See under its frozen section technique tab.
Tuesday, May 24, 2011
Tuesday, May 10, 2011
Hospital Histology
Hospital Histology by Histo-Tech
Author: Ang Sen Hong
May 2011
Foreword
Hi, most of the time, Histology books were written by doctors. So I guess I have to represent from a technician view especially on hands on practical area of Histology which I exprienced over four different hospitals. Hope you all will like this book and I will keep it short and simple, as like all technicians we usually do not like lengthy passages.
Contents:
Introduction
Chapter 1: Specimen reception
Chapter 2: Grossing and sawing specimen
Chapter 3: Decal
Chapter 4: fixation
Chapter 5: Tissue processing and its machine
Chapter 6: Embedding
Chapter 7: Sectioning
Chapter 8: Frozen section
Chapter 9: Slide sending and writing
Chapter 10: Machinery
Chapter 11: Immunostaining
Chapter 12: Special stain
Chapter 13: Other techniques
Chapter 14: Useful habits and ideas
Chapter 15: Work politics
Introduction
This book will mainly talk about Histology in a hospital setting and assumes anyone who reads it already had some histology background so as not to waste tme explaining. It will hopefully share some new ideas or interesting technics used in Histology lab. Some of the info are also from books that I read before like Carson and Bancroft and also info from histonet.org regular discussion forums. It will start off from specimen reception to tissue processing to slide making and end with some comments.
Chapter 1: Specimen Reception
Actually most specimen reception procedures is quite standardized. The goal at this point is to receive specimen correctly according to patient data (two identifiers; name and identitity number), ensure specimen in bottle and receive at the shortest time if possible as is the rate limiting step.
To ensure speed, a few question need to be asked:
1) Is the workspace organised?
2) Is there storage (example: boxes or basket) to keep extraordinary number of specimens?
3) Is the reception procedure simple or choke by too many rules or procedures?
4) Is the computer system user friendly (usually cannot be help, but try asking the computer technical personnel to see if customization can be done). eg: computer response speed to go to next screen.
5) The last question is a test of the reception power that is will the reception area be able to cope or able to clear the specimen in a short time with influx of extraordinary number of specimen especially with limited staff.
Hopefully, specimen reception is straightforward and not lenghty in procedure and the staff is not over multi-tasked (example needed to handle phone calls and do extra billing work) to increase speed, reduce mistake and reduce stress in staff (that it is not particularly over energy draining). Or simply employ more staff to solve the problem.
Specimen reception is seemly unimportant but in truth it is the first station to scan for specimen mixup, mistakes or even specimen loss (which hopefully by God's grace that it will not happen) and is the rate limiting factor to how fast the specimen can be process.
Lastly and still firstly, at this specimen reception first station, clear up any clerical error example wrong specimen, wrong orientation like right side vs left side and other errors before specimen acession into computer system.
Chapter 2: Grossing and sawing of specimen
Grossing of specimen protocol would depend on each individual lab. But as rule of the thumb. cut in a way that the next technician would know how to embed and how much to trim after embedding. In other words, gross in a way with respect to the technical factors that can affect the final outcome. Thus, inform the tech at the next step if special orientation embedding and or minimal trimming after embedding or trim till point of interest after embedding. In addition gross what is needed to be seen to conclude the clinician diagnosis. For example you would not sample an appendix suface only, but rather take its lumen as well to give a full detailed pathological examination. In addition, only one section is needed to conclude the diagnosis eg: white blood cells and inflammation seen microscopically. Thus no need to gross so many section.
Tissue slice thickness should be 3mm to 4mm. (too thick leads to poor tissue processing).
Fix the tissue overnight to see tumour as fixation impart colour, to harden lymph nodes and harden tissue to cut.
For minute biopsies, should be inked. Especially when tissue is white in colour as the embedding wax is also white in colour would make it difficult to see during trimming and sectioning.
With regards to sawing with a electrical saw: The sawing teeth should at least 3 teeth to the sawing suface area. But at times not generally always so. So hold small specimen tight when sawing small specimen. For big specimen, can saw one half, and use a support (such as wooden block or even small unwanted specimen bottle) to hold the sawn surface and other hand to hold the specimen to saw. With confidence and experience, can use both hands to hold the specimen.
The thickness should be 3.5mm so as not to be too thick for decal and trimming/sectioning.
With regards to sawing small specimens, we can place a piece of paper underneath the specimen to support it and saw together.
Lastly, you can thin down the specimen slice by using the electric saw to file out one surface to thin it down.
Alternatively, you can decal the slice and slice it thinner after decal.
Last point to say, sawing is dangerous. But it is also a skill. How good you can saw depend on level of confidence and trying. As rule of the thumb, do not go beyond level of your confidence. Save your fingers today and you will live to saw another day.
Lastly, check correct cassette label before dumping cassette block into formalin after grosiing.
Chapter 3: Decal
Usually for immunostaining, EDTA decal works best than strong acids. Inadditional, EDTA only bind calcium at higher pH at 7-8.
If decal with strong acids, do not decal more than 48 hours in order to preserve nuclear staining and prevent too much tissue loss (as tissue fragmentate off in the strong acid decal solution).
