H&e stain and perls prussian blue technique
H&E STAIN AND PERLS PRUSSIAN BLUE TECHNIQUE
This practical is meant to establish the type of disease which a patient represented with to the doctor. The patient had a history of abdominal pain, abnormal skin pigmentation on both his hands and on his face. He also reported that he had been experiencing fatigue for a long duration of time. The patient's blood showed that he had an elevated blood sugar, a condition knows as hyperglycemia and therefore a diagnosis of diabetes mellitus was made. The patient was then referred to a hospital called John Radcliffe for further investigations. The hospital took a biopsy of this patient which was then sends to the laboratories for cellular pathology so that the pathologists would do the analysis of the histological sections provided. The histological examination was to be done under the microscope using H & E (Heamatoxylin and Eosin) and another technique called the Perls Prussian Blue Technique. These two techniques are explained below.
Hematoxylin and Eosin Stain
This staining technique is one of the most popular techniques in the histological laboratory currently. Usually, it is used by the pathologists when they want to make an accurate medical diagnosis from a tissue which has been biopsied. The technique involves the use of a basic dye called hematoxylin (Joyce, 45). The application of hematoxylin dye on a slide in histology leads to it attaching to some components of the cell. It attaches to cell structures which are basophilic in nature making them have purple blue color. The Eosin dye is an acid which is alcohol based and attaches to structures of the cells which are eosinophilic. The attachment of this dye on such cell structures make them acquire a bright pink color (Mary, Rose, 23).
Basophilic structures of the cell include the nucleic acids. Therefore, all the structures of a cell which contain nucleic acids are basophilic in nature. These structures include the ribosome, the cell nucleus which has DNA (Deoxyribonucleic Acid), RNA (Ribonucleic Acid) and all types of RNA. Since the nucleus of a cell is composed mainly of nucleic acids, it is basophilic in nature attaches to the hematoxylin dye. The cytoplasm is also rich in the nucleic acids since the massager RNA and Ribosomal RNA are also found in the cytoplasm (Kierszenbaum, 56). This means that in a normal cell, the hematoxylin stain will stain both the nucleus and some parts of the cytoplasm, but the nuclei will be stained more than the cytoplasm components since it has more concentration of the nucleic acids which are basophilic in nature (Gartner, & Hiatt, 232).
The structures which are eosinophilic are composed generally of proteins, either extracellular or intracellular. These are mainly found in the cytoplasm of the cell and include the Mallory bodies and the lewy bodies (Young & Stevens, 45). The red blood cells are stained intensely with red because of the abundance of these proteins in their cell. The red blood cells also lack nucleus which is the one responsible for the blue coloration in the other normal cells. Therefore, it takes completely the color of the eosin dye (Ovalle, & Nahirney, 88).
These structures of the cell which take the two different dyes, that is, the eosin and the hematoxylin do not have to be either acid or basic. The classification is based purely on the dye they take and there take its color and not their PH. Therefore, the nucleus of a cell does not have a low pH while the enzymes present in the cell are not necessarily basic in nature. The terminologies are all based on their affinity for the two different dyes. In addition, there are other colors such as the brown and yellow which can be observed in the slide under the microscope. These colors are usually as a result of pigments which are intrinsic to the cell for example the melanin pigment. Apart from the above structures which stain adequately with the above dyes, there are other structures of the cell, for example Basal lamina, which stain poorly with the two stains and hence can not be viewed adequately with the two techniques. In that case, a PAS stain can be used to visualize such structures well
Perls Prussian blue Technique
It uses the principle that the hydrolysis of a mineral acid which is dilute makes it release ferric ions which is from tissue deposits which are protein bound. In presence of ions called ferrocyanide, these ions are usually precipitated as a complex which is very colored and also very insoluble in water called the potassium ferric ferrocyanide (Gartner, & Hiatt, 78). The ferrous ions themselves have no product which is colored and therefore their reaction can not be seen. In this technique, a normal cell will have nuclei which is red in color, erythrocytes which are yellow in color, ferric salts stained deep- blue and lastly asbestos bodies are stained blue or black under the stain (Mitchell, & Peel, 353). The stain is composed of aqueous hydrochloric acid, aqueous potassium ferrocyanide and a neutral red stain.
