I was being attached Histopathology/Cytology with June, Wing Fat and Desmond. For whole of 20weeks, we are being sent to the different stations of the histopathology laboratoy. For the first month, i am stationed at special staining, after which is cytology then processing next it will be embedding and microtomy and lastly we will take shifts to help in all the stations of the lab.
For special staining, i have learnt how to use and care for H&E (Haematoxylin&Eosin) machine and Ventana special stainer machine. It also includes batch collection and mangement of slides. Im also expected to pick up special histological staining techniques to produce good quality stained sections.
I also read up quality management system, environmental management(ISO 1400I requirements), emergency actions, safe disposal of waste, sources of contamination and suggestions to prevent it from the medical technologists in the lab. Some of these can be found in our LMQA notes. We do have to follow good lab management in order to have quality assurance.
Histopathology Lab Process
1) Customer services (hospitals)
2) Histology lab reception (specimen identification and sorting by medical technologists)
3) Accessioning (numbering and sorting request forms)
4) Tissue Processing
5) Microtomy
6) Special staining/routine H&E stains (Haematoxylin & Eosin)
7) Mounting
8) Sorting and labeling of stained stains
9) Dispatch slides
10) Reporting by pathologist
11) Typing
12) Verification
13) Electronic signed-out
14) Reports dispatched-out
Hot plate
- Up to 30 tissue samples slides can be placed on the hot plate
- Sample slides are placed on the hot plate for at least 3minutes
- Small tissue sample slides must be totally dried before placing onto hot plate so to prevent floating
Routine Auto-Stainer
For routine Haematoxylin & Eosin steps involves:
1. Xylene
2. Xylene
3. Absolute alcohol
4. 95% alcohol
5. 70% alcohol
6. Water
7. Haematoxylin
8. Haematoxylin
9. Water
Ventana automated special stainer
- An automated system that is barcode-driven with optimized protocols
- Helps to increase productivity and efficiency
- Image link: http://www.ventanamed.com/ (NEXES special stain)
- Special stain wash solution is required to spray onto the slides to hydrate the tissue samples to prevent them from drying up as it may affect staining
- Example of special stains carried out: PAS(Periodic Acid Schiff), PASD, GMS(Gomori’s Methenanine Silver Nitrate) for fungus, Re (Retic), Fe (iron), Giemsa, Steiner, Alcian Blue
- Slide tray holds up to 20 test slides
- Reagent tray holds up to 25 reagent
1) Key in barcode labels and protocol(test selected) for the slides that are being used
2) Put slides in slide tray
3) Load reagents required
4) Put back reagent tray and open up all reagents’ cover
5) After which, off preheat as preheat is on before machine is used
6) Then click Run
7) Make sure all criteria of pre-run checklist is met
8) Key in total number of slides being processed
9) Run time is shown
10)When the staining process is done, remove reagents and slides
11)Click sign off
12)Slides ready
This is one of the special stains that is being done manually: Test for tuberculosis (TB)- Ziehl Neelsen
TB test/ Zn : stains for tubercle bacilli head : red
Steps
1) Sections to water
2) Commercial TB colour (5mins)
3) Wash in water (running water)
4) Differentiate with 1% acid alcohol (until colorless/light pink)
5) Wash in water
6) Counterstain with 1% Loeffler’s methylene blue (5-10sec) until sky blue
7) Wash in water and go straight to 95% alcohol to control blue color (if very blue, use 70% alcohol)
8) Dehydrate, clear and mount
Sorting of slides
- 10% of the 30 slides are checked for completeness and if they are satisfactory; if they are unsatisfactory or did not meet the guidelines, they will be sent for re-cut (corrective action)
- Is part of quality check
- This is to safeguard the patients’ interest
- Check ready slides against tissue blocks to ensure that both are from the same source
- Request forms that are sent out with ready slides are to be stamped by Amano Pix 3000 with date and time
This is what i have went through in my first week of SIP and this shall be the end of my first post. I shall update more the next time.
Ang Xiao Si Sharon
0503219H
TG02
17 comments:
Hi ya Sharon,
is there any disavantages of using the Ventana automated special stainer and what are some of the types of tissue samples that requires this special stainer? Thanks ya!
Michelle
Hey sharon...
Just want to ask you to clarify what tubercle bacilli head is and if you stain it, it will turn red?
