

Next, we will scan their ID number from the sticker label noted on the urine sample into the machines, Miditron Junior II or Cobas u 411 (Roche). After we scan all their ID number into the machine, we can then press start.

Thirdly, we will need to dip the strip into the urine sample (ensure it is the correct ID number from the machine and urine sample we are using) and then place it into the machine. The machine will then bring the dip stick strip and perform its necessary function (screening of the strip).

A few seconds later, a slip of results containing all 10 components will be shown (Performing a 10 test dip stick method). Of course this is not the end; we will need to load a small amount (15.0ul) of urine sample into a combination cover-slip microscope slide for the medical technologist to view on microscope. This is for microscopic examination, which is also used to verify the results produced from the machine. This combination cover-slip microscope slide also called as Glasstic® Slide 10 with grid chambers by KOVA® is something new, I have not seen in the school lab.



The diagram above is the LIS Window for Urine FEME. The Code nuumber used is 472100 and to print out the worksheet of patients who wants this test (Urine FEME), click report and print. This is a diagram that i drew during the lab.



The picture above is the New Analyzer Machine that we are using for Urine FEME

This picture is taken from
http://labsystems.roche.com/content/products/cobas_u_411/introduction.html on 22 June 2009



The picture above is the Older Analyzer Machine that was used before for Urine FEME

This picture is taken from http://labsystems.roche.com/content/products/miditron/introduction.html on 22 June 2009

This combination cover-slip microscope slide has 10 different semi circle columns, with each having grid chambers. This slide can hold up to 10 different patient’s urine sample. The medical technologist will then view and read the 10 different patient’s urine sample on the 10 grid chambers. Compared to the use of normal glass slide, this combination cover-slip microscope slide save resources as a combination cover-slip microscope is equal to the usage of 10 glass slides.



This is the Glasstic® Slide 10.

This Picture is taken from http://www.cenmed.com/productDetail.asp?productid=17291&catID=&category=5617&mainCat=LNS&cat=5305 on 10th July 2009



This is also Glasstic® Slide 10 but a close up that i drew during the lab.

Urine Culture

The next thing I learnt was to perform urine culture from urine sample. We will have to use two different agar plates, called the TSA (Blood agar) and CLED. First we will have to log in into LIS to check job descriptions of patients requiring for urine culture test. Next, we use a disposable inoculating loop (plastic) to retrieve urine from sample, and then inoculate onto the TSA and then CLED agar plate. Then, incubate the agar plate into the incubator at 37°C.

Open Sample and Distribution to Department

Opening of sample is one of the most important processes in clinical lab. Without helping of opening samples in the admin department, there wouldn’t be any samples to be processed. Also due to the lack of manpower, we have to help out a little. Helping in opening sample is not a very difficult task. It goes like this.

1. Nurse and People delivering/dispatching samples, which usually comes in bulk and packages will go to the admin counter

2. One of our staff will then get the samples and a checklist from them

3. The staff will check against the checklist the amount of samples (how many EDTA tubes how many plain tubes how many urine bottles etc)

4. After ensure checklist is completed (all samples have arrived), some of us will take the bulk of samples and separate them accordingly.

5. We separate the packages by 4 different sites. They are HIV Test, Malaria Parasite Test, VDRL Test and Those that can be open right away.

6. HIV test comes in a single plain tube, Malaria Parasite test comes in 1 Plain and 1 EDTA tube, VDRL comes in 1 plain tube. And usually these are the ones that come in a huge bulk. So they are the last to open

7. The remaining samples can be open right away, like those testing for full blood count or urine test.

8. After which we will send it to the admin people to register, once they register into LIS, print out the label, stick on the sample, they’ll placed it into a basket for us to distribute to the other department for testing

9. Plain Tubes will be sent to Biochemistry department, EDTA tubes will be sent to Haematology department, Stool Bottles or Swab will be sent to Microbiology department and last will be the Urine bottles which will be sent to Urine section, a sub-section of Haematology department.

After the samples are being sent to their individual departments, they will be able to start their test/processing.

This will be the end of my post for now. I will explain more about Urine Drug Test Kits, Microscopic Examination and other related Experiments on my further posts. Do stay tune! =)

This is Jordan Wong Wei Jie (TG02)

Flow Cytometry

Hi everyone, its Gwen here and this week, it’s my turn to share about my SIP experiences. I am in a very pleasant work place and the people here are very friendly. I have also learnt many new laboratory techniques and processes. My research laboratory is doing research of using nano particles as delivery vectors to treat cancer. It aims to reduce the toxicity effects of the drug and to increase the delivery of the therapeutic agents to the tumour. My job basically involves work in the cell culture room. Although I have learnt cell culture techniques before, but during my attachment, I have learnt many new techniques and improved my pipetting skills. In today’s entry, I will be talking about flow cytometry and its preparation steps.