Lastly, fix all tissue before decal in order to protect the tissue during decal, as decal acids can destroy unfix tissue and cause tissue to fragment over time (even in fixed tissue)
Chapter 4: Fixation
The most critical step to all histology work.
Without proper fixation, lead to;
1) antigen loss for immunostain (even though formalin fixation cause cross linking in protein that results the need for antigen retrieval in immunostaining, is more important to retain the antigen for staining than to lose it. Anyway, the cross linking is not a problem in the first place since can be recovered by antigen retrieval and the various antigen retrieval methods that have evolved today makes it nearly 100% possible for immunostaining and modern technology of immunostaining has antigen retrieval incorporated and removed the need of manual and at times unconsistent manual antigen retrieval).
2) poor tissue processing that cause tissue unable to section or section float off slide when staining and other poor tissue processing effects (like morphology preservation).
Reason being fixation fix the antigen and cross-link tissue protein to enable subsequent reagents(alcohol, xylene and wax in tissue processing) to flow into tissue during tisssue processing for proper dehydration, clearing and wax impregnation.
An important factor to take note is the use of heat in formalin. As heat can stop autolysis enymes, heat can both fix tissue via coagulation and increase chemical cross linking reaction by formalin. But temperature more than 68degree celsius cause pyknotic nucleus. By research, a 20min 55 degree celsius formalin fixation is better than a 2 day formalin room temperature fixation. So recommended temperature for formalin fixation is 45 to 55degree celsius.
Other facts about heat in fixation and formalin fixation as follows;
-formalin reacts with DNA at 65degree celsius and 45degree celsius with RNA, while at room temperature only physical entrapment with about 30% loss.
-Acidic fixative remove DNA and RNA.
-formalin fixation takes about 7 days to complete fixation.
-formalin is the standard fixative used in the world currently at 2011as it is still relative compatible with immunstaining, special stain and is a relatively well fixative without much disadvantages than its advantages compared to other fixatives.
-Heat or physical fixation is irreversible, so beware of over cooking the tissue (causing pynokotic nucleus).
-Brain tissue can fix with 15% formalin
About microwave fixation, it can increase staining but increase background with silver stains and immunostains.
Chapter 5: Tissue processing
Usually the only 3 main factors affecting tissue processing is;
1) Fixation (are tissue fixed before processing)
2) Tissue thickness
3) Is reagents in sequence and reagent quality.
Just a few pointers to mention;
1) usually can process the biopsies with the larger tissue slice. Should not have any problems of over dehydration with alcohol in biopsies processing as I presume most duration in each alchol reagents only up to one hour only.
2) usually the first step of processing is formalin reagent to complete fixation. The second reagent can alcoholic formalin (70% alcohol 4% formalin) to ensure fixation and start dehydration as I seen some lab still use normal bufferred formalin for second reagent. As a common problem in labs is under dehydration by alcohols.
3)Alcohol and xylene can age with time, but usually not a problem with enclosed tissue processor, as reagent is not expose to air moisture. (xylene that had aged would usually turn red orangy in colour and alcohol that had aged usually appear the same but tissue sectioning would reflect tissue appears to be soft in sectioning or reagent appeared coloured from repeated use in tissue processing).
4) use the right type of ethanol as certain additives in ethanol can cause colour fading in staining.
5) for processing fatty tissue slice, tissue must not be too thick, preferrably 3mm and well fixed before processing. In additional, an optional step to include a half alcohol and half xylene reagent in the middle of the dehydration reagents in processing to remove some fat content in the tissue for proper processing.
6) Lastly, always remember the 4 main aims of tissue processing is morphology fixation, staining and sectioning and section stick to slide (poor tissue processed section do not stick well).
7) Can use paper to wrap the tissue to flatten tissue.
8) Reagents have to be in sequence otherwise tissue may be destroyed. (tissue cannot adhere to slide and morphology unclear)
Steps in the tissue processing with above points incoperated
1. Neutral buffered formalin 45min
2. 70% alcohol and 4% formalin
3. 100% alcohol
4. 100% alcohol
5. 100% alcohol
6. 100% alcohol (50% alcohol and 50% xylene optional)
7. 100% alcohol
8. xylene (can be replaced with isopropanol)
9. xylene (can be replaced with isopropanol)
10. xylene
11. wax
12. wax
Lastly, certain tissue processor machines come with rapid heating or microwave and shown to process tissue still unfixed or fresh specimen without need to pre-heat the tissue cassettes in heated formalin first. This ease the work and prevent fumes from heated formalin. Inaddition, can even process biopsies and large tissue together in one process and even process fat tissue perfectly. Indeed they work. However, there are also some failures, as by exprience encounter, there was tissue processing of fat that was underprocessed and shrinkage. Thus make sure you evaluate the machine with various tissue (fat especially, soft tissues and other tissues) before purchase.
Chapter 6: Embedding
Embedding is the key to sectioning. But firstly, tissue have to be embedded in right manner to show the pathology. Secondly, is to embed the tissue in the right orientation for ease in sectioning.
Just a few points for embedding the tissue in the right orientation for ease in sectioning;
1) for bony core or strip embed diagonally (reason being if there is a calcified focus on tissue, it will cause a nick on blade and subsequent cause a tear in the rest of the tissue section if embedded vertically.
