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Home , Basophilic, Bluing (fabric), Dye, Hematein, Staining

6/5/2017

Hematoxylin and H&E

Irregular Problems and Solutions

Hematoxylin and eosin (H&E Unstained lacks contrast: all of the stain or HE stain) is a popular staining fixed material have a similar refractive index method in . The two and similar in color. If you viewed an were introduced in 1865 and 1875 unstained tissue section under the by Bohmer and Fischer. everything would appear a It is the most widely used stain in uniform color. medical diagnosis worldwide. The staining process therefore makes use of When a pathologist/mohs surgeon various that stain particular looks at a biopsy or mohs section of components within tissues, so that you can a suspected cancer, the histological distinguish different cell parts from each section is likely to be stained with other. Hemotoxylin and Eosin.

Progressive vs Regressive Staining HEMATOXYLIN

Progressive staining means that the tissue is left in the stain just long enough to reach the endpoint. Therefore it may be necessary to examine the slide at several different intervals to determine when staining is dark enough but not too dark. ( stain) Regressive staining means that the tissue is deliberately overstained and then destained (differentiated) until the proper endpoint is reached.

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History and Logwood Tree

Hematoxylin is the most widely used nuclear stain. Hematoxylin is extracted from the heartwood of the logwood tree (also known as campeachy ) The logwood tree is indigenous to Central America.

So Hematoxylin by itself is not a . Hematein is the actual oxidized product of Hemotoxylin that is a dye. Hematoxylin is relatively colorless and without further How is this done? modifications has little or no value as a biological stain. Can be done naturally by exposing it to atmospheric To produce the functional dye, hematoxylin is oxidized , or by using oxidizing agents such as sodium to hematein and subsequently is bound to one of iodate, mercuric oxide(very Poisonous), several metal including aluminum (Al+3), (Fe+3) permanganate and ; this oxidation process is called and (Cr+3). ripening. Today almost all Hematoxylins are iodized with sodium iodate.

Hematoxylin needs to be used in combination with a Most used Hematoxylins “” – a compound that helps it link to the tissue. The mordant used is typically a metal cation, such as . • Harris It is positively charged and can react with negatively • Mayer charged, cell components, such as • Gill I nucleic in the nucleus. These stain blue as a • Gill II result. • Gill III

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Hematoxylin

Gills Hematoxyllin is the most commonly used On Standing some Hematoxylin solutions will develop a metallic sheen of oxidized dye.(Should be filtered before use) and the I II and III indicates the strength of each, If not filtered, a blue- precipitate may be seen on the with III being the highest strength and provides stained sections. the highest intensity for histological staining of Good Hematoxylin has a winelike smell and a deep -red color. nuclei with shorter staining times. When a few drops are dropped into a container of tap , a bluish black color should be the result. Over oxidized and under oxidized hematoxylin will produce a red to red-brown color. Hemotoxylin is a water based stain and should be rinsed with water.

Filter

Placing a drop of Hematoxylin on filter paper will show a maroon spot with a purple edge, if the solution is ready for use. The absence of the purple border could indicate that the stain is not optimal for staining. Make sure product is not expired also.

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This is what we want..

EOSIN

COUNTERSTAIN

Eosin is the most widely used in the routine staining of sections. Eosin is a fluorescent red/ dye. Eosin is an alcohol based stain and should be rinsed with alcohol. Eosin is an acidic/anionic molecule. Staining intensity is highly dependent on pH levels. Eosin unlike hematoxylin is produced synthetically.

Eosin used properly will show three shades of pink, erythrocytes, , and the of muscle or epithelial cells. Erythrocytes should always be the deepest shade of pink, then the collagen and the lightest being muscle.

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Bluing Agents

Reduced eosin staining intensity is usually the result of an increase in the pH of the eosin solution. • is necessary to convert nuclear coloration Carry over, and inadequate rinsing to from reddish purple to a crisp blue/purple. remove the bluing are usually the cause.. • Bluing enhance the contrast by increasing the crispness of the hematoxylin. Do not go from water to Eosin. • Scott’s Tap water • Tap Water • water • Lithium Carbonate • Buffered solutions (Formalin)

Automated vs Manual Staining Automated

2 types of automated staining: • Linear and Robotic • Linear transfers one slide to the other in a linear fashion with the same amount of time in each container • Robotic transfers the slide into each container for a specific amount of time programed by the user. • There is less carryover of chemicals from one container to the next. • Change less frequently needed.

Manual Staining problems

• Faster than automated • Inconsistent/not as precise Understanding the way the staining works is • Large amount of carryover from container to key to understanding and troubleshooting container. Especially if the basket is used. staining problems. • Needs to be changed out more frequently. It takes time effort on the techs part to learn • Human errors can occur more easily. how to correct staining errors.

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1.

• Overlapping mounting media on the tissue. • Place sections where embedding medium doesn’t cover the other section • Trim embedding medium before cutting.

This blurry area is mounting media on top of the coverslip.. Remove coverslip and then carefully place a new one on.

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Happens when the slide is left out to dry too long before putting coverslip on. Apply mounting media and coverslip. Can dip back in clearing agent also.

Electrocautery causes this extreme basophilic staining of the edge. No fix for this…

Retracted Mounting medium. Make sure coverslip isn’t warped. Make sure no foreign body on slide or coverslip. Skin cut too thick. Cartilage.

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Uneven Eosin stain. Change out Eosin Not washing out bluing solution Not long enough in Eosin.

Nuclei are not blue!! ƒ Not enough time in Hematoxylin. ƒ Hematoxylin overoxidized . ƒ Not enough time in bluing or using cold water. ƒ Increasing the temperature of water, will increase the speed of as with all chemical reactions.

Pale cytoplasmic staining ƒ Check eosin solution pH, can adjust with acedic ƒ Rinse bluing agent out more sufficiently

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Air bubble

Something hard like calcium in the tissue.

Nick in Loose the parts in blade. the cryostat

In Conclusion/Common Problems Dark Nuclear Staining: ¾Sections left too long in hematoxylin. ¾Sections too thick. The Nuclei too pale(Hematoxylin too ) ¾ Sections not stained long enough in hematoxylin Red or Red-Brown Nuclei: ¾ Hematoxylin overoxidized and should not have ¾Ensure the sections are blued been used. properly. ¾ Skipped the bluing stage or bluing solution too ¾Check oxidation status of weak. hematoxylin

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Blue-Black precipitate on top of sections: What you want!!!!!!!!!!!!! ¾Make sure hematoxylin does not have the metallic sheen developing on top. (filter hematoxylin) Pale eosin stain, no distinct 3 different shades of eosin : ¾ Eosin has too much carry over. ¾ Elevation of pH ¾ Not enough time in Eosin. ¾ Change it out

Preview your slides How will all this help? • Be an even greater Mohs Tech/employee. • It is very important to look at your • Allows you to gain great amount of slides before you give to your knowledge. Physician. • Allows you to troubleshoot more • Your time is precious, so when do effectively. it right the first time it makes all the • Avoid recuts. difference in the world!! • Remember you as a mohs tech are worth your weight in gold!!!!!!!!!!!!!

“Genius is 1% inspiration and 99% !!!! THE END QUESTIONS

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