Special Stains Lecture Notes PDF

Special stains 231 MLT Histology  Definition The routine stain is: Hematoxylin and eosin (H&E) Other stains are referred to be as "special stains" because they are employed in specific situations according to the diagnostic need  Haematoxylin...

Special stains 231 MLT Histology  Definition The routine stain is: Hematoxylin and eosin (H&E) Other stains are referred to be as "special stains" because they are employed in specific situations according to the diagnostic need  Haematoxylin Hematoxylin is the oxidized product of the logwood tree known as hematein Since this tree is very rare nowadays, most hematein is of the synthetic variety In order to use it as a stain it must be oxidized - This can be done naturally by air or adding oxidizing agents such as sodium iodate or mercuric oxide  Haematoxylin Hematoxylin will not directly stain tissues, but needs a mordant - a compound that helps it link to the tissue The mordant used is typically a metal cation such as iron, aluminum, or tungsten The variety of hematoxylins available for use is based partially on choice of metal ion used They vary in intensity Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus  Haematoxylin Hematoxylin stains are either regressive or progressive Regressive stain (overstain) - Slides are left in the solution for a set period of time and then taken back through a solution such as acid-alcohol that removes part of the stain - This method works best for large batches of slides to be stained and is more predictable on a day to day basis Progressive stain - Slides are dipped in the hematoxylin until the desired intensity of staining is achieved.  Eosin Eosin the most common dye to stain the cytoplasm in histology It is an acidic dye that binds to basic components of a cell, mainly proteins located in the cytoplasm It gives a bright pink color  Special stains Tissues need special stain 1. Mucin stains 2. Glycogen stains 3. Melanin stains 4. Calcium stains 5. Lipochrome, (lipofuschin) pigments 6. Iron (hemosiderin) 7. Fat stains 8. Connective tissue stains  Mucin stains Acidic mucin Stained by Alcian blue stains Used for diagnosis Carcinoma of colon, carcinoma of breast and intestinal metaplasia Neutral mucin These can be found in glands of the GI tract and in prostate They stain with Periodic acid- schiff reaction (PAS) but not with Alcian blue, colloidal iron, mucicarmine or metachromatic dyes Used for diagnosis “carcinoma of the stomach”  Special stains Tissues need special stain 1. Mucin stains 2. Glycogen stains 3. Melanin stains 4. Calcium stains 5. Lipochrome, (lipofuschin) pigments 6. Iron (hemosiderin) 7. Fat stains 8. Connective tissue stains  Glycogen demonstration techniques 1- Periodic acid- schiff reaction ( PAS) 2- Best’s carmine 3- Hexamine silver technique  Periodic acid- schiff reaction ( PAS) The reaction of periodic acid selectively oxidizes the glucose residues to create aldehydes Aldehydes react with the Schiff reagent Used to detect glycogen, glycoproteins, mucoproteins, basement membrane, mucin, and fungi A predigestion step with amylase or diastase will remove staining for glycogen The nuclei will stain blue while collagen will stain pink  PAS staining PAS staining can be used to: A- Distinguish between different types of glycogen storage disease B- Indicate of certain types of cancer such as carcinomas of bladder, kidney, liver, ovary (adenocarcinoma), pancreas, and lung (adenocarcinoma)  PAS stain Candida in lung, PAS stain.. This is nodular glomerulosclerosis involving a glomerulus in the kidney, as seen with the PAS stain. Glycogen in Ewing's sarcoma, PAS stain. 13  Best’s carmine stain The staining of glycogen is accomplished by hydrogen bond formation between OH groups on the glycogen and H atoms of the carminic acid  Hexamine silver technique The technique is that following chromic acid oxidation, aldehydes are formed from the glycogen This will reduce a hexamine-silver nitrate mixture to a black compound  Special stains Tissues need special stain 1. Mucin stains 2. Glycogen stains 3. Melanin stains 4. Calcium stains 5. Lipochrome, (lipofuschin) pigments 6. Iron (hemosiderin) 7. Fat stains 8. Connective tissue stains  Melanin stains Melanin is normally found in the skin, eye, and substantia nigra of the brain and hair follicles Under pathological conditions it is found in benign tumors, nevus cell tumors and malignant melanomas Demonstration techniques 1-Masson –Fontana “common use one” 2- Schmorl's method 3- The most specific method of all is an enzyme histochemical