2) for tissue with both solid and fat tissue, embed in a way where the blade will first cut the solid area first so as to be able to pick the section first during sectioning. As is often not so feastible to able to section out the fatty area.
3) ensure wax surround the tissue for ease of sectioning.
Next I like to discuss is there are two methods of embedding which I know, namely cold embedding and hot embedding.
Firstly, cold embedding is the normal method to pour molted wax on the metal embedding mould. Subsequently, the wax solidify at the base of the mould as the mould is cold (room temperature) and the tissue is pressed into the soft wax base. This method is fast and relatively good in quality embedding.
Secondly, hot embedding uses a hot mould where the molten wax is pour onto it and the tissue is put into the mould with liquid wax. Next the mould is transferred to a cold surface (such as a cold plate, preferrably with some water on cold plate to maximise contact for heat transfer away from mould and increase speed), which in turn cause a layer of wax to form at the base of the mould for embedding (pressing tissue onto the wax base). This method is useful to orientate multiple tissue pieces and also useful for multiple tiny biopsies or tissue cores, as all tissue pieces could be embedded in the same plane of level. To my opinion, it gives the best quality but is not as rapid as the cold embedding.
So usually I use mainly the cold embedding as speed is more or a concern and hot embedding only on tiny biopsies or cores.
Another pointer is if the core biopsy is crooked, you can put in on the hotplate surface to flatten it.
Lastly, only open one tissue cassette a time to ensure zero mix up in tissue embedding.
Chapter 7: Sectioning
In this modern day, I guess everyone is using rotary microtome and commerical disposal blades, thus what I going to write is of such basis only.
Sectioning, the main skill of the histotech. Can be taught but mastered through learning only.
Sectioning factors include the following:
1) Clearance angle
2) Tissue type
3) Section thickness
4) sharpness and bevel angle of blade
5) Wax hardness which is related to its temperature.
Firstly, clearance angle. Too much tilt cause thick thin section or cause section to lift up from blade when block moves up from its rotary microtome. Too great tilt cause microvibration, undulation and difficulty to ribbon. Usually the clearance angle is adjusted in accordance to the bevel angle of the blade through trial and error.Thus different brands of blades got its own bevel angle that give rise to its own unique clearance angle in the microtome. So once you found the clearance angle, stick to the angle. Typically, the clearance angle is 7.5. You can try from 3 to 8 for sectioning or ribboning but usually most technician just stick to one clearance angle and they do treatment to the tissue blocks such as surface decal and other methods that will be mentioned later.
Secondly, is tissue type. Hard fibrous tissue example fibroids, cervix and others, can use water or warm water to soften the tissue after block trimming. If microvibration occur during sectioning (giving undulated thick and thin section), section 1um thicker to solve as section thicker give stronger section force. Blood can also use water or warm water to soften the tissue after block trimming to give complete section as blood tend to crumple on sectioning. Calcified tissue like bone can do surface decal. Other tissues that may contain calcium or crystal salts affecting sectioning that require surface decal include prostate, gall bladder, skin and thyroid.
Thirdly, is section thickeness. On general, is easier to cut thicker than thinner section. But is usually more easier to ribbon with thinner section. Always remember is better to section thicker to get a complete section than thinner but incomplete.
Fourthly, sharpness and bevel angle of blade. On general any difficult tissue can be section off with a new blade (own comments; with at times, especially very hard tissue like bone or fibrous tissue, can use a less sharp blade to cut as the sharpness of blade can form too extreme pressure at cut surface causing ripping tissue off block than a less sharp blade that "gives up" its cut to the points of extreme hardness in tissue).
Fiftly, the wax brand can affect sectioning due to its wax contents and physcial property (melting point). Use the right brand that allows tissue processing, sectioning and ribboning. Temperature affect hardness of block. The colder the harder but too cold cause the block to crack or brittle. The harder the wax block, the easier to section but lesser to ribbon. So for fatty tissue block would cut at colder temperture that can be achieved by putting on cold plate longer or put into the trough of cold plate (which some cold plate have) which is colder but more importantly, make sure well tissue processing for ease of fat sectioning.
Now I like to talk about difficult tissue to section, namely; skin, blood, calcified and fatty tissue as they usually do not stick well to slide after sectioning. For the above mentioned, we could use polylsine coated slide to adhere section and heat longer (at least 20 min) for slide drying for section adherence.
More so for sectioning, we can use water to soften (or warm water from hotbath that increase speed but beware not to over soak up the tissue with water) for hard tissue and blood or use diluted fabric softner for hard tissue or blood. The key is to mainly impart water to soften the tissue. Can use suface decal on skin and calcified tissue to remove calcium that cause tissue section to float off from slide. For bone tissue can use surface decal, followed by water to soften further for sectioning. For fatty tissue, we can cut thicker even up to 10um and lower the temperature further by placing longer on coldplate or even better in the through of coldplate that is even colder (if that coldplate model has through). Usually for fat tissue requires to section faster (turn microtome wheel faster) or at times normal speed will do. Can do "double cut" if necessary.
"Double cut" means to lower the block to blade till a bit of wax is section and reverse the microtome wheel to lift up the mircotome head and fully turn the wheel to section (This step inevitably increase thickness to nearly X2). The first step acts as a resistance to allow section to form rather than curl up. Alternatively, we can hold the bit of wax with forceps and fully wheel turn to section to allow section to form.
