Many laboratories struggle with poor staining quality and invalid results every day; and they may not be aware of what went wrong in the laboratory processes or how to mitigate the issue.
Pre-analytical preparation is the first step in the specimen journey and it comprises of tissue collection, fixation, grossing of specimen, tissue processing, embedding and microtomy. It plays an important role in the success of tissue staining and pathology reporting. With optimally processed histology, pathologist is able to make a diagnosis accurately in a timely manner, and help the patient receive the right treatment promptly.
View the webinar recording to learn best practice process in laboratory.
Watch Webinar RecordingWhy should you watch?
Learn and share best practice processes in laboratory 
Q&As during the webinar
What you will learn:
Specimen handling, Inclusive of handling of bone specimen 
Fixation
Tissue processing
Embedding
Microtomy
Speaker & Moderator
Speaker
Dr. Tan Soo Yong
MBBS, DipRCPath, FRCPath, DMJ, Dphil (Oxon)
Associate Professor, Yong Loo Lin School of Medicine, NUS
Senior Consultant, Department of Pathology, NUH
International Advisor (West Pacific), Royal College of Pathologists
Moderator
Dr. Richie Jara-Lazaro
Pathologist
Medical & Scientific Affairs
Roche Diagnostics Asia Pacific
Q & A during the webinar
It is important that the oven temperature to be set at 2-4oC after the melting point of the wax as setting the temperature too high may result in cracking of the block during microtomy and causing the paraffin block to turn milky white. It is not advisable to heat the slides on a hot plate but to use an oven instead as sometimes the temperature on a hot plate maybe uneven, and temperature may go higher or lower than the setting.
It is important that the oven temperature to be set at 2-4oC after the melting point of the wax as setting the temperature too high may result in cracking of the block during microtomy and causing the paraffin block to turn milky white. It is not advisable to heat the slides on a hot plate but to use an oven instead as sometimes the temperature on a hot plate maybe uneven, and temperature may go higher or lower than the setting.
Use metal racks instead of plastic racks when drying the slides in the oven. The fumes released from the plastic racks may alter the slide properties, causing slide chemistry issues.
We do not encourage reversed processing once the under fixed tissue is processed. It is too harsh for the tissue and cell morphology will be impacted.
For small biopsies, specimens should be at least fixed for 6 hours. For large specimens, they should be grossed into smaller pieces to allow exposure to the fixative adequately before processing. And for larger pieces and fatty tissues, the fixation time should be longer. Ideally, the thickness of each grossed tissue section should be about 3mm in thickness and fit into the cassette with some allowance between the cassette and tissue.
Not advised to use Bouin fixative for small biopsy. Most antibodies are validated with 10% Neutral Buffered Formalin.
Prolonged formalin fixation may result in decreased antigen detection on certain antibodies, such as HER2 biomarker.
Process this group of specimen on the same day processing by skipping the first 2 jars of formalin fixed in the tissue processor if any. And likely this group of tissue may require a special created protocol with longer antigen retrieving and denaturation time for IHC/ISH. The protocol should be evaluated and validated in-house.
The best way to ensure that the formalin is the correct solution is to ensure that is decant from the commerically produced formalin bottle directly. Otherwise, replace the fixative in the bottle with 10% NBF immediately. However, if the initial fixative is not formalin, you may experience adverse staining result.
Are you asking if recycled formalin can be used in the processor? If your sample workload is high (meaning you have a high number of grossed specimens that goes through the formalin), the water content, liquid and blood will dilute your formalin. Therefore, the tissue will not be properly fixed and the blood pigment found in the formalin will become an artifact to the tissue that will cause difficulty in the interpretation later.
Based on ASCO/CAP guidelines, the time of fixation will be minimum of 6 hours to maximum of 72 hours.
Using fresh 10% Neutral Buffered Formalin (NBF) will prevent formalin pigment as acid will act upon hemoglobin, forming the complex. Avoid re-using formalin that has been used for several rounds of grossing (especially when the grossed tissue are bloody) as this will increase chance of the formalin pigment.
