Microtomy =D

Hi everybody,

Sorry for the very late posting. This would be the final post of the whole 20 weeks! Haha..I would like to talk about Microtomy as it the the last area that i'm posted to.

Subject Title: Lab Technique

Topic: Microtomy

Principle: The principle of microtomy is to sectioned tissue specimens into thin sections so that they can viewed and diagnose under microscope.

Below is a picture of the rotary microtome used for microtomy.



Materials Required

Alcohol Floatation bath 1
Soft Pencil 1
Rotary Microtome 1
Microscopic glass slides 1
Disposable microtome blade 1
Cryoplate 1
Warm floatation bath 1

Steps involved:

1) The block is required to shave first before cutting. The paraffin wax block is secured to the block holder of the microtome and adjusted it to ensure that it clears the knife. THe block holder screws are also readjusted to place the block parallel to the knife. During shaving, while the machine is manually advanced, the block is repeatedly sectioned at 20 microns thickness per slice. Shaving is stopped when the entire surface of the tissue is exposed. The block is then removed from the holder.

2) All blocks should be shaved so that dense and hard tissue can be identified more rapidly and separated from the rest. Those blocks will then pre-treated before sectioning. By doing so, it can prevent nicks and scorelines to the blade. It can also ensure that all staples or sutures are removed from the blocks before sectioning as pathologists might forget to remove them during trimming. Hard bone can also be softened first in 10% commercial fabric softener for 5 minutes, so that the tissue can be sectioned more easily.

3) After the blocks are softened, they are placed face down on the cryoplate to chill the block to allow fast sections.

4) The blocks are secured and adjusted to ensure that it is parallel and clears the knife. It is sectioned at 3-4 microns using the handwheel by allowing the block to advance automatically or manually.

5) The section is then lowered onto the floatation bath. If the tissue has difficulty in spreading, it is placed on alcohol floatation bath first to increase the surface tension before transferring onto the warm floatation bath. The section is then transferred onto a glass slide where the corresponding biopsy number is written on the frosted end of the glass slide.

6) The glass slides are separated according to their respective stains such as H&E, special stains and unstained slides.

In our lab, different biopsies are sectioned at different thickness and different number of slides are required. Below is some examples of different types of tissue biopsies and the thickness and number of slides that are required.



I will stop here for now. Please feel free to ask questions =D
Enjoy your holidays =D

Lok Pui
(0704138G)

Processing :)

Hi people, this is my last post for SIP. Hope you guys are doing fine for your project. :) I have been posted to the trimming room for this month and will be talking about processing for this post.

In the trimming room, pathologists will trimmed large biopsy specimens or specimens that are too big for the technicians to pass. Some of the large specimens are breast, colon, uterus etc. Basically, my job is to assist the pathologists. In the morning before they arrive, my job is to distribute the specimens and write cassettes. Normally for the large specimens around 10-12 cassettes are needed while for those very small 1-2 is enough.

Specimen example colon is labeled with eg.A, B or C etc, according to the alphabet that is written on the form. In other words, if the pathologists uses 14 cassettes for the colon specimen, the cassettes will be written until eg. A14. During the labelling of the cassettes, one have to remain alert and make sure that the correct biopsy number is written, otherwise wrong diagnosis will be given. Following that, i need to make sure that there are enough trimming blades and scalpel, paper towels and paint, so that they do not run out during trimming.

After the trimming is completed for the specimens, the cassettes have to be put into a rack which is soak in 10% neutral buffered formalin. The purpose of this is to fix the specimen. After that, the cassette rack will be placed into the automated tissue processor for processing. There are basically three main steps for processing, which are dehydration, clearing followed last by infiltration.


Dehydration

Principle: It is to remove the water contain inside the tissues so that wax can infiltrate as wax is not miscible with water. (Ethanol is used in this case)


Clearing

Principle: It is to remove the alcohol from the previous step using an agent that is misicble with both the wax and alcohol and for clearing purpose. (Xylene is used)

Infiltration

Principle: It is to replace the clearing agent with embedding medium (Paraffin wax is used)


Zi Shuang
0703383J

Tetraplex PCR

Dear all,

last 10-14 working days left. Hang in there!
Anyway i have mentioned about Real Time PCR and the Molecular probes for Real Time PCR. So lets talk old school, Conventional PCR.

Tetraplex is a panel developed by the scientific officers of the lab. Heard that it is a request from doctors thus resulting in the test being developed.
The panel consist of 4 viruses,
1. Cytomegalovirus (CMV)
2. Varicella – Zoster Virus (VZV)
3. Toxoplasma Gondii (Toxo)
4. Herpes simplex virus (HSV)

Specimen received are either CSF or Aqueous eye fluid (no preference of which eye).

The specimen will go through a manual DNA extraction process to get the DNA.

The difference from what we learn in school as compared to this is that in school, there is only 1 time PCR (1round PCR). However, for Tetraplex, this is a 2 round PCR.
As told by the med techs, the 2nd round is to improve the sensitivity.
Also, for PCR, each patient will have samples, 1 normal sample and another is a spike in.
Spike in is to add positive control to the sample. This is for ruling out any false positive.

After the second round, it will under go a gel electrophoresis.
Gene ruler, the 4 different controls, "spike in" samples and actual samples are added to separate well but on the same slab of gel.

After electrophersis, the gel will be put into the chemimager and we will check for the corresponding base pairs to check if it is pos/neg.

Upon receive a POS, the ministry has to be notified.

Thats all for this post. I'm sorry that i cant go into the details (Supervisor dont approve)...

Cheers.
Tiong han
Tg01

Rotavirus detection

Time really flies, 5 months of attachment is ending in a couple of weeks! Back to my post, i am going to talk about a small, easy-to-use and rapid kit that is used for the detection of rotavirus.

Rotavirus is a major cause of diarrheal illness among infants and childrens. As the name implies, it is a virus and can be classified into Group A-E based on their antigenic group on VP6. Group A is known to be the major cause of human rotavirus disease. The symptoms includes vomiting, watery diarrhoea and fever and may even leads to death due to severe dehydration.



[Rotavirus kit]
Test procedure:

1) Pipette 1 ml of the Rotavirus Extraction buffer into a test-tube.
2) Add around 100 ul or 50mg of stool sample into the test-tube.
3) Mix the mixture in the test-tube using the pipette
4) Let the stool sample to settle for around 3 minutes
5) Pipette 4 drops near the top part of the mixture into a round opening on the Rotavirus kit
3) Read the result on the kit after 5 minutes.


Interpretation:
A blue control band will appear on the test kit.
A positive result will be indicated by a red test band along with the blue control band.
A negative result will only shows the blue control band.
If there is no blue control band, the test will be repeated with a new kit.



Signing off,
Yong Herng
0702243G

HbA1c test

Hey (:

Subject Title: Clinical Chemistry
Topic: HbA1c test

Haemoglobin A1c (HbA1c) Test using Bio-Rad D-10

Note: I will just be focusing on the main procedure of running the samples. I will talk about the Calibration next week.


Principle

This test is based on ion- exchange high-performance liquid chromatography. Samples are automatically diluted on the D-10, injected into the analytical flow path, and applied to the analytical cartridge. The D-10 provides a programmed buffer gradient of increasing ionic strength to the cartridge, where the haemoglobins are separated based on their ionic interactions with the cartridge material. The separated haemoglobins then pass through the flow cell of the filter photometer, where changes in the absorbance at 415 nm are measured.

