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