It depends on the type of background staining that you are referring to. Background staining can be caused by various factors such as using protocols that are not properly optimized (antibody concentration is too high, insufficient endogenous peroxidase quenching, non-specific secondary antibody binding, incubation temperature too high to name a few), poor slide quality, instrument maintenance etc.
It depends on the type of background staining that you are referring to. Background staining can be caused by various factors such as using protocols that are not properly optimized (antibody concentration is too high, insufficient endogenous peroxidase quenching, non-specific secondary antibody binding, incubation temperature too high to name a few), poor slide quality, instrument maintenance etc.
If the controls are sectioned and stored for a while before cutting the patient tissue sections, the slide may be affected by humidity and heat and results in slide chemistry issue, hence the inconsistent staining. If both control and patient tissue are freshly sectioned, and you are running on the Ventana staining platform, check that the slide labels are properly aligned and paste properly on the slide to prevent reagents wicking off. If the tissue sections are too near to the barcode label, it may affect the staining intensity. Another potential cause maybe due to slide chemistry issues, resulting in inconsistent staining. Check that slides are stored in a cool, dry, dessicated area and opened boxes of slides are not exposed to heat and humidity for a period of time, and avoid double dipping. Check the specifications of the instrument you are using is within specifications too.
Chattering and tissue broken can be caused by many factors e.g. tissue being processed with excessive time in the graded alcohols in the processor, alcohol and xylene in processor are set at high temperatures or melting temperature of paraffin in processor or embedder paraffin wax temperature set too high (8-10degrees) above melting point. Also, Microtomy skill and regular maintenance of the microtome play an important part in a good tissue section.
The standard thickness can be at 4µm.
How can you ensure that the purity of these recycled alcohol and xylene are the same as fresh solvents? If the recycled alcohol does not remove the water content adequately from the tissue, and likewise if the recycled xylene cannot properly clear away the alcohol, it will affect the paraffin infiltration and ultimately affect the microtomy sectioning.
Unfortunately we do not have experiencing xylene with chloroform so we are unable to share our experiences.
Q. If we replace xylene with chloroform, what will be effect on processing, IHC & Molecular testing?
Unfortunately we do not have experiencing xylene with chloroform so we are unable to share our experiences.
"Xylene is the last clearing step in the tissue processing and removes any residual ethanol before the paraffin can penetrate well.
If you do not have good quality xylene for the clearing, paraffin don’t penetrate the tissue well, the tissue might not hold up during the microtomy phase. "
Do not use water directly from the tap. Try to use DI water from a water system that is well maintained. Change frequently in the same day if the workload is high. At the end of each day, pour away all the water in the float bath, remove any debris from the water bath and dry it out properly.
Do not use water directly from the tap. Try to use DI water from a water system that is well maintained. Change frequently in the same day if the workload is high. At the end of each day, pour away all the water in the float bath, remove any debris from the water bath and dry it out properly.
Thickness of the tissue section is important when performing IHC/ISH staining. Different antigen retrieving and primary incubation time may vary. The target staining may look weaker on a thinner tissue section. So if you have different thickness tissue section in the same lab, you many require to set many different protocols.
This is what we classified as "Inconsistency in staining". Many factors attributed to this failure in staining. Instrument and/or slide surface chemistry changes can lead to this failure in staining partially. Suggest to call in the Application Specialist that support your lab for assistance.
We recommend to air dry and bake the slides in the oven.
We recommend to air dry and bake the slides in the oven.
By coating the cut slides with a layer of paraffin wax, the wax seals off the tissue section from the external environment. High temperature and air causes oxidation of the tissue sections, and results in decrease in immunoreactivity. Also, a humid environment (which means high level of exogenous water level in environment) will also cause immunoreactivity to decrease.
By coating the cut slides with a layer of paraffin wax, the wax seals off the tissue section from the external environment. High temperature and air causes oxidation of the tissue sections, and results in decrease in immunoreactivity. Also, a humid environment (which means high level of exogenous water level in environment) will also cause immunoreactivity to decrease.
By coating the cut slides with a layer of paraffin wax, the wax seals off the tissue section from the external environment. High temperature and air causes oxidation of the tissue sections, and results in decrease in immunoreactivity. Also, a humid environment (which means high level of exogenous water level in environment) will also cause immunoreactivity to decrease.