Treatment to cutting different tissue
Thyroid (treat against its colloid material) - use surface decal or water or warm water.
Gall bladder and Prostate (due to bile pigments and salts in prostate) - use surface decal.
Bone, fat, skin and blood as discussed above.
Foreign object like clips and sutres - please remove with help of pincer after block trimming or remove before embedding.
Other Pointers;
-Actually, I should have said this in first place. The 3 essential to secctioning is 1)Fixation 2)Tissue processing 3)Embedding.
-To prevent section floating (especially biopies) when drying slide, the best solution is to heat in oven with slide held vertically by rack to allow water to flow out when drying, or next solution is to heat slide with paper in between the slide and hotplate for gentle heating and later transfer to hotplate for completeness in drying the slide.
-After surface decal, can still use water or fabric softener to soften tissue further.
-take note of wind or air flow during sectioning. It can help you in floating your section or disturb you if blowing in wrong direction.
-How good your section is via whether the section produce is smooth and uniform when section out when you see visibly.
-The less stroke you made, the longer your blade will last and faster to get section. (tip: cut a bit more to gain full face during sectioning).
-Cut biopsy then soft tssue, then hard tissue , then calcified tissue, to let blade last longer.
-Extend floating the section on hotwater bath can allow less folds (especially tissue with different tissue zones expand at different rate or circular tissue tubes).
Note: not to prolong floating as can cause tissue to expand beyond actual size.
-At times, unable to ribbon because of wax accumulated behind the blade
-Shave too aggressive can cause microscopic holes especially in very cellular tissue like tonsil and "crispy" tissue after tissue processing like thyroid. Solution is to shave less aggressive, and after shaving, use fine wheel to cut 3 to 5 revolutions (maximum at 20um). If still occur, treat blocks with water or diluted fabric softner or even decal solution to soften the tissue.
-Static electricilty in hands. Solution, just wash hands with water.
-Tissue too fibrous causing microvibration. Solution; can either simply cut thicker or treat blocks with water or diluted fabric softner or even decal solution to soften the tissue.
-common errors causing difficultly in sectioning like dull blade, block too cold/hard affect ribbonning, tissue too hard, loose parts on microtome, microtome wheel turn to fast, blade gum with wax or dirt after repeated sectioning.
-To ribbon; can section thinner, use a new blade, scratch the underneath of block (bottom picture at start of webpage) to disrupt surface to enable ribboning and avoid block too cold/hard and lastly if needed, adjust clearance angle (not too steep angle).
-Some microtome have ribbon knob button at its back. If cannot ribbon, try seeing if the knob button is activated.
- To section; can cut thicker, use a new blade, adjust block hardness via temperture.
-If section curve to one side due to uneven tissue consistency, can ignore it if still can produce full section. Otherwise turn block 90 degree to cut.
(2nd bottom picture at start of webpage)
-Chatter artefact cause by block too hard (too cold) or tissue over dehydrated (soak in water to slove).
-The block to microtome holder position should be the acute angle labeled tip pointing upwards for 1)better hold of block 2)gives a more fixed position when replaced into microtome holder and so less cuts to reach full face.
(2nd picture at start of webpage)
-Wax trap in between the pressure plate of microtome cause blade unable to secure properly causing jagging when sectioning.
-Wax trap in block holder can cause the holder to hold block loosely.
-Own Comments not verified: At times, sharp new blade may not be suitable for cutting hard tissue as the pressure cause by sharp blade is too much, causing to dig out the tissue.
Chapter 8: Frozen section
Basically, frozen section is microtomy. So I would just share a few pointers unique to cryostat microtomy.
-Use maximum clearance angle for sectioning.
-Freeze tissue according to its water content. The more water tissue hold, the more brittle ("crispy") tissue get if freeze longer. Eg: brain, thyroid, parotid and other tissue. Usually freeze lesser and allow block to feeze slowly in the cryostat.
But if we overfreeze, we can use our finger thumb to warm up the cut surface. However, in the first place do not overfreeze. (-15 to 23 degree celsius).
-Freeze longer or use a lower temperature (-18 to -30 degree celsius) for fat tissue. Can 1) cut thicker, even up to 20um ( Always remember is better to section thicker to get a complete section than thinner but incomplete) 2)use antiroll plate to asist sectioning 3)if cannot section, leave the whole block in crystat till fat tissue reaches lower temperature through out the tissue or freeze more with liquid nitrogen to section
-Can also double cut."Double cut" means to lower the block to blade till a bit of media(OCT compund) is section and reverse the microtome wheel to lift up the mircotome head and fully turn the wheel to section (This step inevitably increase thickness to nearly X2). The first step acts as a resistance to allow section to form rather than curl up. Alternatively, we can hold the bit of wax with forceps and fully wheel turn to section to allow section to form.
Can also use the antiroll plate to do double cut. First lower the block to blade till a bit of media(OCT compund) is section and seen in the antiroll palte. Next reverse the microtome wheel to lift up the mircotome head and fully turn the wheel to section (This step inevitably increase thickness to nearly X2). The first step acts as a resistance to allow section to form rather than curl up. Alternatively, we can hold the bit of wax with forceps and fully wheel turn to section to allow section to form.