Thanx, Jo
yoyo~~! wow, reading your post let me recall htech lessons. i'm already quite rusty abt all these.
okay, qn time as u will expect...
1)Principle of stains like PAS, GMS
2)Criteria to check for satisfactory of slides
hope my qn aren't too difficult. take your time to reply! do check my blog for new updates!
Regards,
Chaur Lee TG01
heys sharon...
you say check ready slides against tissue blocks to ensure that both are from the same source. How?
kai lin:)
Hello Sharon!
Have no idea if you've already handled real specimens but I would like to ask..
"In what conditions[environment, pH, fixative?] would these specimens be in for optimal staining to take place and what do you think is the most important factor that would affect staining results?"
Looking forward to your reply!
Thanks,
Alex TG02
Hey hope you are enjoying your time in histology lab.
Anyway I have some queries,
1) Is mounting done manually or is it automated using a machine?
2) Why is the test for tuberculosis done manually and not done with the Ventana automated special stainer like the others?
3) Do you all carry out frozen sectioning for cases that require immediate diagnosis?
Thanks!
Adrian TG01
Hey Michelle,
Here are the disadvantages of Ventana automated special stain machine:
·Expensive to acquire the machine and reagent kits for the different special stains
·When machine break down, will have to do all special stains manually
·Maximum of 20 slides can be run each time
·Some reagent kit such as Giemsa is rarely used and expired, hence causing wastage
Types of tissues that required special stain:
·Usually doctors/pathologists will request for special stains to confirm the presence of disease and make an accurate diagnosis.
·For example, if the doctors/pathologists suspect that patient is suffering from a fungus infection, they will take patient’s tissue to be stained with either GMS fungus and/or PAS haematoxylin.
·Another example would be if the doctors/pathologists suspect that patient is suffering from tuberculosis, they will request for TB/Zn stain.
·While H&E is used for all tissue to be stained and observed under microscope.
-sharon
Hey Jo,
Tubercle bacilli
·It is a microorganism that causes tuberculosis.
·It will be stained red after the TB/Zn stain if there is the presence of it.
·Reagents used: Tb-color carbo reagent, 1% acid alcohol, 1% Loeffler’s methylene blue, 955/70% alcohol
Steps
1.Sections to water
2.Commercial TB color carbo reagent to sections for 5 minutes
3.Wash in running water
4.Differentiate with 1% acid alcohol (until colorless r light pink)
5.Wash in water
6.Counterstain with 1% Loeffler’s methylene blue for 15 seconds
7.Wash in water and go straight into 95% alcohol to control intensity of blue color (if very blue, then use 70% alcohol)
8.Dehydrate, clear and mount
9.Observe positive control slide: red
-sharon
Hey Chaur Lee,
Periodic Acid Schiff (PAS): usually done using Ventana instead of manual
·Function: stains carbohydrate
·Principle: This method depends on production of aldehyde from 1:2 glycol group present in carbohydrate. Carbohydrate oxidized to aldehyde in presence of periodic acid. Aldehyde combined with Schiff’s reagent to form a red dye in situ
Steps:
1.Stain sections with 1% periodic acid for 5 minutes
2.Wash with water
3.Use remaining PAS Schiff’s reagent kit to stain sections for 3 minutes
4.Wash with water for 3 minutes
5.Observe pink colour
6.Stain with haematoxylin for 1 minute
7.Wash with water for 5 minutes
8.Dehydrate, clear and mount
·Control: Mucin positive intestine
·Results: Basement membrane of kidney and other organs- red colour
Most fungi- deep red colour
Glycogen/carbohydrate- reddish pink
Gomori’s Methenamine Silver Nitrate (GMS): usually done using Ventana instead of manual
·Function: Stain for fungus
·Principle: The mucopolysaccharide components of the fungal cell wall are oxidized to release aldehyde groups. The aldehyde groups then react with the silver solution, reducing it to metallic silver, rendering them visible.