Yes you can. Some antibodies that have cut slide stability will not store longer than its limitation period eg ALK. For most other antibodies, storing them in a cool, dry dessicated place will allow storage for several weeks. Antigencity loss will least occur when slides are stored in a dark, low temperature (4 degrees fridge) environment. However, we always advocate using freshly cut tissue slides.
Air-drying slides overnight at ambient room temperature will be adequate.
Sorry, I do not have experience with ion exchange resin method of decalcification. Like most other labs, we had used acid decalcification with poor results. We then switched to EDTA-based decalcification using Osteosoft, which works well for FISH. We did not try anything else since.
EDTA-based decalcifying agent. We use a commercially available reagent called Osteosoft (https://www.merckmillipore.com/SG/en/product/OSTEOSOFT,MDA_CHEM-101728). That works well for us but you may try other brands.
We do not recommend to use acid for decalcification if this sample is likely to be used for IHC and other molecular assay as strong acid affects the morphology.
It will certainly speed up decalcification but you need to watch the heating time. The variables include the type of decalcification agent (for large bone sections e.g. head of femur, most would use strong acids like HCl), the amount of bone present in the specimen, the volume of decalcifying agent in the microwave, which will affect the time needed to heat to a particular temperature, etc. We do not use microwave decalcification in our lab. We tried it before for large bone specimens but sometimes our techs turn on the machine, go off to do something else and forgets about the microwave oven.
After a few disasters, we decided that for specimens like head of femur, there is no real need to rush the acid decalcification using microwave.
For bone marrow trephines, we do not use microwave heating although some labs have tried it. It can work but you need to watch the loss of fluid due to evaporation.
We do not use microwave decalcification, because unlike acid, Osteosoft costs more. EDTA decalcification takes an additional 1-2 nights in our hands, but the results are worth the wait and in any case, the apsirate can give an early indication of the pathology. In the case of a dry tap, the trephine is crucial and we do not want to compromise the quality for speed.
After a few disasters, we decided that for specimens like head of femur, there is no real need to rush the acid decalcification using microwave.
For bone marrow trephines, we do not use microwave heating although some labs have tried it. It can work but you need to watch the loss of fluid due to evaporation.
We do not use microwave decalcification, because unlike acid, Osteosoft costs more. EDTA decalcification takes an additional 1-2 nights in our hands, but the results are worth the wait and in any case, the apsirate can give an early indication of the pathology. In the case of a dry tap, the trephine is crucial and we do not want to compromise the quality for speed.
EDTA decalcifies very slowly. So if you are talking about bone marrow trephine biopsies about 1 – 1.5cm in length using the standard trephine needle, that will take about 2 nights before the decalcification is complete. However, once the bone marrow trephines floats in the EDTA, it means the decalcification is completed.
We do not use microwave decalcification in our lab. We tried it before for large bone specimens but sometimes our techs turn on the machine, go off to do something else and forgets about the microwave oven. After a few disasters, we decided that for specimens like head of femur, there is no real need to rush the acid decalcification using microwave. For bone marrow trephines, we do not use microwave heating although some labs have tried it. It can work but you need to watch the loss of fluid due to evaporation.
It will depend on if the tissue is well-fixed. Slight morphology may be impacted sometimes.
Sorry, I do not have experience with ion exchange resin method of decalcification. Like most other labs, we had used acid decalcification with poor results. We then switched to EDTA-based decalcification using Osteosoft, which works well for FISH. We did not try anything else since.
Not advised to use Bouin fixative for small biopsy. Most antibodies are validated with 10% Neutral Buffered Formalin.
10% Neutral Buffered Formalin is the best fixation for lymph node for IHC and ISH
10% Neutral Buffered Formalin works best
If the biomarker use is for frozen section, no fixation is required. However, if the frozen section piece is to be processed into a paraffin block, it will be best to fixed the frozen tissue section with 10% Neutral Buffered Formalin.
Each specific clone has its own target. It is hard to compare two different clones of the same antibody.
