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.