5th week of SIP

hello! how's everyone? hope you guys are doing great. coz i'm pretty much enjoying my attachment here( somewhere)..hehe
well it has been 5 weeks since we started our SIP..how time flies..
for the past 5 weeks, i was posted to Microbiology department as well as Haematology, micro being my longest stay for 3 1/2 weeks..
so im going to focus on micro for this entry..

the micro department here is pretty much on its own. it has a place by itself unlike the rest of the department. it is always these same people that will be runnig the tests for the patient. they are like our specialist team. unlike the rest of the department, micro is manually run. they are not automated except for blood culture where we use a machine called BATEC. i will talk about it more as in the later part. basically, there are three sections in micro- processing, reading for urine culture and reading for miscellaneous culture.

in micro, we process the order entry on our own. before entering the patient's demographic information, we will make sure that the name on the request form matches the one labeled on the specimen. then we will enter into the LISS. after which we will do the requested tests. there are many tests that is performed by the medical technologist. hence i will only mentioned a few test here( if i mentioned every single test, i believe you people will get bored reading it). so one of the test is urine culture. urine culture require two agar plates- TSA blood and Cled. we divide the TSA plate into 4 and the Cled into 2. the streaking for the urine test is as follows:

streaking on Cled plate

streaking on TSA blood

the next test is MRSA. for MRSA, we only use one plate which is mannitol. we divide the plate into three and the streaking is a follow:

streaking on Mannitol plate

we also do wound culture. wound culture requires 4 plates- TSA blood, Scandler blood, MacConkey and PEA. the streaking is the same as on mannitol.

apart from the test that requires culturing of specimen, here in micro we also performed test using kits. one of the test that uses kit is the test for S.pneumonia and Legionella in urine specimen. the test is very simple to performed.
1) we use the cotton bud and dip into the urine sample.
2)we place the cotton bud in the kit test.
3)we place three drops of reagent into the hole provided, two drops for Legionella (different reagent for different test)
4)we leave it for 15 mins before reading the kit
5)if only the control line appears, it is negative, we both the control line and the positive line appears means its positive

the next test kit is for the detection of Clostridium difficile toxin A and B.
the test is not that easy as compared to the test earlier. basically there are 4 reagent use in this test- dilute, conjugate, wash buffer and substrate.
1)first we put 500ul of dilute and 1 drop of conjugate into a test tube.
2)then we take about 5mg of stool and mix it with the two reagent. ( there must be no clump of stool presence in the mixture)
3) pipette about 1ml of the mixture into the test kit(the small hole) and wait for 10 minutes
4) when 15 mins is up, pipette 300ul of wash buffer into the kit (the big hole) as well as 2 drops of substrate
5)leave it for 10 mins before reading the result
6) the interpretation of the result is the same as the test mentioned earlier.

*i know pictures will help. but i cant find one on the net that resemble the test kit that i mentioned. i will try to get the pics and post it here ASAP. ok..sorry

as promise, i will now talk about the blood culture process. the blood will come in 'blood bottles',
one has a blue cap and the other is gold cap. it is recommended to send the blood in both bottles. if only one bottle is sent, we can still run the test. so after entering the information into LISS, we will incubate the 'blood bottles' in Batec. Batec is a specialized machine for blood cultures. we have to incubate the 'blood bottles' for 5 days. no bacteria grow after 5 days will indicate a negative results. for positive blood, it will shows the results anytime between the 5 days. if the blood is positive, we will then have to culture them on the appropriate agar plates.

blood bottles


all the cultures process will have to be incubated overnight. reading will only be done the next day.

reading of the plates includes identifying the bacteria that is present. plates where colonies are seen (more than 20 colonies) will have to be followed up with various test for identification purpose.
example of the tests are catalyse,oxidase,staph-plus,indole and many more.
each test have its own specificity for identifying the bacteria.


sutiana
0604651J
TG01

4th week comment-replies

due to the generous amount of responses, i have decided to counter each of them in a post.