Each specific clone has its own target. It is hard to compare two different clones of the same antibody.
Yes, Dr Naresh and colleagues have reported good results with their protocol in the Hammersmith Hospital (Naresh KN, Lampert I, Hasserjian R, et al. Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol.
J Clin Pathol. 2006;59(9):903–911) using a weak acid (formic acid in this case). The decalcification time is increased to 6 hours compared to mineral acids, so essentially the processing time is increased by 1 night.
This is a compromise between the loss of antigenicity with usual acid decalcification and the longer time needed for EDTA decalcification. Dr Naresh found that it works well for IHC , ISH using kappa, lambda and EBER probes and by PCR for certain genes.
However, I do not know how good this protocol is for FISH probes and other mRNA targets other than immunoglobulin light chains and EBER, which are found at high copy numbers.
J Clin Pathol. 2006;59(9):903–911) using a weak acid (formic acid in this case). The decalcification time is increased to 6 hours compared to mineral acids, so essentially the processing time is increased by 1 night.
This is a compromise between the loss of antigenicity with usual acid decalcification and the longer time needed for EDTA decalcification. Dr Naresh found that it works well for IHC , ISH using kappa, lambda and EBER probes and by PCR for certain genes.
However, I do not know how good this protocol is for FISH probes and other mRNA targets other than immunoglobulin light chains and EBER, which are found at high copy numbers.
If there is chromogen stained on non-specific area on the tissue, this can happen in many ways. First tissue could be under-fixed and all proteins are then leaked to everywhere after unmasking it. So when you tried to enhance the staining intensity, the background will also be stained concurrently. Sometimes this non-specific staining on tissue can occur if the purity of the antibody is not high.
That is not good news, especially if the specimen had been decalcified with acid. The antigens may well have been lost and it will be difficult to retrieve. With EDTA, over-decalcification is not a problem as it just means more calcium is leached out but the specimens is not damaged. Prolonged contact with acid can destroy the protein antigens. Yes, you can try to increase antigen retrieval time and antibody concentration but what you are trying to do is to enhance the staining of the residual markers that have not yet been destroyed. However, depending on the marker, I doubt you will be very successful.
We do not encourage reversed processing once the under fixed tissue is processed. It is too harsh for the tissue and cell morphology will be impacted.
Similar to the earlier question (4). For bone marrow trephines, we do not use microwave heating although some labs have tried it. It will work but you need to watch the loss of fluid due to evaporation.
We do not use microwave decalcification, primarily because we lose more reagent due to evaporation and Osteosoft costs more.
You have to make sure that the entire specimen is still covered in Osteosoft at the end of decalcification. The only reason to do this is to speed up the turnaround time.
EDTA decalcification takes an additional 1-2 nights in our hands, but the results are worth the wait and in any case, the apsirate can give an early indication of the pathology. In the case of a dry tap, the trephine is crucial and we do not want to compromise the quality for speed. However, if the longer TAT is not acceptable, you can cetainly try microwave.
We do not use microwave decalcification, primarily because we lose more reagent due to evaporation and Osteosoft costs more.
You have to make sure that the entire specimen is still covered in Osteosoft at the end of decalcification. The only reason to do this is to speed up the turnaround time.
EDTA decalcification takes an additional 1-2 nights in our hands, but the results are worth the wait and in any case, the apsirate can give an early indication of the pathology. In the case of a dry tap, the trephine is crucial and we do not want to compromise the quality for speed. However, if the longer TAT is not acceptable, you can cetainly try microwave.
The processing time is the same as other tissues, as long as the sections taken fulfill the usual rules about not stuffing too big or too thick a piece of tissue into the casette. What is more crucial with a large lymph node is that the specimen should be sliced open as soon as possible.
We request lymph nodes to arrive fresh immediately or as soon as possible after resection. It is sliced into pieces of 5mm thickness and fixed in buffered formalin before tissue processing.
We request lymph nodes to arrive fresh immediately or as soon as possible after resection. It is sliced into pieces of 5mm thickness and fixed in buffered formalin before tissue processing.