An accurate index of the mean blood glucose concentration maybe established by the measurement of haemoglobin A1c every 2 to 3 months. HbA1c is formed in 2 steps by non-enzymatic glycation of HbA. The first step is the formation of an unstable aldimine (pre- A1c), a reversible reaction between the carbonyl group of glucose and the N –Terminal valine of the b-chain of haemoglobin. During red blood cell circulation, some of the pre- A1c is converted to form a stable ketoamine, HbA1c. The level of HbA1c is proportional to both the average glucose concentration and the life span of the red blood cell in the circulation. The measurement of HbA1c has therefore been accepted as the clinical management of diabetes.


Materials

1.Bio-Rad D-10 Machine
2.D-10- Haemoglobin A1cRecorder Pack
a) Elution Buffer 1
b) Elution Buffer 2
c) Wash/Diluent Solution
d) Analytical Cartridge
e) Floppy Diskette
f) Calibrator/Diluent Set
g) Whole Blood Primer
h) Samples Vials
I) Thermal Paper
3.Sample Vial Adapter, 10 x 1.5mL
4.Lymphochek Diabetes Bi-level Control, 6 x 0.5 mL
5.Pipettes, 5mL, 0.5mL, 1mL, 7mL
6.Deionised water
7.Disposable gloves



Method

1.Start up the machine if it is in “Sleep” mode.
2.Collect EDTA.
3.Check the amount of blood present.
4.If there is less than about 2 mL of blood in the tube, the sample will be pre-diluted.
5.To pre-dilute, pipette 1500 mL of Wash/Diluent Solution into a vial, followed by 5 mL of the whole blood sample. Use a sample vial adapter for 1.5 mL vials.
6.Invert the tubes about 6 times.
7.Load the EDTA tube(s) into D-10 sample rack and place the rack in D-10. Must ensure that the sample barcodes are facing towards the back of the instrument.
8.Wait for the sample’s laboratory request number to appear on the screen (if they are not pre-diluted) before pressing “Start”.
9.When more than 1 tube is tested, press “Edit” and then, press “Done” for all the tubes and finally, press “Start”.


Results and Interpretation

1. Go to “data” and click “details’’
2. The graph must be above 0.02. If below 0.02, repeat test
3. F must not be more than 3%
4. Total area: 1- 4 million (cannot be more than 5 million)
5. P3 cannot be more than 3
6. Cannot have “unknown” below A1c, if not reject the test
7. The overall results must not be more than 10, otherwise, rerun the test and record “R:” in the worksheet.
8. If got variant window, record “variant window” in worksheet and then
- select sample with variant window
- choose “selected samples”
- Press ‘’Print”
9. At the end of the day, go to “data”, daily summary and press ‘‘Print”
10. If the total area is low (lesser than 1 million), do Manual run (dilution). If total area is very low, take more of sample and vice versa.
a. Take the diluent bottle (ensure that lot number on the bottle is the same with main diluent bottle located behind of the machine)
b. Take the patient’s sample and pipette out 5ul to 1500ul of diluent in the sample vial
c. Invert a few times to mix it
d. Manually key in sample number and run HbA1c test again


Samples from patients with haemolytic anaemia will exhibit decreased glycated haemoglobin values due to shortened lifespan of the red blood cells.

Do not report results if the peak shape of the graph is abnormal.

Expected Value Range

Factors such as duration of diabetes, adherence to therapy, and the age of the patient should also be considered in assessing the degree of blood glucose control.

Histopathology (Processing)

Hi! This will be my second last post =D. I have been posted back to Histopathology lab since the last six weeks. For the four weeks, i went to processing and then went to main lab to do embedding for the next two weeks. As Zi Shuang have posted on embedding, I will talk about processing for this post. For processing, we used two types of tissue processor machine which are Leica and Peloris. For the morning batch of the trimmed tissues, they will be put into the Leica tissue processor. The program used will be extended 16 hours tissue processing whereby it is extended to 7.30am the following morning. For the afternoon batch, tissue sections will be put in the Peloris tissue processor. The program used will be the extended 9.5 hours Xylene free tissue processing whereby it is extended to 8.30am the following day. Thus, for this post I will be emphasizing on the Leica tissue processor.

Subject Title: Lab Technique

Topic: Tissue Processing

Below is a picture of the Leica tissue processor:


The cassettes rack used for this rack is shown below:



Principle: The principle of tissue processing is to remove the extractable water from tissue specimens and replace it with a medium that solidifies to allow sectioning. It consists of 3 stages which are dehydration, clearingand infiltrating. The purpose of dehydrating is to remove water from the tissue using graded alcohols from a lower to a higher concentration. Clearing is to remove alcohol from the tissue with a solvent that is miscible with paraffin wax such as xylene. Infiltrating is to infiltrate the tissue with paraffin wax to allow sectioning of tissues.

Steps involved:



I have observed that sometimes the tissue sections tend to sunk down or bulge up after they are processed and embedded. If the tissue sections are sunk down, it is normally due to the lack of infiltration. However, if the tissue sections are bulged up, it is normally due to the lack of dehydration.

I also observed that the cassette racks and the machine must undergo quick clean to wash off the reagents used for tissue processing. This quick clean process will take about 30 minutes. After this cleaning session, new cassette racks can then be loaded.

Some precautions to take note is that gloves should be wore at all times when you are at the trimming room. This is because you will be dealing with formalin when you are placing the tissue cassette in the formalin container for fixation before they are being processed. As formalin is carcinogenic, gloves can protect your hands from having contact with the formalin. Rinse with water immediately after you had accidentally splashed with formalin. Try to wear mask with shield to protect your eyes as formalin will cause eye irritation which will make you tear.

I will post until here for now =). If you have any questions, feel free to ask me =D.

References:

Picture of Tissue processor
www.dotmed.com/images/listingpics/370518.jpg

Lok Pui
0704138G

cytology (gynae)

Hi everyone, this is my third post. In this post, i am going to talk about processing of the gynaecology specimens in cytology lab.

Basically, the gynae specimens come in two forms, Thin Prep vials or conventional smears. The thin prep vials contain preservcyt solution which is basically a methanol-based medium. The purpose is to preserve the cells to allow proper evaluation under microscope.


Collection of specimen

1)Specimen is collected with a spatula or a cytobrush
2)It is rinsed immediately in the presercyt solution
3)The vial will be capped tightly and labeled with the patient information before being set to the lab

In the lab

1)Once specimen is received, the patient information on the form must be double check with that on the vial and thin prep number is assigned to both of them(eg.0983)
2)The vials are then taken into the processing room to be processed by the Thin Prep 2000
3)Thin prep slides are labeled with the thin prep number and the patient's ID
4)The slide is first inserted into the slide holder
5)This is then followed by putting in the fixative vial which contains 95% ethanol
6)Specimen vial is inserted next followed by slotting in the filter
7)Press program '4'to process the gynae specimens

After processing

1)Once processing is done, the slide holder will drop the slides into the fixative vial
2)The slides in the fixative vial will be left aside for around 15 minutes to adequately fix the cells before staining
3)The stain used is papanicolaou stain which is bascially carried out by the machine
4)Slides are then manually mounted using Depex

Things to take note

1)The slides must be adequately fixed before undergoing staining so as to prevent cells from dropping during that process
2)The specimen vial must always be placed into the Thinprep 2000 before the filter to prevent the filter membrane from being scratched
3)At the end of the day, make sure that grease is applied onto the black rings of the filter cap so as to reduce friction

That is the end of my post, which is generally an overview of how processing of thinprep specimens are carried out. Questions are welcome. :)

zi shuang
0703383J

Real Time PCR Probes

Hi there, i have talked about real time PCR in my previous post. In order to get results from RT-PCR, probes such as Taq man are used.

Taq Man is a RT Probe (the Cheaper one). Taq man is a DNA sequence with binded 2 proteins Reporter protein (at the 5' end) and quencher protein at the 3' end. The quencher protein will inhibit the reporter protein when they are both in the same DNA strand. This is due to the inhibition of proton transfer.

