What is Histology Lab?
A histology lab prepares microscope slides for examination with a light microscope.
Why is Histology important?
Histology is the best way to check tissue. Histology is a collection of thousands of techniques that have been perfected over the years. Doctors rely on histologist everyday for quick and accurate results. Modern medicine depends on histology more and more every year. Over 80% of all diagnosis involve some sort of lab testing.
What is an H&E stain?
An H&E stain demonstrates the Nucleus and Cytoplasm of the cell, and some details of both the Nucleus and the Cytoplasm.
What is Paraffin Processing?
Paraffin Processing converts samples to a chemical state that enables them to be cut very thin to be placed on a microscope slide.
How long does an average specimen take to go from grossing to a finished slide?
A typical specimen will be grossed in, processed, cut and stained in about two days. Non-typical specimens such as decal specimens or very large neuro specimens may take over a week.
What procedures do you recommend for best results?
We recommend that the specimen is submerged in a large amount of 10% neutral buffered formalin as soon as possible after the patient or animal passes or is sacrificed. If you are performing studies on brain or other nervous tissue, the time between death and submersion in 10% neutral buffered formalin should be minimized. 5-10 minutes is recommended for brain and spinal cord specimens. accessing the brain is a difficult task for inexperienced technicians, often times fragments of bone are lodged in the brain during the procedure. These fragments will lead to poor results during cutting of your slides because the bone is mostly composed of calcium. We recommend you rinse the brain off well with 10% neutral buffered formalin to remove any bone dust or fragments before they work their way deeper into the tissue. For a mouse brain, you would want to use about 50ml of 10% neutral buffered formalin per brain. As the tissue is “fixed” by the formaldehyde in the 10% neutral buffered formalin the Ph of the solution changes. If this PH changes too much you will have poor fixation and possibly pigments and artifacts in your sample. The general rule is to use 15 times the amount of fluid as the size of your sample. If you have a 3cm by 3cm sample, you will want to use 45ml of 10% neutral buffered formalin. If you have a 20cm by 20cm sample, you will want to use 300ml of 10% neutral buffered formalin. Using any formaldehyde solution other than 10% neutral buffered formalin will almost always give poor results. 10% neutral buffered formalin is the standard for use in hospitals and veterinary clinics. The buffer in the solution helps your tissue fix properly without having to monitor the PH, as long as you follow the 1×15 ratio of tissue to fluid. Some stain procedures will not work with 10% neutral buffered formalin, but the 1×15 ration still applies if you are using other types of fixatives. Alcohol is often used as a fixative for flourescent staining on frozen specimens, usually methyl alcohol.
If you are submitting organs with capsule like properties, such as Kidney or Liver, Heart and Lung, and especially gastrointestinal tract….. You will always want to cut this capsule to allow the 10% neutral buffered formalin to get access to the inner part of the organ. We recommend making a few small cuts with the tip of a scalpel, one on each side of the organ. For lungs we highly recommend inflating the lungs fully with 10% neutral buffered formalin, use a syringe for this procedure and it will be very easy. If you cut the lung up into 5-10 samples this will also have great results. The inside of the lung has many small details that will disappear after a few hours if the lung is not properly fixed. Gastrointestinal samples will show amazing results if the interior of the organs is properly fixed. A syringe can also be used for this procedure. If you are submitting samples from an animal or person and the stomach or Gastrointestinal tract appears to be full of food, particularly rough food such as mouse feed, grains, vegetation, anything with fibers or just any food in general…. Cleaning out the inside of these organs will give better results. Unless you are studying food to these organs, the food will not be needed and it will often produce poor results on otherwise properly handled samples.
If you are submitting bone, a common mistake is to put the bone immediately into de-calcifying solution. Bone needs to be fixed just like other types of tissue. If it is not fixed it does not cut at all, you end up with a powder instead of a cross-section. Many problems with bone samples are due to the sample not being fixed before de-calcification begins. The de-calcification process draws calcium through the tissue and into the decal fluid. As the calcium moves through the bone it causes major damage to the other bone components if they are not fixed. Another common mistake with de-calcifying bone is to just use one wash of solution. A 1×15 ration of tissue to fluid is the correct ratio for the de-calcification procedure, but the fluid must be changed several times to remove all the calcium. Simple chemical test can be performed on the de-calcificatin fluid to decide the exact end point of the procedure. Alternatively, if you are good at chemistry, you can actually figure out how much acid will be needed to dissolve the calcium by mass in your sample.
We recommend and use the following procedure for de-calcification.
5% Ammonium Hydroxide
5% Ammonium Oxalate
Use equal parts of the 5% ammonium hydroxide solution and the 5% ammonium oxalate solution to make the working solution.
Add 5ml of the decal fluid your specimen is sitting in to 10ml of the working solution.
Observe for any precipitate forming. If you see precipitate (it will be easy to see cloudiness) change the de-calcification fluid and wait a day or two and then try the chemical test again.
After doing the procedure a few times with different specimens you will begin to be able to make educated guesses of decal times needed based on specimen size and mass.
These are the histology specimen preparation steps in chronological order.
processed or frozen
Breakdown of details for each step of histology from the above list:
Receiving a histology specimen is the first step. A histologist or pathologist assistant will remove the sample from its shipping/transport container. The container may be a box, bag or cooler. If the specimen is frozen, it should be placed in a -20 or -80 freeze ASAP. Any written information that is included with the specimen should be reviewed to make sure the specimen and information are correct and matching.
Accessioning a specimen is the second step. A histologist or pathologist assistant will check the actual specimen and note the dimensions, shape, color and any details that the specimen may have. This step is repeated during the grossing step to note any cutting done by the histologist or pathologist assistant and to note what is being submitted for processing or freezing.