-Usually for fatty tissue section fast and harder consistensy tissue section slowly. But at times, tissue may consist of mixture of soft and hard and fatty contents, when section slowly gives a friable section crumbling but ironically gives a complete section when section fast. So try all speed type to section if the logic fails. Irony happens.
-Cut at 7um or higher to stain well. Ironically, at lower thickness, nuclear pattern may not be stained properly.
-Similarly to embedding, encirle the tissue completely with OCT compound for sectioning ease.
-Can do repair work by adding OCT to already form block or even after trimming the blocks to 1)fully encircle the tissue and 2)glue back the tissue if tissue fall off from metal chuck.
-know your instrument, the cryostat heat extractor can be reomove to touch the block if not freeze properly.
-Avoid cryospray as 1)can cause block to crack and 2)aersol created can be a source for infectious bacteria especially Tuberculosis.
-Use 70% alcohol to clean cryostat. Is bacteria-cidal even for TB.
-If tissue section tend to float off that usually happens to fatty tissue or tissue extracted by cauterisation. Solution: 1)Use coated slide or 2) soak the slide in 95% alcohol for 30second, rehydrate in water and stain or 3)air-dry the slide first before staining. Note air dry may change morphology.
Chapter 9: Slide sending and writing
There has always been a debate as in whether to pre label the slides or label the slides immediately after the section is taken from block. In both methods, there are still incidences of wrong labelling. But I have to agree with the histonet forum as it is the responsibility of the technician to take the right slide or ensure the label is correct to the slide before staining or submission of slides.
One pointer is that you can sort the slides before staining to speed up the process. Even better, one can cut via cases, stain the slides in order and these in turn make slide sorting and labeling with ease and faster.
Chapter 10: Machinery
Machinery have proved to reduce time and reduce work for the technician in many ways; from tissue processing to staining to coverslipping, to special stains and immunostains. To even technolgy advance like rapid heating tissue processor to enable fresh tissue and fat tissue to be process and reduce processing time by 50%.
A few pointers I like to point out;
- we can learn the principles behind the machine to find out how to troubleshoot (eg: increase stain time) or use correctly (for example increasing the power of a home microwave does not increase its power but just increases its microwave output frequent).
- we can learn from the machine engineers to learn how to trouble shoot the machine in order to save time (as take time to ask engineer to come).
- we can ask the product specalist to troubleshoot for us. (own comment: there was once my product specialist incorporated a new staining protopcol to enable my immunostain which I not known of).
Chapter 11: Immunostaining
With advance of immunostaining machine that carry out antigen retrieval and immunostaining, so I will discuss mainly on machine immunostain.
Here are some points to take note on immunostaining
-The key to all staining is reproducibility. Obviously, machine stain is better than manual stain esp immunostain machine that do the whole process that include antigen retrieval.
-Do not be surprise that there may be a different protocol for certain antibody staining that can be reveal by the vendor. So contact them if no staining to troubleshoot.
-Antigen retrieval solution important factors are pH and composition. Temperature in antigen retrieval is lesser importance. So if one antigen retrieval solution is not working optimumly, use another pH solution to optimize.
-Limit formalin fixation up to 24hours but theorically it takes 8 days to fix completely by formalin. Furthermore, with antigen retrieval, immunostaining can be done. But just for precaution not to fix tissue for too long.
-Alkaine buffer antigen retrieval cause tissue to float off. So can try 1)use better coated slide like "super-frost" slides 2)use citrate buffer or acid pH buffer antigen retrieval or 3)heat slide at 70 degree celsius for better section adherance to slide (dry slide in oven with slide place vertical so that water can flow out from between section and slide while drying, this gice better adherance). Can dry slide 15minute or overnight. Longer the better. (The high temperature for drying slide should not affect the antigen reason being is being subjected to higher temperature in antigen retrieval).
The last method is to cut thinner at 3um as the floating part may just tear off if it float off during staining and not affect rest of tissue hopefully.
Note: immunostain thickness can be from 2um to 5um.
-Necrosis in tissue cause unspecific staining, this is unavoidable.
- weak antibody can extend timing in antibody-antigen incubation step to enhance staining, up to overnight incubation.
-Heat fixation (formalin) does not affect immunostain at temperature up to 55 degree celsius. Less than 45 degree celsius fixation may lead to poor tissue processing. More than 65 degree celsius can cause vacuolation in cytoplasm and overstain cytoplasm and pyknotic nucleus. So suggested 20min in 55 degree celsius fixation.
-Use EDTA as choice of decal for tissue that require immunostain. Normal decal solution can be used but may cause varied immunostain results.
-If tissue contain too much blood (source of endogenous peroxidase), can incubate slide with hydrogen peroxide or lenghten the hydrogen peroxide step time to ensure endogenous peroxidase is destroyed.
-If background problem, can use a different antigen retrieval to see if helps.
-Control can be taken via needle extraction of old patient blocks if needed.
-section on slide should be in the middle as at the ends of slide is not stain well in machine.