Basic: PCR processes are Denaturation, Annealing and Amplification.
At denaturation, DNA will become Single stranded DNA strands. At Annealing, the probes will bind to complementary base pairs along the targetted DNA strand. At Amplification, Taq Polymerase will bind to the DNA strands and produce copies of the DNA strands. The Taq polymerase will move down the targetted DNA strands to produce complementary strands. Once the Taq Polymerase will "free" the reporter protein once it hits the probe (Breaking up the probe). The released Reporter protein will fluores to give a signal. A reader in the machine will pick up the signal and the signal will be converted to picture form which is shown on the computer screen. So as the PCR cycles continues more reporter proteins will be released thus forming a graph.

Another kind of probe which is used is the molecular beacon (slightly more expensive i heard). Molecular beacons are in a hair pin structure with the 2 proteins at the ends of the DNA seqence. It will denature at the Denaturation step to form SSdna. Will bind to DNA at annealing and the reporter protein will be activated to give off the fluorescence signal.

The two probes above are the more commonly used probes due to the cost.
For pictorial form, please refer to the link below
http://www.iba-biotagnology.com/images/naps/rt_pcr.gif (Taq Man)
http://www.pentabase.com/Portals/0/EasyBeacons%20mekanisme.jpg (Molecular beacon)

Credits to
Dr. Chan for teaching me about RT-PCR on first week of SIP.

Cheers
Tiong Han
Tg01
0703762E

cytogenetics[THERMOTRON & TECAN harvester]

Hello everybody,
in the blink of an eye, we are ending our attachment in less than 2months time. Hope everyone is still doing managing well. Anyway, back to the topic, i will be focusing about the two machines that are used in the cytogenetics lab. Cytogenetics is the study of chromosomes structures, its function and behaviour.

TECAN harvester






[The reagents]



As the name implies, it is a harvester however it is a robotic harvester that is involved in the harvesting of the cells. There are three major steps in the Harvesting procedure. The first step is Mitotic arrest,so as to arrest the cells in the metaphase stage because chromosomes are best studied at the metaphase stage as it is the clearest and at its most contracted state. The tecan harvester will add in Colcemid, a mitotic inhibitor that helps prevent spindle fiber formation, a process by which sister chromatids are pulled to opposite poles for incorporation into the 2 daughter cells. In addition,it also promotes chromosome condensation.

The second step is Hypotonic treatment whereby the cells are treated with a hypotonic saline solution to increase the cell volume so that chromosomes can spread out to allow easier identification.

The last step in harvesting is Fixation whereby the cells are fixed with addition of 3:1 methanol: glacial acetic acid so as to remove water from the cells and preserve them, by hardening the membrane and prepares chromosomes for the bending procedure.




[Drying of slides]

After which the slides are dried in the THERMOTRON before they are heated, stained and karyotyped.

hOPE this will give everybody some idea how harvesting of cells is carried out in the cytogentics lab that i am attaced to.

cheers,
Yong Herng
0702243G

SUBJECT TITLE: Chemistry

Name of Test: G6PD Screening Test

Principle

Glucose-6-P reacts with NADP+ to produce gluconate-6-P and NADPH and H+ in the presence of G6PD. The NADPH produced in the reaction fluoresces under long-wave UV-light. If there is a marked deficiency of this enzyme, or if G-6-PD is lacking entirely, no fluorescence will be observed.

Materials

1. Timer (not necessary)
2. G6PD Reagent
3. Negative Control (Commercial Blood)
4. Pipette
5. Pipette tips
6. Filter paper (absorbent paper)
7. Gloves
8. Sample in EDTA tube
9. Hitachi Cups
10. Tube rack (to hold the sample tube & Hitachi cups)
11. Marker (for labelling purposes)

Method (using 1 patient’s sample)

1. One Hitachi cup was labelled with “Negative Control”.
2. Another Hitachi cup was labelled with the patient’s laboratory request number e.g. “08-48”.
3. “Negative Control” and “08-48” were written below respective circles on the filter paper.
4. 100ml of G6PD Reagent was pipetted into patient’s tube labelled e.g. “08-48” and “Negative Control”.
5. 5ml of patient’s EDTA Blood and Negative Control were pipetted into the labelled cups “08-48” and “Negative Control” respectively and mixed.
6. The pipette tips were remained in the cups.
7. Using the same pipette tips, 5ul of mixture from each cup was pipetted onto each circle on filter paper respectively as shown below as the 1st drop.
8. It was left at room temperature for 5 minutes.
9. After 5 minutes, the 2nd drop was pipetted and left for another 5 minutes.
10. After 5 minutes, the 3rd drop was pipetted and incubated for 10 minutes at 35°C.
11. After incubation, the filter paper was observed under long wave UV light using equipment shown below.
12. Result was recorded.

Note: EDTA tube would be passed to Haematology Section for ABO typing if the patient is an infant. Otherwise, the tube would be filed back.


Results and Interpretation

If fluorescence is seen under UV light, the result is recorded as “present” on the worksheet. For the Negative Control, there should not be any fluorescence seen. However, if fluorescence is seen using the patient’s blood, it indicates that the patient has the enzyme G6PD and therefore, it shows that the person is not suffering from G6PD Deficiency.

Before reading the results under UV light, ensure that the filter paper is dry. The sample obtained from a normal or slightly reduced G6PD activity will show a strong fluorescence. If there is absolutely no fluorescence after 10 minutes of incubation in all the 3 drops, it suggests that there is a total or significant deficiency of G6PD. If there is fluorescence in any of the 3 drops but the other 2 drops do not fluorescence, incubate for another 5 minutes and observe again.

During the test, ensure that the blood is well mixed with G6PD reagent. Otherwise, it can result in false positive result. In other words, the patient does not have G6PD Deficiency, but the result shows that he/she has.


By: Rebecca (0703363B)

Cytology (Non-gynecology)

Hi everybody, This week is my turn to blog again =) I will talk about the things I have done in cytology as many people have been posted about histology stuff. For the first week in cytology lab, I had learnt to process gynecology specimens using the Thinprep 2000 processor. For the next 2 weeks, as we are not allowed to process non-gynecology specimens, so I had observed my trainers on how they process non-gynecology specimens, such as sputum, BAL, fluid, CSF and FNA. However, my supervisor allowed us to process our own urine or sputum samples so that we can learn how non-gynecology specimens are being processed. For the last week, I had learnt how to screen by screening their teaching slides.

Subject title: Lab Technique

Topic: Urine Papanicolaou Stain

Principle: The principle of this staining is to see detect whether the patient has any abnormalities in the kidney, bladder or the urethra. Such abnormalities can be infection, malignancy, reactive or carcinoma. Normal people should be negative which consists of urothelial cells. However, squamous cells might be also present in the urine as they shed from the trigone. Squamous cells consist of superficial cells, intermediate cells, parabasal cells and basal cells.

Steps involved: Mid stream urine is collected to ensure that the specimen is free from unnecessary contaminant. After the urine is collected, it is poured into a test tube and centrifuged at 2000 rpm for 10 minutes. The supernatant is poured away, leaving the cell pellet whereby it is dissolved in a few drops of Shandon fixative. The mixed solution is then put aside for 5-10 minutes to ensure that the cells are fixed. It is then cytocentrifuged where the mixture is being transferred onto the slides. The slides are then placed in 95% alcohol to fix the section so as to prevent the cells from dropping while staining. They are left aside for 15-20 minutes before putting into the staining machine for staining. The slides are mounted using Depex. They are screened to see whether there are any abnormalities such as infection or malignant.

Results

Normal urothelial and cervical cells are present. Below is a picture of normal urine papanicolaou stain.



However, if patient has any abnormalities such as infections, the papanicolaou stains will be different. Below are pictures on various types of infections.