The practical was carried out using three sections which had been biopsied previously. They were labeled A, B and C. All of them were embedded in paraffin wax. Section A is from the patient while section B is slide to serve as control so as to see if the procedure of staining is properly working. The two satins sections were to be stained using the Perls Prussian Blue. Section A is from the patient which was got by biopsy. Section C was also from the patient got by a biopsy. It was to be stained with the hematoxylin and eosin. It is similar to section.
Health safety was observed throughout the practical. All the times during the practical, gloves were properly worn. This was to avoid the contamination of the hands with the various dyes which are known to be poisonous and can cause poisoning. In addition, the eyes were also covered with safety glasses to prevent any chemical from splashing into the eyes. Therefore, the eyes were properly covered. The hands were kept far away from the face since the hands had been contaminated with the chemical. This was meant to minimize the possibility of the dye reaching the eyes. The fingers were never put into any of the chemicals since it can lead to burns or erosions of the fibers. After doing the practical, the hands were washed to ensure that there were no remnants of the chemicals on the hands.
De-waxing also known as hydration of paraffin was done on all the slides so as to remove the wax on to which the slide was embedded after the biopsy. The de-waxing is meant for removal of all the wax so that it will not interfere with the staining technique. Wax would also prevent the proper viewing of the slide under a microscope. This is done by the following.
On each staining bench, six tanks are put. The first, the second and the third tanks are filled with Xylene. The fourth tank was filled with Ethanol. Fifth tank is filled with industrial methylated spirits which is hundred percent. Lastly tank six is filled with seventy percent Industrial Methylated Spirit. The three sections are laid on the staining rack which is dry. A wet rack was avoided because it would lead to deposition of water in the Xylene. They are placed on the benches corresponding to the tanks.
The sections were then placed in to the tanks from tank one to tank six. In each of the tanks, the slides were placed there for exactly two minutes. This was meant to wash all the wax in the sections to avoid its interference with the staining procedure. The slide was then rinsed with running water from a tap. This is to remove all the Xylene left on the slides. The slide was then rinsed again with distilled water to remove any of the chemicals remaining in them. The whole procedure was repeated for all the slides from section A to section C.
After rinsing all the slides with the distilled water, they became ready for the staining by the two techniques, the hematoxylin and eosin and the Perls Prussian blue Technique. Great care was taken during the whole process of de-waxing since Xylene is a very poisonous substance. It is also very flammable and it can cause fires very easily. Xylene is also known to be an irritant and can cause a lot of allergic reaction on the body (Telser, & Young, 4). For such reasons, there was a lot of care when handling the chemicals. The slides were also rinsed properly to avoid the chemical being carried by the sections.
After the de-waxing, the slides were then stained using the following method:
The slides were placed in hematoxylin dye for five minutes only. This time was enough to allow for the dye to be attached to those tissues which are basophilic.
After five minutes, all the slides were removed from the hematoxylin dye. The excess dye was rinsed with water which is distilled to prevent the dye from over staining.
The slides were dipped in acid alcohol for three times, each time taking approximately one second. This is to remove excess hematoxylin and therefore to enable the viewer to differentiate the parts which have taken the dye and which have not taken it. The dye which has not been attached to cell components or structures is washed away the acid alcohol. Again, the sections were rinsed thoroughly in water from tap to remove the alcohol from the slide. This was done to ensure that the acid alcohol does not remove all the dye present in the slide and hence causing the structures to loose the dye which they have already acquired.