method called DOPA-oxidase 4- Bleaching techniques 5- Formaldehyde-induced fluorescence  Special stains Tissues need special stain 1. Mucin stains 2. Glycogen stains 3. Melanin stains 4. Calcium stains 5. Lipochrome, (lipofuschin) pigments 6. Iron (hemosiderin) 7. Fat stains 8. Connective tissue stains  Demonstration of calcium Abnormal deposits of calcium may be found in any area of the body Calcium appear deep blue-purple with H&E stain Von Kossa method is used for deposits of calcium or calcium salt In this method, tissue sections are treated with a silver nitrate solution and the silver is deposited by replacing the calcium reduced by the strong light, and thereby visualized as metallic silver Special staining (von Kossa) showing black colored calcium deposits  Special stains Tissues need special stain 1. Mucin stains 2. Glycogen stains 3. Melanin stains 4. Calcium stains 5. Lipochrome, (lipofuschin) pigments 6. Iron (hemosiderin) 7. Fat stains 8. Connective tissue stains  Lipochrome (lipofuschin) pigments These are the breakdown products within cells from oxidation of lipids and lipoproteins They are the wear-and-tear pigments found most common in heart, liver, CNS, and adrenal cortex Lipochrome can be stained by 1-Sudan black B 2- Lipochrome may also exhibit a strong orange autofluorescence in formalin-fixed, unstained paraffin sections 3- Schmorl's methods. 4- Long Ziehl-Neelsen acid fast Lipochrome pigment in the cytoplasm of hepatocytes, H&E stain.  Special stains Tissues need special stain 1. Mucin stains 2. Glycogen stains 3. Melanin stains 4. Calcium stains 5. Lipochrome, (lipofuschin) pigments 6. Iron (hemosiderin) 7. Fat stains 8. Connective tissue stains  Iron (hemosiderin ) Hemosiderin (storage iron granules) may be present in areas of old hemorrhage or be deposited in tissues They present in reticular endothelial organs such as spleen, live,r and bone marrow Hemochromatosis refers to a condition of iron overload associated with organ failure  Perl's Prussion blue reaction It is the classic method for demonstrating iron in tissues The section is treated with dilute hydrochloric acid to release ferric ions from binding proteins These ions then react with potassium ferrocyanide to produce insoluble blue compound ferric ferrocynide (the Prussian blue reaction) Hemosiderin, liver, iron stain  Special stains Tissues need special stain 1. Mucin stains 2. Glycogen stains 3. Melanin stains 4. Calcium stains 5. Lipochrome, (lipofuschin) pigments 6. Iron (hemosiderin) 7. Fat stains 8. Connective tissue stains  Fat stains The oil red O (ORO) stain can identify neutral lipids and fatty acids in smears and tissues The ORO is a rapid and simple stain It can be useful in identifying fat emboli in lung tissue or clot sections of peripheral blood Fresh smears or cryostat sections of tissue are necessary because fixatives containing alcohols, or routine tissue processing with clearing will remove lipids  Fat stain – ORO stain Oil red O stain of fat emboli in lung Oil red o staining for adipogenesis  Special stains Tissues need special stain 1. Mucin stains 2. Glycogen stains 3. Melanin stains 4. Calcium stains 5. Lipochrome, (lipofuschin) pigments 6. Iron (hemosiderin) 7. Fat stains 8. Connective tissue stains  Connective tissue stains Trichrome stain Helps to highlight the supporting collagenous stroma in sections from a variety of organs Helps to determine the pattern of tissue injury Aid in identifying normal structures, such as connective tissue capsules of organs, the lamina propria of gastrointestinal tract, and the bronchovascular structures in lung Chronic active hepatitis with collapse in liver, trichrome stain. Scleroderma with fibrosis of submucosa in stoma trichrome stain. Cerebral abscess in brain, trichrome stain. 30  Connective tissue stains 1- Gordon & Sweets’ method Reticulin stain 2- Gomori’s silver method It is useful in parenchymal organs such as liver and spleen to outline the architecture Delicate reticular fibers, which are argyrophilic, can be seen A reticulin stain occasionally helps to highlight the growth pattern of neoplasm's  The demonstration of reticular fibers Normal liver at medium Normal spleen at high magnification, magnification, reticulin stain reticulin stain 1- Gordon & Sweets’ method 2- Gomori’s silver method  Staining of elastic tissue fibers 1- Verhoff’s haematoxylin 2- Orcein methods 3- Aldehyde fuchsin  Thank you  “If you have a dream, you can spend a lifetime studying, planning, and getting ready for it. What you should be doing is getting started.” –Drew Houston