Bacterial infection



Fungal infection



Viral infection



Patient might also be having urothelial carcinoma. Below is the picture of this example.



Things to note
Scums need to be removed every morning to ensure the quality of the slides are maintained.
If the specimen is malignant, all the staining solution must be filtered or stained to prevent contamination.

If anyone has any questions, feel free to ask me =)

Lok Pui
0704138G

References:

http://www.bostwicklaboratories.com/HOME/getdoc/f3882049-3705-4478-81b4-11112e4eb64b/Urinary-Tract-Infection.aspx

www.cytologystuff.com

The Staining :)

Hi everyone, it is my turn to post for this week. For this month, I have been posted to the routine staining area; thus, I have been exposed to various special stains like CAB, PAS, PASD, MT, Orcein, Fe, Ziehl Neelsen etc.
Today, I will introduce on PASD and Fe stain.

PASD (Periodic acid Schiff diastase)

Function: It helps to differentiate glycogen from other carbohydrates

Principle: Glycogen is digested by diastase where no reaction will occur with Schiff’s reaction afterwards.

Procedure:
1) Dewax and bring section to water
2) Treat section with diastase
3) Wash well with water
4) Treat with 1% aqueous periodic acid for 5 mins
5) Wash in water
6) Treat with schiff’s solution for 5 mins
7) Wash in water
8) Counter stain nuclei with haematoxylin
9) Blue in water
10)Dehydrate, clear and mount in DPX

Result:
Any presence of glycogen will be stain magenta on the PAS stained slide while for the PAS/D stained slide, there will be absence of it.

Note:
A positive control must always be used




A picture showing presence of glycogen stained magenta (PAS/D stain is used). This suggests that the patient is suffering from glycogen storage disease



Fe (Perl's reaction)

Function: Demonstration of iron and haemosiderin

Principle: Dilute hydrochloric acid solution release ferric iron that is tightly complxed to protein as hemoglobin. The ferric iron will then reacts with potassium ferrocyanide solution to produce an insoluble blue compound which is ferric ferrocyanide.


Reagent preparation:

Perl's reagent
20% hydrochloric acid -- 5 ml
20% potassium ferrocyanide --5ml


Procedure:

1) Dewax and bring section to water
2) Treat with perl's reagent for 20minutes
3) Rinse in SRW
4) Counterstain with nuclear fast red for 3 mins
5) Wash, dehydrate, clear and mount in DPX

Result:

Iron -- dark blue
Nuclei -- red




Perls Prussian blue stain showing hemachromatosis in cardiac muscle

Control: A positive control must also be used in this case


Staining is a very challenging job whereby you need to be able to multitask, however, i learn a lot of many different stains and the different things that need to be observed under the microscope.

Feel free to clarify any doubts and i will try my best to answer them :)

References:

http://images.google.com.sg/imgres?imgurl=http://img.medscape.com/pi/emed/ckb/pediatrics_genetics/941088-941089-941632-941708.jpg&imgrefurl=http://emedicine.medscape.com/article/941632-media&usg=__r1ZzgRvxBDwxsQKDdIiuE03bghw=&h=346&w=573&sz=37&hl=en&start=2&tbnid=x8c9fQVuwvM8JM:&tbnh=81&tbnw=134&prev=/images%3Fq%3Dperiodic%2Bacid%2Bschiff%2Bglycogen%26gbv%3D2%26hl%3Den

http://images.google.com.sg/imgres?imgurl=http://www.gladstone.ucsf.edu/gladstone/files/histology/Perls-Prussian-blue-stain.gif&imgrefurl=http://www.gladstone.ucsf.edu/gladstone/site/histology/section/2288&usg=__GqcH8PvwJO0Uffc2OAIeVfzDNgU=&h=331&w=504&sz=44&hl=en&start=7&tbnid=RpRio2w7otbr2M:&tbnh=85&tbnw=130&prev=/images%3Fq%3Dperls%2Breaction%26gbv%3D2%26hl%3Den%26sa%3DX




Cheers,
Zi Shuang
0703383J

Ahhh- CHOOO

I'm at a molecular lab. My current entry was very generic because i didnt mention anything about the current outbreak. Now the situation is getting better so i think it is okay to touch on the touchy subject.

In the Molecular lab, our assays are base on Polymerase Chain Reaction (PCR). There are 2 kinds of PCR, Real Time PCR (RT-PCR) and the conventional PCR. I discuss extraction of Flu sample using RT-PCRin this entry. Will talk more about the probes on the next entry and after that will be conventional method.

As all may know, at the initial stage of the outbreak, everyone is very worried. Everyone with sore throat and fever wants to know if they are infected with the famous "swine Flu". Culturing of the virus takes a long time. The faster way to get the confirmation will be detection of the virus DNA. DNA works by having complementary base pair (A--T, G---C) the number of dashes indicates the number of bonds between the different complementary base pair.

Lets start on how we (the staffs) go about testing.

Test ordered: H1N1 PCR
Specimen Type: Swab in VTM media (If specimen is for PCr and Culturing)
Dry Swab (For PCR. swab nose then throat.

Protocol: If samples is from the campus (Within the hospital), Inform the lab by calling the lab, before despatching. This is a highly recommened step to do especially if it is a VIP sample or urgent sample.
From outside campus, liase with lab incharge. SHE/he will say either we receive from you or no we are not going to do it for you.
Once we receive the sample (In the lab), information will be matched from the order from and the specimen. If it matches (matches 99.9% of the time), swabs will be sent into the Biological Safety Cabinet (BSC). Order form will be keyed in.
2 tubes will be label, 1 2ml Tube and 1 1.5ml eppendorf tube. the 2ml tube will be for preextraction and the eppendorf tube will be needed for post extraction.

500ul (Micro litres) of PBS will be dispensed into the 2ml tube and swabs will be broken in the tube and tube vortexed. Swab will then be removed (this step is to dislogde the virus into the PBS solution). If it is in a VTM media, about 1.5ml of media is dispensed into the 2ml tube.

Extraction is done by a machine. it is able to run 24 sample at 1 time. For respiratory virus, for this case it is considered, the lab uses off board lysis (lysis will be done out of the machine.)
Lysis buffer will be dispensed into a different wells. 1 well for 1 patient. samples are added and incubated for 10mins. this allows the virus to be lysed. Magnetic beads are then added to the wells. The machine does extraction by using magnetic beads. DNA is -ve charge and the machine is able to manipulate magnetivity to extract DNA.

There is about 55ul of elute/DNA buffer. so 55ul of elute is transfered to the 1.5ml tube.

For real time PCR, it enables you to amplify and get the results at the same time.
Samples wil be added to cappillary tubes which contains a DNA master mix, (F-primer, R-primer, Taq Polymerase, dNTP and some other things which is from the commerical kit (TAQ MAN PROBE) which willl flouresce when to give a reading.)
The capillary tubes are put into the lightcycler. 50 cycles of PCR will be runned. PCR- Denaturation 95ºC, annealing 56ºC, elongation 72ºC. At the end of each cycle, a sensor will read the amount of light given out by the probes to give a reading. If it is a postive case, a sigmoidal curve will appear. Then we will bless him (just kidding). Positive or negative cases, reports will be sent out. If it is a urgent sample, we will call the consultant or doctor.

ALong with this test, a FLU A RT-PCR is runned on another machine as a confirmatory test. IF Flu A Pos, h1n1 pos = Good luck home quarrentine/ observation. If Flu A pos, h1n1 neg= no h1n1. If Neg Flu A, pos h1n1 = troubleshooting to be done by lab personnel, results inconclusive.

RT-PCR will enable the lab to get results in 4-6hours, unlike culturing which may take days. Therefore, molecular techniques is still more prefered in the campus.