All the slides were placed in eosin and left there for duration of one minute each. This was to ensure that the slide stays enough time in the dye so as to be stained by the dye adequately. After the one minute, the sections were removed from the dye (eosin) and the excess eosin washed off with tap water. The washing was not done for a long period since eosin is a water soluble dye which can easily be carried away. Washing for a long time would have washed all the eosin from the slide leading to the eosinophilic parts of the cell not being stained by the dye.
After the rinsing with running water or water from the tap, the slides were quickly rinsed again with seventy percent (70%) IMS. After this rinsing, the slide was placed in one hundred percent (100%) IMS while agitating the section. The section was sloshed up and down for duration of one to two minutes. This removed any air bumbles which had been trapped in the slide. The slides were again placed in Xylene taking a lot of care because Xylene is very toxic and flammable and agitated again for another two minutes
After the agitation of the section on the slide, a cover slip was placed on the slide by putting a drop of a mountant on the glass which is to be used as the cover slip and covering the slide section. The slide was then removed from Xylene and then the slide was touched gently on the side with the section and cover slip. At each stage, it was ensured that there was smooth slipping of the solvent over each slide. It was observed carefully and if the slide looked like it is smeary, the stage was repeated so as to get the best results. The above slides had been adequately stained with the hematoxylin and eosin stains and ready for observation under the microscope.
Perls Prussian Technique
This was done on section A and B only. Care was taken when dealing with the reagents since they were very harmful. The reagents to be used were Xylene which can cause fires and is very poisonous, IMS (industrial methylated spirit) which is also very flammable, DePeX mountant which is toxic, potassium ferrocyanide and neutral red both of which are harmful.
The staining process involved the following steps:
Ten milliliters of hydrochloric acid which is two percent (2 %) in concentration was measured. To the measured two percent of hydrochloric acid, it was added two percent potassium ferrocyanide. The contents were mixed thoroughly by shaking hem well. The slides were then placed on a rack for staining near the sink. The above solution was carefully filtered onto the slides and the slides left to stand with the solution for about fifteen minutes. After fifteen minutes, the slides were carefully rinsed in distilled water to remove the excess solution from the slide which has not been attached. Zero point one percent of neutral red was filtered onto the slides and left to stand for a minimum of five minutes so as to give the slides adequate time to attaché to the dye.
Five minutes later, the slides were rinsed thoroughly and carefully in distilled water to remove any excess dye which has not been attached to the cell components. This was to ensure that the dye does not interfere with the viewing of the slide. Using a filter paper, the slides were blotted so that they dry. In seventy percent of industrial methylated spirit, the slides were quickly rinsed while at the same time agitating the slide by making a slosh up and down. The slides were then placed in absolute industrial methylated spirit (one hundred percent) and further agitated for another one to two minutes.
The slides were also placed in Xylene and agitated again for about two minutes to ensure that there was no gas trapped in the slide. The slides were then covered with cover slips by placing a mountant upon the cover-slip which is to be used to cover the section on the slide and the slide removed from Xylene and then was gently touched on the side of the slide with the section to the cover slip. In steps j, k and l, it was checked if the solvent had smoothly slipped over the slide. It was checked if the slide was looking like it is smeary and the procedure repeated in such a case.
The experiment was well done. Results were as follows:
Section B was normal with the normal structure of the liver. It has the normal iron pigment of the liver which is blue in color. This shows that the liver has iron stores in it. It contains several pigments which are the storage form of iron in the liver. Section A showed some large blue coloration in the cytoplasm. This is aggregation of iron in the cell. The iron pigment appears blue in color with the Prussian blue stain. This means there is excess of iron stored in the liver of that individual.
Iron overload in the body leads to increase in the iron store of the liver. In the normal organism, excess of iron is stored in the year. The increase in the iron stores is referred to as Haemachromatosis. In this condition, the increased iron stores in the body lead to increase in the liver iron. The iron is stored in the intracellular compartment of the liver.