Steps
1.Dewax, bring sections to water( Pre-warm working methenamine silver solution at 56 degree Celsius)
2.Oxidise in 5% chromic acid solution for 30 minutes
3.Wash and bleach in 1% aqueous sodium bisulphate until section is colorless
4.Wash in tap water, then to type II water
5.Place in working methenamine silver solution in oven at 56 degree Celsius until section turns yellowish brown
6.Rinse in type II water and tone with 0.02% gold chloride for 1 minute until section turns grayish
7.Wash and fix in 5% aq. Sodium thiosulphate (hypo) for 5 minutes
8.Wash, counterstain with light green colouring solution for 1 minute
9.Wash, dehydrate, clear and mount in DPX
·Control: Any tissue containing fungus, Aspergillosis and penumocystis
·Results: Fungi- sharply delineated in black
Mucin- dark grey to black
Background- green
Criteria to check if the slides are satisfactory (guidelines for assessment of H&E control)
·Nuclear component
ØCrisp: chromatin well defined
ØAdequate: chromatin is still visible, but not well defined
ØInadequate: nucleus not stained/too dark
·Cytoplasm component
ØAdequate: cytoplasm shows 3 shades of Eosin stain
ØInadequate: cytoplasm are weakly stained/not well differentiated
·Nuclear cytoplasm contrast
ØAdequate: provides a good contrast between shades of blue and red
·Dehydrate/clear and mount
ØAdequate: no residual water, no residual alcohol and no bubbles on slide
ØInadequate: any one of the above present on slide
General guidelines to grading of slides
·Expected cells to stain: (0) not stained
(1)weak demonstration of more or equal 25% of cells
(2)good demonstration of more than 75% of the cells
(3)excellent demonstration of cell
·Background : (1) absent
(0)present
·Counterstain : (1) adequate
(0) inadequate
-sharon
Hey Kai Lin,
Check between tissue block in cassette and ready stained slides:
1.Batch number
2.Biopsy number
3.Block number
4.Medical technologist’s initial
5.Tissue orientation, size and shape
6.Test request and request form
7.Number of slides required for test requested
-sharon
Hey Alex,
Optimal Staining
·Tissue must be well fixed ( pH 7 to 7.4)
·H&E (Haematoxylin & Eosin is not really affected by pH)
·Ideal processing time for different tissues (normally processing time for most tissues is 13 hours while gastric tissues is about 2 hours considered as rapid staining because they are fixed at mm/hr0
·Trimming done by pathologists must be of ideal thickness for different tissues
Most Important factor affecting staining?
·There is no most important factor affecting staining because there are generally alot of factors affecting it which are equally important.
·For example, Processing time, trimming thickness, reagents (brand variations), fixation and etc
·Right from the beginning (the very first step) to the last step will contribute to the quality of staining
-sharon
Hey Adrian,
Mounting
·It is done automatically in the lab
TB test
·It is done manually because the lab doesn’t have the Ventana reagent kit for TB stain.
·The steps for the TB stain are generally simple and fast to be carried out manually.
Steps
1.Sections to water
2.Commercial TB color carbo reagent to sections for 5 minutes
3.Wash in running water
4.Differentiate with 1% acid alcohol (until colorless r light pink)
5.Wash in water
6.Counterstain with 1% Loeffler’s methylene blue for 15 seconds
7.Wash in water and go straight into 95% alcohol to control intensity of blue color (if very blue, then use 70% alcohol)
8.Dehydrate, clear and mount
9.Observe positive control slide: red
Frozen sectioning for cases that require immediate diagnosis?
Regarding this queation, i will let you know again. So sorry about that because i have not learn anything about frozen sectioning yet.
-sharon
Well elaborated and thanks for your prompt and concise response!
- Alex :)
Hey Adrian,
Frozen sectioning for cases that require immediate diagnosis·
-Frozen section diagnosis is a means of rapid preliminary diagnosis made on fresh unfixed tissue.
·The rapid diagnosis helps the surgeon to make an intra-operative decision regarding the extent of surgery.
·It allows the margin of the tumor to be examined to see whether the tumor metasized.
·The final diagnosis is dependent on the paraffin blocks and haematoxylin and eosin stain.
·Frozen sectioning alone takes around 5 to 10 minutes. Factors affecting it will be the complicity and size of the tissue and number of tissues required.
·Frozen sectioning lab is usually found in or near the operating theatre.
-sharon
hi sharon!
just wanna know, why is Haematoxylin used twice by the auto staining machine?
Ying Ying
TG01
hi sharon
Ventana automated special stainer.
What other types of specimens can used using the automated stainer?
Vinodhini
TGO1
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