Grossing is the step that gets the specimen grossly cut for histology. Cassettes come in different sizes, so the correct cassette size is chosen during this step as well. A common size cassette is better if possible, but some times it will not be big enough for samples such as eyeballs or turtle eggs. Specimens that should not just be cut to fit into a cassette! For specimens that will have a surgical margin to check, these margins are often split up into different cassettes. The surgeon will often mark the sides of the specimen and each marked side can be examined by a pathologist.
Processing the specimen is usually done one of three ways:
1. paraffin processing
2. snap freezing
3. polymer embedding
For each of these steps procedures number in the thousands. A standard procedure for normal size specimens will be discussed for each. Keep in mind that the bigger the specimen, the longer each step will be in the procedure. If the specimen is small each step will be shorter. Expect the steps to increase just like discussed for fixation. You want a 1 to 15 ratio of tissue to fixative solution, likewise you want this ration for processing specimens. If you overload the machines, or containers if hand processing, the tissue will have horrible results and take extra time and care in the following steps. This slows workflow in the lab and all your histology and pathology friends will not be happy with you if you do this. If the blocks are not processed properly and then need to be cut a year or two later, you may not have detail of membranes or nuclei, nd they also may not stain with IHC!
Paraffin processing is the most universal histology procedure. In all labs, this procedure is probably the one procedure that every lab does almost identically. If you learn it once you will not have a problem when going from lab to lab, you can always have expertise on this part of histology. If you are a histology student, or a pathology student, this is something you should learn to avoid difficulty when running your processor. Different manufacturers design processors differently, and the control panel will always try to be more flashy and user-friendly as new models are sold, none of these machines will give you better results. Hand processing is the supreme and best way to process, though the equipment is more extravagant than a simple processor. Hand processing is dangerous and needs to be approached like a true safety hazard. An expert will have no trouble, but a new histologist should be trained and watched before performing hand processing alone. Almost 100 percent of all labs do not hand process. It is old-fashioned and does take up a large amount of technician hours. Only a lab with excellent workflow can hand process. For this reason, and the fact that many many histology labs are constantly understaffed in hospital settings, automated processors are the preference of histologist and pathologist everywhere.
Processors are machines that have to process the tissue and then clean themselves. Reagents in these machines are usually— Formalin, alcohol, xylene and paraffin. Some labs use substitutions for these steps, for various reasons such as safety, waste disposal, cost, time, processor manufacturer specifications, tissue types, melting temperature preferences, decalcification needs, species specific needs and many other reasons. The basic steps of Formalin, alcohol, xylene and paraffin relate to the basic needs of processing. These needs are as follows – Fixation, Dehydration, Infiltration. Fixation preserves the tissue. Dehydration removes all water from the tissue. Infiltration is the most complicated part of processing, and in the case of paraffin is usually performed with xylene followed by paraffin. The xylene can mix with both the alcohol and the paraffin and that is why it was chosen. It has no other purpose. Xylene substitutes have been around for many years now, and still many labs prefer xylene. Xylene is flammable and it is a neurotoxin, this is not shown as a hazard in the way that histologist use it. In recent years safety has been a focus in most labs and new protocols have been put in place to make sure a safe working environment.
After processing embedding is the next step in histology. Embedding is an important and time sensitive step. Paraffin embedding involves use of embedding machines and cold plates. The embedding machines are paraffin dispensers with two large warm bins for specimens and molds. Cold plates, sometimes attached to embedding stations, are steel plates with a freezing pump system. Cold plates cool the paraffin rapidly to speed up the entire embedding process.
When you are embedding or directing someone else to embed remember that the mold should be warm, not cold, for good cutting. You want to overflow the mold, keep the cassette hot and put the cassette on the mold before the overflowing paraffin pours out of the mold, this will make sure the cassette holds the paraffin in place well and will not have air bubbles. Air bubbles ruin blocks and can cause future levels cut from the block to mismatch early levels due to the need to re-embed the specimen because of an air bubble. Small air bubbles will form in any tissue that has a cavity, such as a heart. These cavities can be filled most of the time, and care to do so will make sure proper cuts.
Another point of embedding is removing excess wax from the blocks. Most labs now use a warm block melter that is angled to allow the paraffin to drip down into a container. New histologist will melt on block at a time, and use care not to touch the warm plate to the cutting area of the block. More experienced histologist may melt several at a time to save time. This technique does not affect cutting, but the largest block sizes will not be melted 100% in the angled block face where block ID data is written. For this reason the writing surface is often again cleaned while cutting by more experience techs.
From this point on in the study guide, histology lingo will be used. If you do not know what a term means at this time, a glossary will be included at the end of the guide.
Block- an embedded sample ready to be cut, often has 2 parts, a cassette being attached to it for fitting to a microtome and labeling purposes. For frozen this can also mean an embedding frozen piece of tissue.
Grossing- the specimen being examined and recorded
Histology- the study of tissue.
Histotechnology- the lab science of preparing tissue for microscopic examination.
H&E- a stain that shows detail of the cell, contrasting nuclei from cytoplasm. This is by far the most common stain in the world for tissue. H&E stands for “Hematoxylin and Eosin”. Usually this stain follows similar protocols with the most notable variation being the addition of Phloxine to the Eosin. Phloxine will stain a deep deep red on some tissue components and is preferred by some doctors. Hematoxylin is a reagent prepared with much care, and needs to have time to ripen during the last phase of preparation. The process has been compared to making wine, though it is in fact a much less time-consuming task, and you cannot drink it. The hematoxylin and eosin are always filtered and before the first use.
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