The experiment was done well and the expected results were got. The control section was positive therefore the staining procedure was done well. The section also showed the normal liver contents including the iron inclusions which provided a basis for the comparison between the normal tissues and that of the patient. This would help in the diagnosis of the condition. The pathologist has a normal section which he or she can refer to and compare with the patients section making it possible to establish if there is any abnormality in the section from the patient. If the sections are similar, there is no problem but if they are different, then it means that the one for the patient is abnormal.
In cases where the control section is not stained, the staining technique was not good and the whole process of staining and viewing the sections should be repeated. Results in which the control specimen is showing no features of the normal cell are rejected. With the results of the normal section showing the normal inclusions of the iron in the liver tissue, then the staining was okay and therefore the results are acceptable
Other structures also seen are the nucleus which is stained blue black and cytoplasm pink red. This is an indication that the nucleus has substances which are basophilic in nature. Therefore they take the dye of hematoxylin. The cytoplasm is pink red in color. This is the color of eosin and thus has taken the color of eosin by attaching to it. Other structures which were visible under the light microscope are hepatic portal vein, hepatic artery, bile ducts and liver sinusoids. These are part of the normal liver.
The patient can be said to have been suffering from early haemachromatosis. Haemachromatosis is a disease or condition in which there is an accumulation of excessive amounts of iron in the body. Unfortunately symptoms for haemachromatosis do not usually appear until organ and tissue damage has already began. However, insidious and vague symptoms such as joint pain, fatigue, lack of energy, abdominal pain and skin color changes are considered as early symptoms of the condition and these symptoms were presented by the patient. The excess iron is transported into the liver by a transport protein called transferritin and stored intracellular by the hepatocytes. The histological appearance will be that of a blue coloration of the hepatocytes. The cells will contain a large mass of the dark blue material in the cytoplasm. This is different from the less amount observed in the normal cells of the bluish material which represents the normal storage iron in the liver which is as a result of hemolysis in of red blood cells.
The experiment is good enough to diagnose the disease. The appearance of the iron pigment in the intracellular compartment of the cell which is more than the one of the normal liver cell (control) is diagnostic of haemachromatosis in the patient. Since the control section showed that staining of the slides was good as it showed the normal histological findings then the results are accurate. The experiment was satisfactory and has no areas which need improvement. There was normal staining and all materials were available. The experiment was done according to the set standards and therefore it was very accurate. As proved by the presence of the normal histological findings on the control section B, there was no problem with the staining technique. In conclusion, the patient can be said to be suffering from haemachromatosis.
The nucleus is blue because it has contents and structures which attract the hematoxyline dye and therefore taking the color of the dye after staining. The cytoplasm is pink red meaning that it has taken the eosin color. This proofs that the cytoplasm has eosinophilic substances. These eosinophilic substances include enzymes which are mainly composed of proteins. The other structures viewed under the microscope in section C included the liver sinusoids, the bile ducts, the hepatic portal vein and hepatic artery and were all normal excluding the possibility of liver cirrhosis in the patient which is also associated with hemochromatosis. Usually the complication of haemachromatosis is the cirrhosis of the liver thereby distorting the normal parenchyma and leading to the end stage liver failure leading to other organ failures such as the brain.
Treatment for haemachromatosis is usually very straight forward and inexpensive depending on the stage of the condition. In early and less complicated stages of the condition, the initial aim treatment is to rid the body of excess ironand this is done through phlebotomy. His means that blood would be removed from the patient he same way it is drawn from donors at blood banks. Depending on how sever the iron overload is, a pint of blood will bw taken once or twice a week for several months to a year and occasionally longer. The blood ferritin levels will be tested periodically to monitor iron levels. The ai of this is to blood ferritin levels to the low end of normal.
Once iron levels return to normal the maintenance therapy begins and this involves giving pint of blood every 2 to 4 months for life. Some people may require more often phlebotomy depending on the severity f the condition. Also advice on diet should also be seeked and followed.