Flow cytometry can be done to test for various purposes. In my case, it is to investigate the cellular uptake efficiency of the various treatments that will be mentioned later.

Firstly, the cells must be in confluence so that they can be seeded on to 12-well plates. For seeding, the cells in the flask first need to be trypsinised. So, the used media must first be removed and the flask must be washed with PBS. This is to remove any residual media that may inhibit the effect of trypsin. Next, trypsin must be added and then the flask is incubated for 5 mins at 37°c. After 5 minutes, the flask is viewed under the microscope to ensure that the cells are no longer in clumps and are single-celled. Media (twice the amount of trypsin added) is then added to the flask and pipetted up and down to inhibit the action of trypsin. If the cells (previously seen under the microscope) are still in clumps, the media would be pipetted more vigourously against the wall of the flask. After ensuring that the clumps are gone, the cells are then pipetted into a 50 ml falcon tube.

10µl of the cells are then pipetted into a hemocytometer and the number of cells is counted, and then averaged. This is to determine the amount of cells and fresh media that is to be added to each well to achieve the wanted cell concentration. After the total amount of cells and fresh media have been calculated for all the wells to be seeded, the calculated amount of cells and fresh media are added into a new 50 ml falcon tube and pipetted up and down to mix it well. The cells (with fresh media) are then pipetted/poured into a reservoir. I ml of the cells are then pipetted into each well of the 12-well plate. The plates are then incubated overnight. Throughout the whole process, it is very important that there must be no bubbles present as the bubbles can cause asphyxiation. The remaining cells left are subcultured into a new flask to continue the cell line.

The next day, treatment is prepared according to a table that my mentor provides. The table determines the amount of polymer, apoptotic protein, drug, buffer and media to be added based on the different conditions and/or concentrations that were set by her. For example, the condition can be addition of the free drug, the drug with the polymer, the apoptotic protein only, the apoptotic protein with the free drug and the apoptotic protein with the polymer and drug. For flow cytometry, the drug and apoptotic protein contain fluorescent dyes that are highly sensitive to light, thus all the lights in the cell culture room must be turned off. The laboratory that I am working at deals with certain light sensitive drugs and I had an experience of working in total darkness except for a torch light. It was really an unforgettable incident. After adding the treatment to the cells, the plates were incubated for another 3 hours at 37ºc.

After incubation, the cells will then be washed with PBS and then detached with trypsin. The effect of trypsin is then neutralized with PBS and the cells are pipetted to a 15 ml falcon tube. The cells are then centrifudged at 1500 rpm for 5 minutes, with the temperature set at 4ºc to prevent degradation of the cells. The supernatant discarded and the cells are resuspended with PBS and then transferred to two polystyrene flow cytometry tubes. The tubes are then stored in an ice box and sent for flow cytometry. The flow cytometry machine is found in a shared facilities area and the machine is operated by a trained technician.


Taken from:http://www.eppendorf.com/int/index.phppb=072836f7d878666d&action=products&contentid=1&catalognode=9537
This is the multichannel pipette that i mention above.


Taken from:www.bcm.edu/cbass/?PMID=8386
This is the flow cytometry machine that i saw.


Taken from:www.medsci.org/v06p0051.htm
This is an example of how a flow cytometry result will look like but it is not my result.

That’s all I have to share this week, see you all again next time.

Collection and culturing of corpus cavernosum tissue from rat

Hello! Im back to share with you how to collect corpus cavernosum (CC) tissue, which is this spongy erectile tissue that forms the penis in the male rat, from the rat to culture your own cells!

Firstly, we give rat anaesthesia intraperitoneally and wait for around 5 minutes for the rat to lose its body sensation before starting to dissect the rat.

A perineal incision (the region between the anus and scrotum of the male rat) is done on the rat using a surgical scissors and the opening is held stable using hooks. Then, using the arterial forceps and toothed forceps, we locate the bulbocavernosus muscle, which looks like a small white triangular pouch. The corpus cavernosum is within the pouch and therefore, we have to retrieve the whole pouch by cutting it out using the scissors and toothed forceps. 3 cuts is made, one at the penile stem, the other 2 at the left and right crus to remove the entire bulbocavernosus muscle.