THE CODEC 5 said...

Hi,

So far in your attachment, have you met any difficulties? If there is, can you share?

By the way, you mention that if the orientation of the tissue is not done properly, then it will hinder the sectioning. So what happen if this really happens? Does your lab redo it or just leave it as it is? The samples obtained are limited right?

Thanks. That's all.

Xin Yi
TG02

xin yi-
so far, i believe that it has been a pretty smooth-sailing ride on my part. nothing lethal whatsoever. haha. though initially, i had a minor issue on adapting to the working environment and understanding the interpersonal dynamics. but time has truly rectify my concerns.

well, if the large tissue is not properly orientated(meaning not of the same level), the tissue block has to be shaved deeper so that a full face can be achieved. however, if the thinner tissues are not embedded to the paraffin wax at the same level, they will have to be re-blocked. this is because shaving the thinner tissues more will risk losing the patient's tissue.

~immortals~ said...

olla!!!!
aint it abit gruesome to see all those parts of the body??

anyway, yew saed that orientation of the tissues are crucial, such as the orientation of the cyst.

so my question is, why is the cyst wall the main interest?

and why must the cyst wall be at the base of the mold, and not be anywhere else?

mayafirhana
tg02

maya-
haha initially, it put me off a bit. but after a while, i am pretty much accustomed to it. A cyst is an abnormal, sac-like structure within a tissue that can contain fluid, air, or semisolid substance. it can be present at anywhere in the body and varies in size. The outer portion of a cyst is termed the cyst wall. i believe that the cyst wall is the most crucial part to produce a diagnosis. the cells at the wall will show if it is benign or malignant. oh, it has to be at the base of the mold because if you recall embedding right, you will always section the surface of the tissue which is at the base of the mold. do you recall? hehe =)

~immortals~ said...

Hi Tira!!

U mention that the specimens for surgery are sent to the specimen recieving room. How long are they usually kept there before being processed?

Another quest: What are the possible errors that might arise in the embedding process?

opps my name is AmiR!!
TG02


Amir-
hello Amir!! Specimens FROM surgery are sent to the receiving room to be correlated with the forms. subsequently, they are sent to the trimming room. depending on their sizes, they are either trimmed by the pathologists or immediately placed into the cassettes and fixed in formalin. and after that, they are placed in the processor. so the duration from being in the receiving room to being processed varies. larger tissues, longer duration. hmm errors during embedding..when the paraffin wax has not completely cooled down and it is immediately being sectioned, the tissue will break. that's when re-blocking has to be done.

tg01 group 2 said...

Hi Athirah,

Some questions to ask you,

1)You mentioned that the forcep is hot and I understand it is sterile.
Wouldn't the heat from the forcep breaks the tissue?

2)What do you use to press the tissue flat down on the mold and how do you ensure this is done properly?

Thankz!

Han Yang
TG01

hey han yang,
1) oh no. it would not. the temperature of the forceps is about 65-70 degrees celsius. At this temperature, the tissue will not break. The heated tips of the forceps actually helps to prevent the tissue from sticking to the tip and hence prevents carry over which could lead to contamination.

2)we use forceps and a flat-surfaced tip made of pure metal.

BMT said...

Athirah!
Ok I'll bug you with a question. Which I'm sure will be arguably easy for you to answer since you seem to be a pro at Histo already. =P

Anyway, must all tissue samples (like you have mentioned the cyst) be placed at the base, or can they be placed in the center?

Elyana
0606676E
TG01

dear elyn-
it has to be placed at the base so that sectioning can be done efficiently. imagine if it is placed at the center of the mold, the process of sectioning will be prolonged.

~immortals~ said...

miss nurathirah,

You mentioned the use of an electrically heated forceps for the transfer of the tissues into the mold.

I would like to know at which temperature are the forceps heated to, as i believe that a temperature which is too high may damage the tissues.