-Antibody suitable from one vendor machine may not be suitable for another vendor machine due to incubation temperature, timing and antigen retrieval buffer difference. May need to optimise the parameters or ask your vendor to troubleshoot or get another brand of antibody.
-Manual heat antigen retrieval; wait till temperature cool near room temperature before transfer to water as slide dry up in seconds if too hot , causing non-staining.
Chapter 12: Special stain
Mainly I will discuss on special stain factors and pointers rather than machine special stain as currently there is a limit to the number of type of special stains machine offer and is not profitable to run exotic stains of only a few cases.
Factors;
-1)fixative effects 2)primary dye properties and mode of staining action 3)Desired result 4)Possible errors in procedure 5)tissue itself. Different tissue stain differently.
6)pH 7)temperature 8)dye concentration 9)salt in dye that can increase or decrease staining intensity
Technical variable skills
Due to various factors that can affect staining, a set of knowledge (which i term "Technical variable skills") is needed to undo effects on staining or even modify staining to desired result.
Things to consider include;
-fixative used for fixation.
-fixation if prolong (months), under, inproper type of fixative will affect staining.
Note: fixation can imparts chemical groups to tissue to enhance staining. Prolong fixation with formalin, saturates with formaldehyde groups causing weak staining.
-primary dye their purpose, mechanism of staining and its properties.
-Desired stain results.
-Sources of errors and precautions in staining and corrective actions.
-staining depends on 1)affinity of dye to tissue, 2)rate of loss (eg: during differentiation, loss in subsequent stain reagents steps and dehydration).
-Basic dye more soluble in alcohol (So dehydrate fast or air dry to dehydrate). Acid dye less soluble in alcohol.
-Section thickness (thin section stain faster, thick section stain longer but stain darker)(For detection substance of interest staining, thicker section increase sensitivity but decrease in specificity as morphology is vague in thicker section). eg: too thick is difficult to differentiate Gram positive and negative bacteria.
-Decolourisation best result is by dipping in reagent in coplin jar instead of flooding the reagent horizontally on slide.
-older blocks section may need longer to stain, as formalin deteriorate with time and become acidic.
-can change counterstain if desired or even no counterstain.
-counterstain colour may change the colour of primary stain eg: blue with yellow dye gives green colour.
-alkaline solution tend to cause tissue to float off.
-manual vs machine stain; because different tissue stain differently so with manual stain can stain at different timing to desired intensity. If tissue section tend to float off, can stain manually for more care. For machine stain, it stain more consistent or reproducible results.
Remember different tissue stain differently, so adjust stain time accordingly if needed.
-Heat slide longer on hot plate (at least 20min or more) before dewaxing for staining to prevent tissue section float off.
-Can use coated polylysine slide to prevent section float off.
-Differentiation to desired colour state in terms of 1)substance of interest 2)background 3)constrast
-Stain solution that has precipited means stain has deteriorated.
-Uneven staining can be solved by using dilute stain and stain longer and over time, different tissue parts will stain differently via its own affinity for the stain.
-air dry the slide as an alternative to dehydration especially if section is partially floating off. But once dried, may not be able to restain again.
-timing is critical in certain special stain. eg: minimum time in iron alum step of retic stain to ensure sensitize of tissue, maximum time in oxidization step of special stain to prevent over oxidisation.
-Even embedding media can affect staining (rarely).
-old reagent can cause poor staining except for some reagent that need to aged, eg: PTAH reagent.
-Dye defective or impurity can affect staining.
-Silver stains are tricky generally. (quite a number of factors involved that will be discussed later).
-improper dye storage lead to decomposition and affect staining. (especially if dye powder colour has changed).
-Stain precipitation may mean stain deterioration.
-weak staining due to loss of substance of interest eg1)calcium loss in fixative or eg2)modified substance cannot stain.
-unexpected staining by depoosits. Eg: carbonate deposits stain in Feulgen nuclear stain.
-Other problems like tissue loss in GMS due to overheating and black deposits in Von Kossa due to contaimnated glassware.
-Remember to press the timer,
Differentiating principles
>basic dye use acid solution to differentiate and vice versa.
>use mordant solution for differentiation
>Solubility, example eosin can be differentiated with high water content % alcohol. (eosin is soluble in water).
>oxidiser solution to oxidise the colour. (not really use to differentiate. Usually to bleach colour)
>Can water, alcohol, weak acid like 1% acetic acid, acid like 3% sulphuric acid and acid alcohol to differentiate of strenght from weakest to strongest respectively.
Note: At times actic acid can fix the stain than differentiate it, eg:light green.
More Pointers;
Silver stains and microwave staining tips
-Tissue pathology affect staining eg: unavoidable background in necrosis in silver stain
-Microwave staining tend to cause background.
-Microwave factors -1)power 2)time 3)volume of liquid to microwave. Note: increasing household microwave power just increases microwave frequency and not its power level.
-microwave staining is harder to control than oven but faster.
-check slide regularly in microwave silver staining to desired intensity.
-Adjust microwave timing accordingly as different household microwave is of different power each.
-silver stain break fown when overheat, causing non specific staining, ie. background. Ideal temperature is 40 to 50 degree celsius but is impractical as gives a long staining time.
-is ok to overstain but not to point of obsuring.
-washing thoroughly in silver stain procedure is important to prevent background.
