Preparing slides for histological examination, whether for research or diagnostic purposes, is a critically important process, as the quality of preparation will directly impact the ability to accurately analyze tissues under a microscope. While there are reliable routines that many histology labs follow, such as neutral buffered formalin fixation and paraffin embedding, there are still many things that can go wrong during processing as well as many ways to improve and optimize processing procedures. The following five tips are things every histotechnologist should keep in mind while preparing samples for microscopy in order to avoid common problems and produce high quality slides for downstream examination.
1. Identify Common Artifacts and Their Causes
From initial sample removal and collection to fixation, embedding, sectioning and everything in between, there are many points in the preparation process where something may go wrong and lead to problem artifacts in the final slide. Understanding the potential causes of specific types of artifacts gives you a starting point to reflect on your current procedures and understand what steps can be taken to prevent the same problem from occurring in the future. Knowing the nature of different artifacts can also let you know if the affected sample can still be salvaged with additional steps or reprocessing. Additionally, knowing which tissue types are more susceptible to specific problems can help to optimize procedures for those specific tissues.
Artifacts may be due to overprocessing, underprocessing, mechanical damage, temperature problems, contamination and more, with each problem having its own telltale signs and some problems being clearly noticeable even before sections are placed on sides, such as tissue that is sludgy and mushy due to insufficient processing. Underprocessing artifacts, such as smudged or “muddy” nuclei, or tissue that disintegrates and separates during flotation, are a sign that more time may be needed in your processing schedule, or that your grossed sections are too thick for reagents to properly penetrate the sample.1 Signs of overprocessing, like microchatter (numerous parallel microcracks) from sectioning of tissue that has become hard and brittle, tell you that your processing schedules are likely too long and should be shortened.2
Another common artifact that may be related to excessive fixation time is formalin pigment, which appears brown or black under the microscope. Blood-rich tissues like spleen and bone marrow are most susceptible to this problem, as it is the result of acid formaldehyde hematin forming when acid formaldehyde reacts with hemoglobin.3,4 Recognizing this artifact allows you to know when your formaldehyde fixative may not be properly buffered, although buffered formalin solutions can still have this problem when fixation times are too long. Properly identifying this artifact is also useful, because formalin pigment can be removed through treatment with saturated alcoholic picric acid solution if caught prior to staining.
Other notable artifact types include holes in the tissue, giving it a “moth-eaten” appearance, which is often caused by poor knife quality or poor technique in trimming or sectioning; large circular air bubble artifacts with radial cracking and/or uneven staining, from poor flotation technique; and the appearance of “Swiss cheese” like vacuoles interstitially and in cell cytoplasm resulting from freezing and ice crystal formation during transport, which could be prevented by incorporating up to 10% alcohol (such as ethanol) to the formalin solution to lower its freezing point.5,6 In summary, understanding the causes of artifacts is the first step toward implementing a solution.
2. Ensure You’re Using Suitable and Well-Maintained Tools and Reagents
Poor handling techniques or improper processing schedules may be the cause of some preparation problems, but other problems could come from the tools and reagents you’re using to prepare your tissues. Low-quality, poorly maintained or incompatible products can become sources of contamination, mechanical damage and other artifacts that will interfere with downstream analysis, even when everything else is done right. Firstly, reagents should be of sufficient purity and renewed regularly to prevent problems with contamination and reagent carryover. Secondly, items like forceps, paraffin molds and knives, as well as surfaces and water baths, should be cleaned well between samples to prevent cross-contamination.
One important consideration is selecting the right cassette type relative to the size of your sample. Attempting to take a “one size fits all” approach to cassette selection can lead to problems ranging from poor processing, to tissues falling out of the cassette, to tissues being squished and distorted by the bars of the cassette.7,8 Keep in mind that tissues shrink during processing, so proper cassette dimension should be chosen to ensure that especially small samples will not fall through the perforations after shrinkage. If sample sections are too large to fit appropriately into any available cassette, you may be cutting slices that are too thick, or your lab may need to invest in larger cassettes. Size must also be taken into consideration for paraffin molds, as there should be an adequate margin around the tissue so that the tissue does not touch the edge of the mold.
Knife blade maintenance is also important both for initial trimming and for microtomy, as problems with knife quality and configuration can be a source of mechanical damage artifacts. Dull knife blades can cause crushing, holes, chatter and other defects; if a microtome blade is clamped loosely or there is some obstruction in the microtome causing excessive vibration, this can also lead to chatter and other damage. When such artifacts are seen on slides, and aren’t the result of other problems such as overprocessing, you should consider switching to a new, sharper blade, ensuring the microtime knife is tightly secured in the knife holder, and ensuring the microtome is cleaned and free from any obstructions or defects.