I would greatly appreciate if you could enlighten me on this matter.

thanks!!!!

fellow med tech,
rusydiana

Rusyyy-
the temperature of the forceps is about 65-70 degree celsius. so far i have not witnessed any tissue damage at this temperature. also the electronic forceps have overheat protection, to ensure that the temperature of the forceps do not go beyond 70 degree celsius.

BMT said...

Hi:) can i ask u if the machine used to fill the wax is the same as the one we have in our sch lab?thanks:) because i am curious how does it fills the wax:)

Rachael
Tg01

hello there rachael-
honestly, i can't remember exactly how the embedding machine in school looks like. haha. well since there are many types of embedding machine. however, i can assure you they consist of the same parts; the wax dispenser, the cold plate, the hot surface, the mold storage area.


i'd appreciate it if you'll correct me if i'm wrong. thanks.

Nurathirah
Tg01

4th week

hello! i've been posted to Histopathology. Honestly, i feel that it has been an intriguing month. i feel blessed to be embarking on this mirthful journey with Ernest and Ying Chee. Being part of the Histopathology Lab has helped paint a picture of what Histopathology is all about. Htech finally makes more sense now. Since i am in a routine lab, i shall focus more about embedding for this week. However these are the events in Histopathology Lab:

1. Specimens from a surgery are sent to the Specimens Receiving room. These specimens are kept in containers filled with formalin. Each specimen is allocated a biopsy number.

2. Specimens are transported into the trimming room. The larger specimens such as breast, colon, liver, lungs are trimmed by pathologists. On the other hand, the smaller specimens are handled by the medical technologists as they do not require trimming of any sort. The medical technologists have to solely place the small tissues into filter papers and subsequently into cassettes. This is to ensure that they are not lost when placed into the tissue processor. The area of interest of the larger specimens are trimmed and placed into cassettes. For example, for a breast specimen, one of the most important area is the lymph nodes. Hence the lymph nodes are extremely sort after by the pathologists. The pathologists will practically be frisking for the lymph nodes as they are rather minute. The cassettes are then shut and placed into a container of formalin. This is done by the medical technologist assisting the pathologist. Also, biopsy number is written on each cassette containing any tissue by the med tech. The med tech must also record the name of the pathologist performing the trim and the number of cassettes used for the case.

3. The container of formalin filled with cassettes will be placed into the tissue processor for 9.5 hours.

4. The tissues are then ready for embedding. This is the part which i would like to discuss in my entry. This is because i have been observing how embedding is done for about a week and ulteriorly i was given the chance to embed the specimens myself. With close supervision, for sure.

a) Depending on the size of the tissue, a suitable mold is chosen.Retrieved July 19, 2008, from,

http://www.leitzmicro.com/website/products.nsf?open&language=english&path=/website/products.nsf/(allids)/77de5e12c4f02672c1256a72004c1348

b) The mold is partially filled with wax. The mold is placed on the cold plate and the tissue is orientated in the mold. This has to be done simultaneously to ensure that the wax does not cool down completely before the tissues are properly orientated. If that happens, the position of tissue in the cooled wax will be uneven and this will hinder smooth-sailing sectioning during microtomy later. The mold is placed on the cold plate to provide a "third hand". When the wax is partially cooled, the tissue will not be floating around. By the way, the tissue is picked up from the cassette and into the mold using an electrically heated forceps. As it is hot, the chances of tiny tissues getting stuck on the forceps are arguably zilch. Henceforth, using those forceps hinders contamination.

Retrieved July 19, 2008, from,

http://www.leitzmicro.com/website/products.nsf?open&language=english&path=/website/products.nsf/(allids)/0215b39eb7698a5641256ab60036fc0f


Even the way tissues are orientated is crucial. Take cyst for example, the cyst wall is the part of interest. Therefore, when embedding, the cyst wall must be at the base of the mold. The senior med tech termed is as "block standing". When embedding, i believe the most important reminder to note is, to ensure that the tissues are pressed flat down. Of course, this has to be performed gently to prevent any smashed tissues as a result. The cover of the cassette is discarded while the base of the cassette is placed on top on the mold. Wax is dispensed for the second time above the cassette.

c) The mold is then placed on the cold plate until the wax is fully cooled. When the wax has wholly cooled down, the tissue block can be removed from the mold easily.