To retrieve the corpus cavernosum from within the pouch, we have to cut away the muscles and tunica albuginea surrounding the corpus cavernosum. The corpus cavernosum is then minced and centrifuged in PBS for washing. PBS was then aspirated and the cell pellet was mixed with DMEM by pipetting. Then the mixture would be added to culture flasks that was pre-loaded with fresh DMEM. The flasks would then be placed in the carbon dioxide incubator for growth.

Renee
TG02
0703634F

Radioimmunoassay - laboratory technique

Hihi…! I’m Zhang’e! Today, I am going to share what I have learnt for the past 2 weeks!

I’m attached to endocrine laboratory and from the word ‘endocrine’, you all should know what I am studying about. And ya, its hormone! My lab actually collaborates with the Singapore zoo to conduct projects on the animals. The purpose is to determine the hormone levels of the animals using their feces, urine or plasma/serum. For example, the cortisol level will allows us to know whether the animal is in stress or progesterone level to determine the ovulation cycle for reproduction.
So for the past 2 weeks, I was handling with white tigers’ urine. There are three white tigers in the Singapore zoo and they are Omar (the male tiger), Winnie and Jippie (female tigers). I have to determine their cortisol levels and the technique that I used is a modified method of radioimmunoassay (RIA), called RIA-SPA (scintillation proximity assay).

Radioimmunoassay, sound familiar? Ya, we have learnt the theory before but not practical. I am taught from basic what RIA is about and started to do the assay. RIA is a technique used to determine the concentration of an unknown sample. It employs the use of radioisotopes to label antigens and these labeled antigens (known concentration) will compete with the non-labeled antigens (the sample or standard) for the limited number of antibody binding sites. There are 5 steps in RIA: Preparation, purification, competition, separation and computation. The principle of RIA-SPA is the same as that of RIA, the only difference is that RIA-SPA eliminates the need for a separation step and the addition of scintillation fluid.

Separation is needed in conventional RIA to separate the bound fraction from the free fraction. Bound fraction includes bound labeled antigens and bound non-labeled antigens. Liquid scintillant is then added to the bound fraction. Only the labeled antigens will emit radiation which causes the scintillant to fluoresce. If there is no separation of the fractions, the scintillant will also bind to the free-labeled antigens, leading to inaccuracy of results. The fluorescence will be measured in a beta-scintillation counter (when beta-emitting isotope is used) or gamma-scintillation counter (when gamma-emitting isotope is used). 3H is a weak β-emitter in an aqueous environment thus it is brought close to a scintillant in order to fluoresce and be measured in β-scintillation counter. In the absence of scintillant, the radiation energy of the radioactive substance is dissipated and lost in the solvent.

In RIA-SPA, SPA reagent is used. It contains second antibody bound to fluomicrosphere. Fluomicrosphere is a bead that contains scintillant. The bound antigens (both labeled and non-labeled) will be immobilized on the fluomicrospheres and because of the close proximity between the bound labeled antigens and fluomicrospheres, the radiation produced by the labeled antigens will stimulate the embedded scintillant to emit light. The bound non-labeled will not cause the scintillant to emit light as there is no radiation produced. For free labeled antigens, the radiation energy is absorbed by millions of surrounded solvent molecules and is dissipated as heat. This does not affect the fluorescent process thus separation of bound and free fractions is unnecessary.


Cortisol test
Preparation of reagents
1. Sample: Urine sample was extracted using dichloromethane. The purpose of extraction was to remove steroid-binding protein that will interfere with the measurement of hormones.
2. Standard: The standard, which was provided in a bottle, was diluted into varying concentrations. Standard is a known concentration of non-labeled antigens (cortisol).
3. Labeled radioactive antigen: ³H Cortisol was used in this assay. A fixed concentration of labeled antigen was used and the radioactivity was set around 10,000cpm.
4. Antibody: Known concentration of rabbit antibodies was used.
5. SPA reagent: It contains second antibody bound to fluomicrosphere (embedded with scintillant).

Competition
The assay was first performed using the standard, antibodies and labeled antigens to determine the dose-response curve. The standard, with varying concentrations, was added to the respective assay tubes (in duplicates). It was followed by the addition of labeled antigens (³H Cortisol), antibodies and SPA reagent to all the tubes. The tubes were incubated overnight.

Computation
After incubation, the tubes were counted in a beta-scintillation counter. This counter would measure the radioactivity of bound labeled antigens. The results obtained were plotted into a dose-response curve.



This is an example of a dose-response curve. Dose refers to the concentration of non-labeled antigens and response refers to the amount of bound labeled antigens. Because of the competition binding to the antibodies, the amount of bound labeled antigen is inversely proportional to the amount of non-labeled antigen. Higher amount of non-labeled antigens will displace more of the labeled antigens from the antibody thus resulting in less bound labeled antigens (lower response).