3. Temperature Control is Crucial
We all know that temperature can have drastic effects on biological tissue, reagents and chemical reactions, and controlling temperature during each step of the histological preparation process is key to producing high-quality slides and preventing major artifacts and distortions. As mentioned, unintentional freezing can lead to the formation of ice crystals that will turn your tissue into Swiss cheese. Excessively high temperatures, for example, during transport or grossing, can speed up decomposition of unfixed samples and even cause the tissue to become “cooked,” shriveled and dried out, which is very difficult to reverse.2 Room temperature is generally considered satisfactory for fixation, although heat does increase the speed of fixation and must be carefully balanced to avoid damage and uneven fixation. Temperatures up to 36-45°C may be employed for a limited time period. Even for microwave processing, maximum temperature and exposure time should be limited, as gasses in the tissue could vaporize and cause a similar “Swiss cheese” effect to ice crystals.5
Temperature control is especially critical during paraffin wax embedding, as well as during subsequent floating and section drying. The temperatures of the hot plate on your embedding center, the paraffin wax reservoir, the water bath and the slide drier should all be checked frequently.8 Excessive heat can cause thermal damage to tissue, which can be evident in slides with shriveling, cracking and pyknotic nuclei. A water bath too close to the melting point of the wax will cause sections to break apart, while sections may not readily flatten on water that is too cold, leading to compression and wrinkles; a temperature around 4-5°C below the melting point of the wax is ideal.
The cold plate temperature is also very important; it must be cold enough to solidify the paraffin, but too cold and the paraffin block may be too hard to cut into nice ribbons. In extreme cases, if the blocks are allowed to freeze before cutting, the tissue can separate from the paraffin and the wax could even crack. The block should be maintained at a balanced temperature before cutting; blocks that are too warm will experience excessive compression during sectioning while blocks that are too cold and hard can suffer from artifacts like chatter. Neither condition favors keeping your microtome blade clean and sharp either!
4. Don’t Rush!
There is always pressure to get things done faster in the lab, but true efficiency does not sacrifice quality for speed. While it is extremely important to begin fixing fresh tissue samples as soon as possible, excessive rushing that involves rough sample handling, improper processing schedules, carelessness with contamination and overall poor technique will only result in problems requiring additional time to resolve. First, samples should always be handled with care to avoid mechanical damage, as well as in a manner that prevents cross-contamination between samples or human contamination like skin or hair. This includes taking the time to clean surfaces and tools, skim water baths between samples and replace reagents when needed.
As mentioned previously, using an excessively short processing schedule or a schedule involving excessive heat in the hopes of speeding up fixation can lead to underprocessed, overprocessed and/or damaged tissues that may ultimately be unusable. An appropriate sectioning speed is also crucial – even when using an automated microtome. Setting the microtome to the highest possible speed is rarely the best option, as this can also lead to chatter artifacts resembling venetian blinds.5 Lastly, it may be tempting to float sections from multiple blocks at once to increase throughput, but this runs the risk of cross-contamination and inaccurate identification of specimens, especially if they are of the same tissue type.
References
- Carson, F. L.; Hladik, C. Histotechnology : A Self-Instructional Text; Ascp Press: Chicago, 2009.
- "Troubleshooting & Reprocessing Difficult Paraffin Blocks" by Geoffrey Rolls, Leica Biosystems Knowledge Pathway. https://www.leicabiosystems.com/us/knowledge-pathway/troubleshooting-reprocessing-difficult-paraffin-blocks/
- "Artifacts in Histological and Cytological Preparations" by Geoffrey Rolls, Neville J Farmer, and John B Hall, Leica Biosystems Knowledge Pathway (2021). https://www.leicabiosystems.com/us/knowledge-pathway/artifacts-in-histological-and-cytological-preparations/
- Chatterjee, S. Artefacts in Histopathology. Journal of Oral and Maxillofacial Pathology 2014, 18 (4), 111. https://doi.org/10.4103/0973-029x.141346.
- "60 Minutes: 20 Histology Tips," Webinar Presented by Clifford Chapman, Leica Biosystems Knowledge Pathway (2020). https://www.leicabiosystems.com/us/knowledge-pathway/60-minutes-20-histology-tips/
- "How to Prevent Freezing Artifact in Fixed Tissue Samples," Article by Rachel Corn, Kansas State Veterinary Diagnostic Laboratory (2016). https://www.ksvdl.org/resources/news/diagnostic_insights_for_technicians/february2016/freezing-artifact.html
- "Tips & Tricks to Better Histology in Tissue Based Research," Webinar Presented by Fiona Tarbet, Labroots. https://www.youtube.com/watch?v=42rUgUvgb3Q
- "101 Steps to Better Histology - a Practical Guide to Good Histology Practice," eBook by Geoffrey Rolls, Leica Biosystems Knowledge Pathway. https://www.leicabiosystems.com/knowledge-pathway/101-steps-to-better-histology-a-practical-guide-to-good-histology-practice/