5) The tissue block is sectioned using the microtome.

6) The tissue is placed in the waterbath for fishing.

7) The slide is dried on the hot plate.

8) The slide is loaded into the H&E autostainer.

9) Slides are sent for sorting to be sent to the various pathologists for examination.



Nurathirah
0606561I
Tg01

3rd week of SIP

Name: Hardina Bte Hamzah

Topic: Histology
There are many processes that take place in Histopathology Laboratory.
Firstly, specimens will be sent by the portal assistant. There are steps to follow when receiving the specimens. We need to check the information on the requisition form against specimen bags or bottles to ensure it tallies. The specimens will then be given specific numbers as identification. We must ensure that the specimen bottles or bags are labeled properly to ensure accuracy. Formalin is present in each packaging to preserve the specimens.

The specimen will be inspected by the medical technologist or assistant pathologist after receiving. The gross description of the specimen is recorded. The specific parts of the specimen will be taken and placed in cassettes to be processed. Specimens that have been placed in cassettes will be loaded into a tissue processor. All the reagents needed are present in the tissue processor. For example, reagents like formalin, alcohol, xylene and wax. The specimens will undergo dehydration to remove water and immerse in xylene to remove excess alcohol. Then they will be exposed to hot liquid paraffin turning soft, moist tissues into a sample miscible with paraffin. This process will only be done at the end of the day as it takes 14 hours to finish.
When the tissue processing is done, drain out all the excess wax present in the chamber before taking out the specimens. Embedding is done after tissue processing. Wax is half-filled into embedding mould and the tissue is placed inside. The tissue is pressed down so that all the parts of the tissue will be exposed when it is cut into thin sections. Then, the base of the cassette is placed on top of it and more wax is added. The mould will be put on ice to cool is faster. After it cools, the paraffin block is removed from the mould. After embedding is done, the baskets will be loaded into the tissue processor and flushing will occur. This process will remove the excess wax and clean the baskets.

The paraffin block is cut into thin sections (3µm) by using microtome. We must be very careful when using microtome as the blade is sharp and can injure our fingers. After sectioning, the section is placed into a water bath with the temperature between 43-50°C to allow the section to expand. Then, fishing is done by orienting section properly onto slide. We must maintain the water bath at the correct temperature and free of debris to prevent contamination. The work area has to be cleaned daily to ensure cleanliness.

The slides with the tissue on them will be placed in the oven of temperature between 85-90°C to melt the wax and fix the tissues on the slides. Then, the slides are loaded into the autostainer to undergo H&E staining. Firstly, it will hydrate the tissues (1. Xylene: to remove wax, 2. Alcohol: to clear xylene) and stain with haematoxylin. Then, wash the slides to remove excess haematoxylin. The tissues are then stained with eosin and later undergo dehydration (1. Alcohol: to remove water, 2. Xylene: to clear alcohol). Depex are then placed on the tissues followed by coverslips. This is done by a machine. This process is mainly automated.
These slides are then labeled and re-checked against paraffin blocks to ensure no mistake is done before sending them to pathologist.

Topic: Cytology
Conventional Pap Smear
Pap smear is the smearing of cells from the cervix and staining it with Pap stain. The pathologist will then observe the slides to see if there are any cancerous cells or infection.
Firstly, I will explain on how to obtain the sample and make a smear.
1. Insert a broom-like device into the vagina.
2. Ensure that it comes into contact with the ectocervix.
3. Gently push the device and rotate clockwise for 5 times to ensure that enough cells are collected.
4. The doctor will then smear the cells onto a glass slide thoroughly and spray-fixed the smear before sending to the lab.