Disclaimer: I will not be able to find out the quantity of reagents added. Above knowledge is base on my observation in the lab and hope i dont offend anybody.

Cheers,
Yeo Tiong Han
TG01

P.s. I will get pictures from the lab. Have to ask permission first. 15/08/09

HISTOooing [ Reticulin stain & Frozen section]

Aloha everyone,

i was attached to HISTOPATHOLOGY on week 3 for three days. During thse 3 days, i managed to perform several technique such as tissue embedding, frozen section and special staining and observed many eye-opener stuffs. I got to personally see organs such as uterus, breast, kidney, as well as post-mortem of a stillbirth fetus(22weeks) being dissected and examined by pathologist. It was quite downhearted to see a dead feteus as from what the pathologist said, the baby has brain rupture which may have cause the stillbirth.

Anyway,I did a retic control for RETICULIN STAIN;
Reticulin is a type of fiber that provides structural support in tissues such as liver and kidney. The fibers in our normal liver are in well-defined strands however, an abnormal tissue such as a necrotic liver will have a discontinuous pattern. The positive control that was used is a normal liver, the reticular fibers should look black, when i see it under the microscope, it looks likes small little black dot, and the nuclei will be pinkish red.

Picture of reticular fiber:
http://neuromedia.neurobio.ucla.edu/campbell/connective_tissue/wp_images/34_reticular_fibers.gif

[Picture of retic control]


Next, i also managed to try out how frozen sectioning is like. Firstly frozen section is different from the paraffin sections, frozen sections comes without fixatives,or formalin. Wherever, there is frozen sections, i will follow the medical technologist to the operating theatre (OT) to obtain the fresh tissue excised during surgery.

FROZEN SECTIONING is a rapid diagnostic process whereby the pathologist will make a fast and rapid diagnosis to the surgeon whether the tissue is bengign or malignant,or to evaluate whether the tumour has been completely removed, is like the pathologist acts as a consultant to the surgeon to determine the extent of further surgery at the time of surgical procedure.

For example, if a tumour appears to have metastasized(spread),the suspected metastasis will then be sent for frozen section so that the pathologist will inform the surgeon whether or not to continue the surgery as there is no point in continuing the surgery if the tumour had metastasized.

So after the fresh tissue are obtained from OT, a small amount of OCT (optimum cooling temperature)compound is added onto the tissue and freezed immediately by liquid nitrogen. Thereafter which it is sectioned via a cryo-stat. The interesting part is the tissue section produced will be picked upon by a glass slide, is like you gently use the glas slide to touch the tissue section, the tissue will be condensed onto the slide and produce a smear. As compared to paraffin section it is different, whereby you need to fish it as we did during our histology practical. Next, the section are stained with Haemotoxylin and eosin (H&E), after which the pathologist will be called upon to examine and report the results. The whole procedure is a fast one, around 10 minutes or so, as it is an intraoperative procedure whereby the surgery is still ongoing so diagnosis has to be fast.


[Picture of OCT compound]

[cryocut]

[Picture of interior cryocut]

[Place Where staining is carried out]

[ Place where pathologist cut & examine the tissues]





Note:The above pictures taken 've been granted permission by my lab staff.=]



Signing off,
Yong Herng
0702243G

6th week of attachment

Hi this is my 2nd post here. Sorry for the delay in my posting. I was supposed to post yesterday. Anyway, I am permanently stationed at Biochemistry/Immunology Section for these 5 months.

Subject Title: Clinical Chemistry

Name of Test: Urea Breath Test

WHAT IS UREA BREATH TEST?

The urea breath test (UBT) is a test for diagnosing the presence of a bacterium, Helicobacter pylori (H. pylori) in the stomach. H. pylori causes inflammation, ulcers, and atrophy of the stomach. The test also may be used to demonstrate that H. pylori have been eliminated by treatment with antibiotics.

___________________________________________________________________________________________________________________

PRINCIPLE

The patients are given urea labelled with 14C or 13C orally. In the presence of H. pylori organism, urea is converted by the bacterial enzyme urease to 13CO2 and ammonia. The 13CO2 is absorbed in the bloodstream and gets transported to the lungs exhalation in the breath. This results in an increase in the ratio of 13CO2 to 12CO2 in expired breath in the Post-Dose breath sample. In the absence of H. pylori, the Post-Dose breath sample has essentially the same amount of 13CO2 as the baseline breath. The exhaled CO2 is trapped, processed and analysed. 14C is a beta emitting radioisotope and can be detected using liquid scintillation counting and 13C is a stable, non-radioactive isotope that is measured using a mass spectrometer (infrared spectroscopy). The difference in the 13CO2 levels are calculated too.

____________________________________________________________________________________________________________________

MATERIALS needed (For 1 patient)

1. Two breath bags
2. One Urea tablet
3. UBiT- IR300 Machine
4. 100ml of water
5. Disposable Cup(s)
6. Timer

____________________________________________________________________________________________________________________

METHOD

[Patient’s Preparation before Test]
a) The patient must fast without drink or food for at least 6 hours before test
b) If the patient is a smoker, he/she must not smoke 2 hours prior to test
c) The patient must stop all antibiotics/antibacterial drugs at least 4 weeks before test [e.g. Amoxicillin (Amoxil, Moxam),Bismuth tricitrate (Denol), Clarithromycin (Klacid), Fasigyn (Trinidazole), Metronidazole (Flagyl), Tetracycline (Tetrex, Mysteclin, Achromycin), and any other antibiotics]
d) The patient must stop all Proton Pump Inhibitors at least 1 week before test [e.g. Losec (Omeprazole), Somac (Pantoprazole Sodium Sesquihydrate), Zoton (Lansoprazole) and Nexiam]
e) Lastly, the patient must stop all H2 Receptor Antagonists at least 1 day before test [e.g. Cimetidine (Tagamet, Sigmetadine, Magicul), Famotidine (Amfarnax, Pepcid, Pepcidine), Nizatidine (Tazac) and Quick EzeRanitidine (Zantac, Rani 2)]


[Patient: Steps DURING the test]
a) Breathe into “Pre-dose” or “Baseline” blue breath bag WITHOUT taking the urea tablet
b) Within 5 seconds, swallow the UbiT tablet (under fasting condition) with 100ml of water. The tablet must not be chewed, crushed or dissolved.
c) Lie down on your left side for about 5 minutes (to allow the tablets to react more with H. pylori)
d) Remain seated for another 15 minutes (to enable CO2 gas from being exhaled from lungs properly)
e) Breathe into “Post-dose” or “Sample” bag

These 2 bags with the Laboratory Request Form will be sent to the Clinical Laboratory for analysis.

[Medical Technologist: Procedures in the Laboratory]
a) Register the sample at administration section
b) Passed on to Biochemistry section
c) Check the labels and paste the labels onto the bags if not pasted yet
d) Conduct test using the machine UBIT-IR300
e) To use the machine:
1. Press “Yes”
2. Enter Sample ID/ Patient’s Lab Request Number
3. Press “ENTER”
4. Press “Yes” to run/start
5. Insert “Baseline” bag at Pre knob
6. Insert “Sample” bag at Post knob
f) Verify results

____________________________________________________________________________________________________________________

RESULTS AND INTERPRETATION

A value of 2.5% or higher indicates that the patient is positive for H. pylori infection. If the isotope is detected in the breath, it means that H. pylori is present in the stomach. If the isotope is not found, H. pylori is not present. When the H. pylori is effectively removed by antibiotics, the test changes from positive (isotope present) to negative (isotope absent).