-red blood cells can stain up with silver stain.
-waterbath is faster than oven for heating. But fastest is still microwave.
-dip slide up and down in microwave staining to ensure temperature evenesss.
-all argentaffin substance is reducing and similarly, all reducing substance is argentaffin.
-alkaline silverr solution tend to cause tissue to float off.
-Silver stain solution can break down over time causing background and decrease staining power and so may need to stain longer.
-Polylysine coated slide do not cause background.
-Gold chloride and sodium thiosulphate step in most silver stain will decrease the silver stain a bit.
-Try not to use tap water to wash as phosphate in tap water can react in silver stain.
Sodium thiosulphate as reagent can 1)remove iodine 2)remove unreacted silver and 3)fix reduced silver.
Retic
-Iron alum step is critical step. Do not decrease this step timing as iron alum used to sensitize tissue for silver staining.
-Wilder Silver solution must have turbid for complete silver impregnation for staining.
-the wash step after silver stain is important to reduce background.
-Troubleshoot no staining with fresh newly prepare reagents.
Masson Fontana
-desired result should be argentaffin granules dark brown and clear background. If there is background means is overstained.
Metachromatic dye eg: Tol Blue
-factors afftecting staining due to its basic dye 1)pH 2)dye concentration 3)temperature.
-No alcohol dehydration as can lose stain.
Hematoxylin and eosin staining
-Hematoxylin by adding acid to it gives more specific staining but lenghten staining time.
-"Blueing" (washing in slight alkaline solution) is to convert soluble reddish purple hematoxylin to insoluble blueish purple hematoxylin.
-To restore hematoxylin basophilia in tissue like over decal tissue 1)can use iron hematoxylin or 2) treat slides win 5%periodic acid for 30minute and wash before staining.
-care not to agitate in ammonia (alkaline) solution in blueing, as alkaline solution cause tissue section to float off.
-Hematoxylin age with time and stain darker.
-If eosin becomes weak, can add more eosin solution and acetic acid.
Note: not to over add acetic acid as eosin may reach an acidic pH that stain everything across and not just cytoplasm.
Ziehl Neelson stain
-use 1%acid alcohol for more uniform decolourisation.
-do not allow carbol fuchsin stain to dry up or it will form insoluble compound, causing staining artefact.
-Avoid tap water as may contain mycobacteria causing false positive.
-Phenol and alcohol in carbol fuchsin enhance staining especially if the acid fast bacteria got waxy capsule.
-Decal can affect staining causing false negative.
Modified Fite stain
-use 1% sulphuric acid 5 to 10 minute instead of 1% acid alcohol decolourisation and constantly agitate slide to prevent background. This increases sensitivity but also increases false positive.
Gram stain
-old or dead bacteria may have damaged cell wall causing false gram negative result.
Warthin Starry
-If overstain can treat with iodine and sodium thiosulphate to remove some stain.
-higher pH give denser stain.
Giemsa
-at higher pH gives denser stain.
PAS
-Can use kidney as control as gives strong positive compared to liver tissue glycogen stores.
-Can use cervix for glycogen control.
-PAS no staining can troubleshoot with new Schiff's reagent or new preiodic acid.
-mild PAS; using 0.01% perioduc acid instead of 0.5% to stain N-acetylsialomucin but glycogen and other substances are not stain.
-Schiff reagent incubation step should be 10 to 15 minute and not 5minute.
GMS
-Chromic acid step can bleach stained slide. Thus can use stain slide to do GMS if no choice.
-bacteria and calcium deposits also stain up in GMS.
-hold slide vertically to when washing off sodium thiosulphate step. This can remove off the background.
- tissue loss in GMS due to overheating.
Alcian Blue
-use pH1 0.1N HCl to to rinse and differentiate. Can use appendix as control.
Alcian blue-PAS
-After staining with Alcian blue, rinse with water and not acetic acid as acetic acid affect PAS staining. (periodic acid reaction).
Colloidal Iron
-When preparing the colloidal iron solution, the water must be boiling to ensure total conversion of Ferric chloride to Ferric oxide.
-Cut two slides. One for test and one for control.
Congo Red
-Amyloid is protein, not carbohydrate.
-Amyloid is metachromatic with Tol blue stain and metachromatic with crystal violet (crystal violet in HCl solution) or metachromatic with methyl violet.
-Control slide age with time and lose amyloid. So do not cut too many amyloid control slides.
-section thickness 8 to 10um.
Masson Trichrome
-can use aluminium hematoxylin instead of iron hematoxylin for nuclear stain as acetic acid use in Masson Trichrome staining is not strong to differentiate off the aluminium hematoxylin.
CAB
-At pH1.3 it stains most intentive. Can use HCl to prepare.
-To prepare pH2.5 can use acetic acid to prepare.
EVG
-Can restain if over differentiated, provided had not used alcohol wash step to wash away iodine.
-agitation of slide (dipping slide up and down) during differentiating is important.
-Van gieson reagent counterstain step can remove a bit of elastin stain.
-Can cut 2 test slides for differentiation at differnet intensity. As EVG differentiation is tricky.
PTAH
-PTAH reagent stain better if used 2 weeks after preparation.
-Potassium permangate step time critcal as too short gives too blue result and too long gives little blue with more red result.