Assay was then continued on the samples. All the reagents and steps performed were the same as mentioned above (sample was used instead of standard). After measuring the radioactivity of an unknown sample, the result can be compared and interpolated from the standard curve and the cortisol concentration in the urine smaples can be determined.



Beta-scintillation counter
Retrieved on June 06, 2009 from website http://www.gla.ac.uk/media/media_61475_en.jpg

The end! =D

Zhang’e
0704086H
TG02

Order Entry

Hello! It's my turn to share what I've learnt during my first 2 weeks of internship(:

I'm attached to a clinical laboratory in a hospital. It's a really small and cosy laboratory so we have almost all the different discipline in the laboratory. The people here are really friendly, helpful and approachable. To me, it feels like one big family of medical technologists working together. Although it's a serious environment, we still manage to have lots of fun somehow(: We do our work seriously when dealing with patient's samples and have fun when there’s no sample for us to do. Enough of the fun-filled working environment and now to what I do there.

For the first 2 weeks of attachment, I'm assigned to do order entry. The doctors will order the test, then phlebotomist will take the blood from the patient and I will enter the tests and the rest of my colleagues will do the tests. I receive the test forms from the tubes via the pneumatic tube system. Initially, the sound of dropping pneumatic tubes was quite scary as it comes suddenly without warning. After awhile, I know when the pneumatic tubes are coming by hearing the different sounds made.

There are a lot of tests and there are different codes for different tests and I need to remember all of them in order to be efficient. But it's pretty easy to remember the tests codes because it's usually those few tests that you'll see many times a day and the test codes are very much related to the tests. It's okay if I cannot remember the rare ones too since there's a file with the different tests codes for me to refer to. Not only do different tests have different codes, it also varies for in-house tests, corporate screening tests and send-out tests. However, all that I need to know are in the files beside me so it's pretty easy doing order entry after awhile. For the first few days, I find it quite stressful as I don’t know when to order a separate section for the different tests and so a lot of modification needs to be done, which slows down the whole process a lot. For the urgent tests, time is important because we need to deliver the results within that short turnaround time.

Other than different test codes to remember, I also need to learn and remember the different blood tubes used for the different test. For this, we already learnt during clinical chemistry case studies and lectures that certain contents in the blood tubes can contaminate the blood depending on what tests we are doing. For example, blood is taken in an EDTA tube yet a potassium test is ordered. In this case, we need to call up the wards and ask them to send us another tube of blood and inform them that we need to reject the specimen. We also need to ask for the staff nurse whom we talk to regarding the wrong tube used so as to ensure traceability.

For order entry, it’s not just ordering of tests but also to place the send-out tests and histology or cytology specimens at the front desk area for the other order entry staff to do their work. Furthermore, if there’s blood banking forms, we need to order the tests and walk to the blood bank area to pass it to them to do their work. I also need to check the specimen received for corporate screening against the packing list and then sign and fax it back when I’m done. After ordering the test using the forms, I need to do counterchecking to ensure that what I’ve keyed in is correct and that I did not miss out any tests. Most of the time, I’ll need to stand and walk around to get the pneumatic tubes from the pneumatic system and pass the various forms to various people so it’s quite tiring. The only time I get to sit is when I’m the one doing the order entry. All these is what order entry is about.

Next, I’ll be talking about some tests that I’ve done on Friday at the urinalysis section although I’m not stationed there. I won’t be giving the full account of it but what I know from the limited time I have there.

One of my nice colleagues taught me how to identify microorganisms and the different cells present in the patient’s urine. I find it very interesting cause I didn’t manage to see all those in school during our clinical chemistry laboratory session. After which, he also taught me how to use arterial blood gases (ABG) test using the roche equipment. The ABG tests is done behind the order entry area, thus, I’ve seen it many times but didn’t get to try it until Friday. It looks really simple to mix the blood by rolling it between 2 hands. However, it wasn’t that easy for me probably because it was my first time and with others staring at me, it’s pretty nervous. Next, he taught me how to do the blood ketone test using the test kit, which is really easy since the reader will give you the result after you drop a drop of blood on the test strip. Lastly, I get to try doing the syphilis test, which is something like what we do in medical microbiology laboratory. It resembles the staphylococcus test kit. Add the reagent then the serum and spread it within the circle then use the rotator to rotate it for you for 8 minutes before observing for agglutination. Presence of agglutination means positive for syphilis and no agglutination means negative result.

That’s all from me for now(:

Goh Michelle
TG02