P.S. I have a picture of the machine but somehow something seem to be different at blogger.com. I can't seem to bold my words too and suddenly, there is no usual icons for me to press in order to attach files or pictures ): Anyway, you can just go to this link shown below:

http://www.gribbles.com.my/ubt2.html

Signing off,
Rebecca Chew (0703363B)
God bless (:

Answers to Questions

QN 1: what type of tissues did you usually stain?

Siti

ANS: For our lab, we don’t usually know what we are staining as the specimens were already trimmed by the pathologists and some specimens are really very small, so it is very difficult to be identified. However, I have been to the trimming room for the first week, I will name some organs that are being trimmed and then sent for staining. The most common types of tissues are breast, colon, cysts, uterus, kidney and liver and the lymph nodes.

QN 2: if you have diagnosed a tissue which is malignant,will the staining solutions in the auto-stainer machine be contaminated and affect the subequent tissues(which may not be malignant)that are going to be stain?

Yong Herng

ANS: For our lab, we do not diagnose any slides, unless they are checking the quality of the slides. By right, the tissues are already fixed on the slides using hotplate, so the chances of it to contaminate the staining solutions is quite low. However, if the tissues are not fixed on the slides properly, some tissue parts might wash away and stay in the staining solution. This might result in the production of floaters in the subsequent slides which might lead to misdiagnosis. Thus, staining solutions should be filtered daily or change in alternate days.

QN 3: Do you have any microscopic pictures on how a normal and malignant tissue differs in morphology and staining? Thank you.

Li Yinliang Alex

ANS:

The picture on the left is an example of tumor cells of the liver where the cells become irregular in shape and there is an abnormal increase in the number of nucleus.


The picture on the left is an example of normal cells of the liver.

Reference:
1) IHC World, normal H&E staining,taken on 1st july 2009, from
http://www.ihcworld.com/imagegallery/displayimage.php?album=3&pos=16

2) Atlas of pathology, H&E staining of tumor liver, taken on 1st July 2009, from
http://www.pathologyatlas.ro/chronic-myeloid-leukemia-liver.php

QN 4: after examine the stain under microscope,do you store the specimens for a day or two or just simply throw it away?

Nyzah

ANS: Are you referring to the specimens that are trimmed or the slides that are stained? For the specimens that are trimmed, they are stored for about 2-3 months. For the slides that are stained, they are stored for about 10 years.

QN 5: What is photo-oxidation and how exactly does it affect the microscopic images of the slides?

Siti

ANS: Photo-oxidation is where Haematoxylin oxidized due to the presence of visible light and oxygen where it will produce a layer of oil-like substance floating at the top of the Haematoxylin solution. This oil-like substance is also known as scum. As it is floating on top of the solution, it will stick onto the slides when they are lifted up, hence, affecting the quality of the stains.

QN 6: Erm for the staining right, isnt Haemetoxylin being used to stain so what is the reason of using Lithium Carbonate? Is there other function of Lithium Carbonate?

Jennifer

ANS: Yup..Haematoxylin is being used to stain the nucleus blue but Lithium Carbonate is used to maintain the blueness or make it bluer. From what I have learnt so far, I don’t think that there is other function.

H & E Staining :)

Hi everyone!

Finally its my turn to post this week! I am attached to histopathology lab for my attachment (same as Zi Shuang). As what she said last week, our lab is separated into various areas, such as embedding, shaving, trimming, staining and sorting. For my case, I have been doing staining for my first posting. I find that staining is a very challenging task as it needs alot of concentration and the capability of multi-tasking because we are required to do heating, H&E staining and special stains at the same time. If we are lucky enough, we also have to do different types of special stains manually. For the past 5 weeks, I have done mainly H & E Stain and Special Stains using machine. However, I have also done manual special stains such as Alcian Blue, Mucicarmine, Ziehl Neelsen, PAS, PASD, Perl's Iron, CAB, VB and Orcein stains. As we have been doing H & E staining manually during our HTech practical, I will introduce to all of you on the H & E staining machine.

Subject title: Lab Technique

Topic: H&E Staining

Principle: The principle of H & E staining is to demonstrate different parts of the tissue components in contrasting colours, such as pink, blue, deep red, orange red and etc. Haematoxylin can be oxidized to Haematein by using natural oxidation or chemical oxidation. It requires a mordant, which is usually a metal, to enable Haematein dye to be well demonstrated on the acidic nucleus to give a blue colour. Eosin has the ability to distinguish between the cytoplasm of different types of tissues, connective tissue fibers and matrices.

Steps Involved: Slides are firstly collected from the trimming and fishing area and placed on the edge of the hotplate for air dry. The tissue sections are fixed on the slides by heating on the hot plate for 3 mins. The slides are placed in the staining rack and transferred to the H&E staining machine where dewaxing and staining are being carried out. After they are stained, they are directly transferred to the auto mounting machine for mounting.


Picture of H & E staining machine


Picture of Auto-mounting machine


Picture of Hot plate


Picture of Staining rack

Result:
Nucleus: Blue
Cytoplasm: Pink
Muscle fibers: Deep pinky red
Collagen: Pale pinky red
Fibrin: Deep red


Picture of slides produced

Procedures for H & E staining machine

1 Xylene 2 mins
2 Xylene 2 mins
3 Absolute alcohol 1 min
4 95 % alcohol 1 min
5 70 % alcohol 30 secs
6 Running water 30 secs
7 Haematoxylin 3 ½ mins
8 Haematoxylin 3 ½ mins
9 Running water 30 secs
10 0.5 % acid alcohol 2 dips
11 Running water 30 secs
12 Lithium carbonate 30 secs
13 Running water 30 secs
14 Blue in running water 3 mins
15 Eosin 25 secs
16 70 % alcohol 1 min
17 95 % alcohol 1 min
18 Absolute alcohol 1 min
19 Absolute alcohol 1 min
20 Absolute alcohol 1 min
21 Absolute alcohol 1 min
22 Xylene 1 min
23 Xylene 1 min
24 Xylene 1 min
25 Depex -

Some points to be noted:
To ensure that the slides are well demonstrated and differentiated, a control must be stained to assure that the machine is at its tip top condition.
Lithium carbonate must be replaced when it had turned purplish in colour. This is because Lithium carbonate needs to maintain a pH value of 9-10 to stain the nucleus blue, so if the pH value drops it will affect the demonstration of the nucleus.
Slides must be ensured that they are facing out of the staining rack so that the slides are mounted at the correct side.
Haematoxylin and Eosin should also be filtered before use and stored in the dark area to reduce photo-oxidation, debris and dust which will affect the quality of staining.

Clinical interpretation: The H & E slides can be used to diagnose the presence of tumors by looking at the abnormality in size and shape of the respective tissue cells.

I will stop here from now. If you have any doubts, pls feel free to ask me any questions! :)

Lok Pui
(0704138G)

The Embedding :)

Hi everyone, I have been assigned to a histopathology lab for my attachment. The main lab itself is divided into areas for different purposes like embedding, shaving, trimming, fishing, staining and sorting of documents. For my first post, I will talk about embedding.

The principle of embedding is where tissues are embedded in paraffin wax so that they are hard enough to withstand cutting into thin sections. On the other hand, it also makes them soft enough to allow the blade to cut through, with little or no damage to both the blade and tissues. The equipment used is an embedding centre, which is shown in the picture below.


Picture of a tissue embedding centre


There are three main compartments, which consists of the wax dispenser, cold plate and the heated storage area for metal moulds. Wax dispenser as the name implies, dispense appropriate amount of melted wax into the metal mould used. Cold plate allows the wax to solidify, the heated storage area stores 4 different sizes of metal moulds, depending on the size of tissues to be embedded. Also, in the picture above, there is a black ‘knob’ on top of the metal cover. Lift it up and there is a space to contain metal racks which is used to put cassettes. (The cassettes contain tissues inside).

The procedure for embedding is very straight forward.