Sudan Black
-Sudan black is better than oil red O as 1) stain fat better and 2)stain phospholipid as it is slightly basic dye.
Iron stain (Perl's stain)
-over decal of tissue can lead to loss of iron causing weak result.
-if bone marrow is heated directly on hotplate, can cause tissue to float causing background as iron stores may float too. The best solution is to heat in oven, or next solution is to heat slide with paper in between the slide and hotplate for gentle heating and later transfer to hotplate for completeness in drying the slide.
-do not use tap water as iron in tap water can cause background.
Von Kossa
-black deposits in Von Kossa due to contaimnated glassware.
Mast cells
-stain with Tol Blue.
Plasma cells
-stain with methyl green pyronin.
General bacteria stain
-giemsa, methylene blue.
Other pointers
-Is primary dye positive or negative charge or is its staining base on solublity
-Old tissue block may stain longer. Eg: control blocks.
-Acidic formalin decrease nuclear staining
-Prolong formalin fixation for months tissue need to stain longer.
-Thicker section stain darker and thinner section stain faster.
-Degenerate tissue may only stain weakly or cause background.
-Sodium Thiosulphate used to remove pigments like mercury can interfere with staining.
-Hydration of tissue important to pick up stain.
-Prolong wash in water or alcohol can lose stain.
-Prepare reagent that need boiling in a boiling water bath for better control of temperature.
(1st picture at start of webpage)
-section on slide should be in the middle as at the ends of slide is not stain well in machine.
-Staining smears should stain longer as smear slide is thicker.
Chapter 13: Other techniques
Cell block
-To make cell block, you can centrifuge the fluid to obtain cell pellet and add liquified gelatin or foodgrade gelatin (gelatin with water and heat to dissolve), and allow to cool and harden and process with normal tissue processing.
-The best method is to centrifuge, decan, add 95% alcohol to fix cell pellet for 10minute and centrifuge again, decan, add 95% alcohol to fix cell pellet for 10minute and centrifuge again. Lastly, decan to obtain cell pellet. This usually gives greater cell yield and could obtain cell pellet with or without gelatin. (Optional step to add 95% alcohol to fluid in first place).
Colour restoration of specimen for photography
-Soak the specimen in 95% alcohol 30min to 12 hour to restore colour. But any longer, colour may fade and is irreversible.
Manual coverslipping
-Do not lay slide over the coverslip with mountant or mountant may squeeze out. Quickly overturn the slide once the slide is coverslipped.
Removing hematoxylin precipitate the fast way
-by just skimming off the precipitate form at the stain surface due to oxidation. (But best is still to filter).
Chapter 14: Useful habits and ideas
Some thoughts to share
-Section blocks by cases. Is more effective.
-Allocate personel to work at lunch hour even 1 person. Is effective especially rarely any interference during lunch hour.
-Ensure all blocks tally at end of day. As slides get lost easily, at least you still have blocks to recut.
-stay back to check before going home. eg: tissue processing set correctly, hotplate and oven off.
-Not sure, please ask.
-Safety first.
-Salvage a situation, use alternative. Is more effective.
-Identify critical steps be it in learning or doing.
-Honesty. Not react to fear. (Especially is a mistake that can be rectify or the damage is not job threatening).
-Proactive.
-Usualy things not done because lack of follow up.
-Keep instruments near to you especially within hand reach. Arrange the intruments via memory. Is effective and faster.
-Can visit histonet.org to see their forum. Is useful.
-Less things in mind, faster and easier to work.
-Negative thoughts is obstruction. Think positive and think easy.
(But if you sink into depression in work. Can consider alternatives. Remember you are born into the world with God looking after you. Wherever you go, God has prepared a place for you.)
-Not changing is wrong.
-mistakes amplify in a process
-Human factor important as is man is working, not machine.
-Comunicationis poor in lab. (This I do not know how to change). But if relationship is great and harmony, things get done easier.
Some Critcal Areas
-Transcription errors
>Read name and identity number when passing specimen.
>Ensure the cassette is labelled corrected when grossing the specimen.
>Read block and slide number when completed sectioning.
-Procedures
>Change forcep when passing biopsy and visibly check forcep is clean to prevent tissue contamination.
>Embed one cassette at a time. This error proof the process from wrong tissue contamination and clerical error from picking the wrong cassette cover.
-Rule of thumb
>It is the responsiblitity of the technician himself to ensure the right cassette label to specimen, right slide to tissue block and right slide label pasted on the slide and others. Visibly go through the label before doing. Hopefully, this can solve transcription and clerical error.
>Ensure tissue processor is running before you go home. Check thoroughly not haste. Have a calm mind to do so that you remembered that you remembered you had done the checking.
Chapter 15: Work politics
Actually I am not the best position to speak about work politics. But I just like to stress on a few points. Man cannot live on his own. We all need each other.
The first thing first is to complete one's own work and at the same time think of others too. Build healthy relationships. I believe all employers or supervisor aim is to first see competence in work and good character (honesty) and harmony in work place. For goal of all workers is independence in work. Lastly, strive for interdependence for greatness. Success is not base on individualism but by team work.
I pray for those after reading will have harmony and excellence in their workplace and career. Cheers
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