I presume you mean for IHC staining? Do you have a problem with weak staining for H/E using the same specimen? If you get weak staining with both H/E and IHC stains whilst the controls are good, then perhaps it may relate to specimen fixation.
Check the formalin and fixation times. If H/E stain is fine but you get weak IHC staining, especially when this is an intermittent problem, then perhaps it has got to do with the fluidics.
The charged slides you use may have been exposed to humidity and temperature and as a result, the reagents during IHC do not flow properly.
Use a new pack of slides and if you open a pack of unstained slides but have many slides remianed, store in a dark, cool and dry environment. Our brain specimens are treated exactly the same as other specimen types.
Check the formalin and fixation times. If H/E stain is fine but you get weak IHC staining, especially when this is an intermittent problem, then perhaps it has got to do with the fluidics.
The charged slides you use may have been exposed to humidity and temperature and as a result, the reagents during IHC do not flow properly.
Use a new pack of slides and if you open a pack of unstained slides but have many slides remianed, store in a dark, cool and dry environment. Our brain specimens are treated exactly the same as other specimen types.
Yes, our sentinel lymph nodes are identified using methylene blue. Processing such specimens with routine cases is not a problem. The colour does not run and stain other specimens. If you have a problem, perhaps the dye used is different.
We are not able to do much if the sample has not been properly fixed or processed. In general, if the tissue is over-fixed, you can overcome the block in immunogenicity by increasing antigen retrieval time using a higher pH buffer instead of a lower pH for antigen retrieval. What over-fixation does is to increase the covalent bonds in the cross-linkage and that makes it harder for the epitope to open up, and therefore the immunogenicity might reduce. By heating longer, you might be able to break these bonds and therefore allow the immunogenicity to improve.
However, if the tissue is under-fixed and the formalin quality is not good, and the antigen has been degraded as a result, there is very that we can do about that.
However, if the tissue is under-fixed and the formalin quality is not good, and the antigen has been degraded as a result, there is very that we can do about that.
In general, for formalin fixation, the penetration is about 0.5 – 1mm per hour, so the fixation time should be about 8 hours or so in general, but it depends on the specimen. If the specimen is very small for example 2mm, then 3 to 4 hours of fixation might work. On the other hand, if you are handling big specimens like brain, the fixation might take weeks. It really depends on the type of specimen and you can overcome the problem of a big specimen if you cut it down to smaller pieces of tissue. That will enable the fixation to be better. As a rule, overnight fixation is at least 8 hours.
Yes it can, be but EDTA decalcifies very slowly. So if you are talking about bone marrow trephine biopsies about 1 – 1.5cm in length using the standard trephine needle, that will take about 2 nights before the decalcification is complete. For trying to decalcify head or femur, the big pieces of tissue, then the decalcification will take significantly longer period of time and that may be unacceptable in terms of turn around times. For these cases, you can try acid decalcification as it is not so crucial for these specimens to have perfect morphology and immunohistochemistry. On the other hand, with regards to bone marrow biopsy, it is crucial to have perfect morphology as IHC and molecular assays to be needs to be performed. Hence we will use EDTA.
I presume you are asking on what is the best storage method to preserve the immuno staining. You should cut the slides just before performing the immunohistochemistry stain. This will ensure that best results of the immunohistochemistry. If you have to store unstained cut slides, you have to make sure that you store them in a cold but yet dessicated environment. In general, we do not perform IHC on cut slides that has been stored at room temperature in our tropical climate for more than 1 week. This is lab dependent as perhaps lab conditions are air-conditioned and with low humidity.
Stained slides storage can be kept for a longer period and how long can these be kept, and how long before the slide fades depends very much on the environment that they are placed in. In air-conditioned environment, they can be kept for 2-3 years.
Stained slides storage can be kept for a longer period and how long can these be kept, and how long before the slide fades depends very much on the environment that they are placed in. In air-conditioned environment, they can be kept for 2-3 years.
We treat the cell block much like tissue. For cell block, the cytology specimen are collected in ethanol or formalin fixative. For these specimens, we keep them at least for 4 hours, sometimes routinely we just fix for 8 hours.
More Q&As are coming, please stay tuned.