1) One cassette is taken out from the racks and opened
2) The lid is detach and thrown away into a biohazard bag
3) A metal mould of the appropriate size is chosen and filled with melted wax
4) Tissue is transferred into the mould using a heated forcep
5) It is held down by the forceps to prevent it from floating around
6) A metal press is used to press down the tissue so that the surface is on even
plane.
7) The mould is covered with the other part of the cassette and press down to
secure it
8) The top is partially filled with melted wax again
9) It is then left on the cold plate to allow the wax to solidify




Picture of some cassettes




Picture of metal moulds

‘The procedure looks pretty easy, so what is so hard about embedding’, is my first thought when I observed the procedure. Only when I attempt it did I realize that it is not as easy as it seems. The trick to embedding is that every little thing that you do during the process has a tiny principle behind it, which will eventually reveal whether the tissue has been embedded properly. For example, the orientation of the tissue is especially important. This is because it will determine if the relevant areas of the tissue that needs to be studied has been exposed fully. Different types of tissues need to be orientated in a different way.

If there is more than one tissue, it has to be arranged in the same way/pattern for easier studying under microscope. Another thing to take note when there is more than one tissue is that they must be placed closed together. The reason being that wax cannot expand but tissues can. Thus, when this happens, the tissue is constraint to a limited space because of the wax and it will be ‘squeezed’ together, forming a lot of folds.

Another tricky part is where to place the mould during orientation of the tissues. Can it be placed on the cold plate? The answer is no. This is because the wax will solidify very fast on it. If there is only one big tissue, it will not really pose as a problem. However, if there are around three or more tissues, by the time the third or fourth needs to be put in, the wax is already partially solidifying. Thus, when you attempt to press down the tissues, the whole thing will be something like a ‘mash potato’ (it really looks like that).
Although it is not preferred on the cold plate, it can still be carried out nicely, provided that the person has lightning speed (no joking really). In conclusion, speed plays a major factor during embedding and practice will make perfect. There are still other precautions, but I will stop here for now.

Any questions pertaining to embedding will be gladly welcome.

Cheers,
Zi Shuang (0703383J)
TG01

References:

Picture of tissue embedding centre, Retrieved on 18th July 2009 from

http://images.google.com.sg/imgres?imgurl=http://www.gmi-inc.com/images/EG1160_duv_pro_b_sh%255B1%255D.jpg&imgrefurl=http://www.gmi-inc.com/Categories/histology.html&usg=__UeZDfSiguhmad3l39c-ohrfU3O0=&h=350&w=500&sz=20&hl=en&start=8&um=1&tbnid=cHWzwlCJJKy2kM:&tbnh=91&tbnw=130&prev=/images%3Fq%3Dshandon%2Bembedding%2Bcenter%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26um%3D1


Picture of cassettes, Retrieved on 18th July 2009 from

http://images.google.com.sg/imgres?imgurl=http://www.proscitech.com.au/cataloguex/img/R/rch42.jpg&imgrefurl=http://www.proscitech.com.au/cataloguex/online.asp%3Fpage%3DR4&usg=__hd28LbblhsHqxdMYYtEjE8qNDaQ=&h=219&w=320&sz=36&hl=en&start=35&tbnid=g1i_6q4zg1QQ1M:&tbnh=81&tbnw=118&prev=/images%3Fq%3Dcasettes%2Bhistology%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D18


Picture of metal moulds, Retrieved on 18th July 2009 from

http://images.google.com.sg/imgres?imgurl=http://www.largro.com/MBMPic1.jpg&imgrefurl=http://www.largro.com/mbm.html&usg=__xqeT05lsc160vtvJrEhkCumuia8=&h=141&w=200&sz=6&hl=en&start=25&tbnid=MS50N43X1Tkc9M:&tbnh=73&tbnw=104&prev=/images%3Fq%3Dmetal%2Bmoulds%2Bhistology%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D18

Laboratory Management And Quality Assuarance

Safety level of labs and safety in the lab [EDITed 13/07]

Hey everyone, it is the 3rd week of Sip. Due to the current outbreak of the Virus that we all know, the lab that i am with has changed most of their routine work to focus on the "NOT TO BE MENTION-ED", thus, i am not able to give more details or information on the routine work steps.

So i will talk more about safety of the Labs.

There are different levels of biological safety rating for labs, ranging from 1 to 4. BSL 1 labs handle the common, least pathogenic substance/microbes. Little protective gear has to be woren in the lab (little NOT none). BSL 2 lab handles with substances/microbes that are slightly pathogenic as compared to BSL 1 labs. There is even a biological safety cabinet (BSC). The BSC works by having a certain air flow current and HEPA filters. HEPA filter can remove particulates which includes microorganism from the air. Also, in a BSL 2 lab, more personal protective equipment (PPE) has to be woren. PPE such as yellow gowns, n95 mask. A BSL 3 lab handles with the super dangerous and more pathogenic substance/microbes. More protective PPE such as face mask, yellow gowns, n95 face mask has to be woren. BSL 4 lab handles with high risk and life threatening organisms. Both accesses to BSL 3 and 4 labs are very high monitored, access to these labs are prohibited.

If you were to work in a lab that has higher BSL, you will definitely require more training. Also, more PPE will be required in the different labs.

There are 3 classes of BSC available.

Class 1 BSC offers worker protection and enviroment protection. It does not provide product protection as "dirty" air constantly enter the BSC from the front and may risk contaminating the specimen. It is often used for low to moderate infectious agent. The air in the BSC will pass through the HEPA filter thus it protects the enviroment by filtering out the infectious particles.

Class 2 is further divided to class A1, A2, B1 and B2.
All Class 2 BSC will provide worker protection, enviroment protection and product protection. Class 2 BSC can be differetiated from Class 1 BSC by features such as opening with maintained inward airflow, airflow in the BSC is unidirectional ad is filtered throught the HEPA filters and the filtered air goes to a room or a facility exhaust system.
Class 2A BSC have 70% recirculated air and 30% air that is removed, while class 2B1 BSC have 40% of the air being recirculated and 60% that is removed. Class B2 has 0% air recirculation and 100% air removal. Class 2A has its HEPA filter on the top and filtered air flow downwards. Class 2B has its HEPA filter fixed a the bottom, air from the enviroment flows to the filters first before going into the working chamber.
The difference in class 2 A BSC 1 and 2 is that in 1 75FPM (Air Flow in feet per minute) while class 2A 2 has 100FPM.

Class 3 BSC is the total containment cabinets. it is gas tight and provides the highest level of protection to the enviroment, personnel and product.

Next, safety is a important issue in the laboratories. Every labs should be equipped with at least a fire extinguisher, for my case, there is 3 in the 3 different areas in the lab. Even the pantry has one too. One of the first few things that happen to all staffs when joining a lab is a safety briefing, after that, safety manuals will be passed to them for reading. This shows that safety is definitely not an issue to be joke with.

In a recent fire evacuation drill, all staff knew where to go to in case of an evacuation. There was also fire extinguisher demonstration where a volunteer demonstrated on how to use a fire extinguisher to fight a fire.

With that knowledge on safety, i believe those who are not sure of their safety features in their lab should go check it out.




Picture of a Biological Safety Cabinet (BSC Class2)

Cheers,

Yeo Tiong Han
TG01

Observations always involve theory. ~Edwin Hubble


Reference
BioSafety Levels,The Ohio State University, Retrieved on 10 July 2009 From
http://academic.marion.ohio-state.edu/sciencefair/BSL.htm

Picture of the BSC Retrieved on 10July 2009 from :
http://images.pennnet.com/articles/cr/thm/th_0804crcertification04.jpg

Intro to Biological Safety Cabinets, The Baker Company, Retrieved on 10July 2009 from:
http://www.bakerco.com/resources/intro.php

BOOM BOOM BOOM!