However, several problems have been discovered when using the conventional Pap smear.
After the cervical sample has been collected using the broom-like device, the cells will be smeared onto the slide. However, over 80% of collected sample is discarded and higher chances that the cancer cells will not be smeared onto the slide. After staining the slide, it will then be observed under the microscope. Due to the smearing technique, the cells tend to clump together and will have difficulties to identify cells. In addition, as 80% of collected sample is discarded, there will be missing cells which restrict from producing an accurate diagnosis. It will not reflect the patient’s actual condition.
Therefore, a new method is introduced.

Liquid- based Cytology (LBC)
After cervical sample is collected using the broom-like device, the device will be rinsed into a vial containing preservatives. This ensures that 100% of collected sample is present in the vial and no cells are left on the device. This sample will then be processed using a processor. This process is automated. The processor will stir the solution in the vial to disperse the cells. Cell will be collected onto a filter by suction. Cells will then be transferred onto a slide by making the filter come into contact with the slide. This will produce a thin layer of cells and minimize the clumping of cells. With this, it will allow the pathologist to detect any abnormalities.

2nd week of SIP

Name: Nurdyana Abdul Rahman
Topic: CELL-DYN RUBY

Picture taken from: http://www.abbottdiagnostics.com/Products/Instruments_by_Platform/default.cfm?system=cell-dyn&suffix=ruby

HEY people!How's your SIP?I hope its going great aite. As for me, I was attached to the Haematology Lab for the first 2 weeks. Most of the tests there are done by analysers which makes the job of a Medical Technologist much easier. However, Medical Technologist is still responsible for the proper sorting out of the samples and maintaining the machinery so that the result obtain would be accurate. Here is one of the analyser that I use to perform Full Blood Count(FBC).

CELL-DYN RUBY

The CELL-DYN RUBY is use to perform full blood count and retics. It uses the optical technology which is Multi-Angle-Polarized Scatter Separation (MAPSS). It helps to differentiate the different white blood cells and at the same time also measure the full blood count such as MCT, MCV, Hb, WBC, RBC and etc.

Upon receiving EDTA blood sample, checking of clot in the blood is done manually before the EDTA sample is placed into the CELL-DYN RUBY. EDTA sample is checked to see if clot is present because in presence of clot, it will affect the overall full blood count. If blood is found to be clotted, the sample is rejected and a request for a new EDTA blood sample is done. Sometimes, blood are clotted in EDTA tube because it has not been mixed thoroughly. Wherelse for sample that are not clotted, they are placed into a rack which will then enter the CELL-DYN RUBY. Results will directly be uploaded into the LISS.

As for retics, the amount of reticulocytes are counted. The test is slightly different from FBC. It requires prior preparation before loading it in the analyser. 20microlitres of the sample is added to the reticulocyte reagent tube and mixed well. It is then incubated before it is run. Incubation should be between the range of 15minutes to 2hours.

In the laboratory that I am attach to, there are two of the same analysers. Which means there are 2 CELL-DYN RUBY. Two analysers are use together at the same time so to ensure that the turnaround time is shorter. Maintainence of analyser are done once a week. Since there are two analysers running at the same time, correlation is done twice daily. Once in the morning and the other in the evening. Correlation is done by using the same EDTA sample and running it in both machine. Since it is the same EDTA sample, both machine will have to produce similar results to indicate that they are working properly and according to standards. This is also part of the maintainence of the machine. Autovalidation is also done. Every 10 samples, a random sample is collected and result of that random sample is printed form LISS. Peripheral blood film is done and stained. It is then viewed under microscope to manually count and observe the cells. This is to ensure that the analyser is working well.

I hope with this information you guys have learnt and now know another brilliantly invented analyser that makes life of a Medical Technologist easier. Good Luck for your coming weeks!!!

Peace,
Dyana.