Moshi Moshi everyone,

For the first three days of my SIP,i was attached to Haematology lab. The first thing i was told to do was to learn how to make a peripheral blood film. There is a very senior med tech, Mr Yeo, who demonstrated and explain the technique to me. He make around 5 slides for me to see and i thought to myself; 'seemed easy to me, should not be a problem'. But i was wrong, i tried it myself and it doesn't turn out to be easy as i either make a edgy or too thick or too thin film. Then i remembered why during one of the E-lecture for haematology, Mr Poh mention practice until you know to how to make a blood film... because most of the staffs there mentioned that they take some time to practice to perfect it. A well made blood film should be thick at the drop end and becomes thinner at the other end. Most importantly, the glass spreader should be clean and dry because if it is wet,it may cause lysis of red blood cell.


Although i only have 3 days at Haem lab but i think i learnt quite a few tests; Kleihauer Betke test and dengue serology

Kleihauer Betke Test.
is a test used to access the degree of Foetal Maternal HAemorrphage(FMH) via detection of foetal red blood cells in maternal peripheral blood.

Method:
1) 3 different smears are made: a)negative control
b)Test sample
c)Positive control
2) Air dry the smears

3) Fix the smears in solution C( ethanol) for 10mins

4) Stain the slides in elution solution ( consist of solution A,B &C)for 20sec and rinse with tap water.
*solution A- 0.75% Haemotoxylin in 96% ethanol
solution B- 2.4g of ferric chloride in 0.5% hydrochloric acid
solution C- 80% ethanol

5) Counterstain the slides with Eosin B for 3 mins, wash with tap water and air dry.


Results:

Foetal red blood cells will appear as darkly red stained cells while adult RBC will appear as palely stained ghost cells.
If there are more than 10ml of foetal RBCs in maternal circulation, it is clinically significant.

Dengue serology:
I was fortunate to perform the test under the guidance of a staff. The test is carried out in the biohazard safety cabinet. There is this commercial kit called Dengue Duo Cassette, which allows presumptive differentiation between primary and secondary infection via detection of IgM and IgG antibodies respectively. This kit is easy and also convenient as the result is known in 15mins.

Picture of a Dengue Duo Cassette:

Taken on 4july 2009 from: http://www.pacbiotekindo.co.id/products/dengueduo_cassette.html

Principle: The principle is based on binding of dengue-specific IgM or IgG antibodies present in the patient sample to the anti-human IgM or IgG antibodies embedded on the cassette membrane. There is also a control to ensure the test is carried out correctly.

Method:
1) Add 10ul of serum to the circular well via a micropipette
2) Allow the sample to absorb entirely within the circular well
3) Holding the buffer bottle vertically, add 2 drops of buffer to the square well at the base of the cassette.
4) After 15mins, read the result

Result:
Any trace of pink line in the test area shows a positive result.
Primary infection: Pink band in the IgM & Control region
Secondary infection: Pink band in the IgG,control region and/or IgM region.

For now that is all about it,hope everybody will have a glimpse or some idea how dengue is detected=)

Signing off
Yong Herng
TG01
The Sesame

1st Week of Attachment (:

Hi everybody! (:

I am attached to this Hospital where we get ourselves rotated to different departments/sections/disciplines. But eventually, I think we'll be in the section where we are first rotated to. Anyway, the different departments/Sections are:

1. Haematology
2. Biochemistry/Immunology
3. Microbiology
4. Administration - Involved in registering the specimens, etc.

So, I'm attached to the Biochemistry/Immunology Section.

____________________________________________________________________

Subject Title: Clinical Chemistry

There are a few tests I learnt this week. But for this post, I'll just post on one of the tests which is conducted everyday.

Name of test: Microalbumin Urine Qualitative Test

This test is usually carried the most in the morning (as morning is the time when specimens come in) and in the late afternoon at about 4.30pm (where it is also the time when specimens arrive).

Principle(s)

Microalbumin Urine Test is used to check whether the patient's urine contains albumin and creatinine. As you know, Albumin is a plasma protein which is found in the blood. It is filtered by the kidneys. Under normal conditions, albumin is not supposed to be present in the urine as the kidneys would have filter the albumin and albumin would be reabsorbed back into the body. Only very low amounts of albumin is present normally. So, if albumin is seen in the urine, this would indicate that the kidneys are not effective in filtering albumin and kidney damage is plausible.

The Clinitek Status Microalbumin Machine consists of a test table. The test table contains a sensor which will detect the reagent strip and the machine can then start the test.

Reagent Strips are used. They are firm plastic strips that contain 2 reagent areas testing for albumin and creatinine in the urine. These strips are read using Clinitek Status® Analyser. A albumin-to-creatinine ratio is determined too in mg/g or mg/mmol. The reagent strips provide semi-quantitative results and these serve as screening test for microalbuminuria.

Chemical Principles:

Albumin = test is based on dye binding using high affinity sulfonephthalein dye. At constant pH, blue colour is developed due to albumin. Colour ranges from pale green to aqua blue.

Creatinine = test is based on peroxidase-like activity of a copper creatinine complex which catalyses the reaction of diisopropyl benzene dihydroperoxide & 3, 3', 5, 5' - tetramethylbenzidine. Colour ranges from orange through green to blue.


Materials used for this test

1. Urine Sample in Dry Container
2. Microalbumin Machine (Clinitek Status)
3. Paper towels (These are to put on the table to prevent spillage of urine on table directly)
4. Biohazard Bag (To discard the used reagent strips)
5. Clinitek Microalbumin Reagent Strips
6. Gloves


Picture of Clinitek Status® Analyser:









Taken, 28th June, 2009, from
http://images.google.com.sg/images?q=clinitek%20status&oe=utf-8&rls=org.mozilla:en-GB:official&client=firefox-a&um=1&ie=UTF-8&sa=N&hl=en&tab=wi


Picture of Clinitek® Microalbumin Reagent Strips:








Taken, 28th June, 2009, from
http://images.google.com.sg/images?hl=en&client=firefox-a&rls=org.mozilla%3Aen-GB%3Aofficial&um=1&sa=1&q=clinitek+reagent+strips&btnG=Search+images&aq=f&oq=


Method

1. Check that the Patient’s name on sticker label on the cap is the same as the label pasted around the bottle. If it is wrong, it is given back to the administration section.

2. Press "Strip Test" followed by "Enter New Patient'' on the analyzer.

3. Key in Patient's Lab Request Number and press "Enter".

4. Take out one reagent strip from the bottle and dip it briefly and completely into the urine

5. Place it on the test table and ensure that the tip of the strip touches the sensor.

6. Press "Start" on analyzer.

7. Record Results

8. Press "done"

9. Discard the used strip

10. Wipe the test table with paper towel after every test for each urine sample to prevent urine buildup.


Results

Expected results:

Albumin = < 20mg/L (Normal)
20mg/L - 200mg/L (Microalbuminuria)
> 200mg/L (Clinical Albuminuria)

Creatinine = <10mg/dl (Normal)
10 - 300mg/dl (Microalbuminuria)

Albumin-to-Creatinine Ratio = < 30 mg (Normal)
30 - 300mg/g (Microalbuminuria)
> 300 mg/g (Abnormal & Clinical Albuminuria)

Note: We are to print out a worksheet first before we conduct the test (any test for that matter). On the worksheet, in this case, if the urine is normal, we record "Negative" or "Neg". If the urine is abnormal, we record ''Abn".

For Abnormal or highly abnormal urine, the following steps are applied:

a) Pour the urine into a tube
b) Spin down using Cnetrifuge machine
c) Order for quantitative test using Cobas Machine.


Done By:
Rebecca Chew
0703363B