DAMS - LAST LOOK: Pathology

LAST LOOK Pathology LAST LOOK Pathology Largest Medical Education Institute in the Country Published by Delhi Academy of Medical Sciences (P) Ltd.HEAD OFFICE Delhi Academy of Medical Sciences (P.) Ltd. 4-B, Grovers Chamber, Pusa Road, Near Karol Bagh Metro Station, New Delhi-110 005 Phone : 011-4009 4009 http://www.damsdelhi.com Email: info@damsdelhi.com ISBN : First Published 1999, Delhi Academy of Medical Sciences 2018 DAMS Publication All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic, mechanical, including photocopying, recording, or any information storage and retrieval system without permission, in writing, from the author and the publishers. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials.

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	Histotechniques and Special stains

TECHNIQUES IN PATHOLOGYHistopathological Techniques- All histological procedures can be divided into a similar series of steps. For the paraffin method these steps are as follows: I. Tissue resection II. Fixation III. Dehydration IV.

Infiltration and embedding in paraffin V. Sectioning with a microtome VI. Mounting on microscope slides VII. Clearing and Staining VIII. Preparation of permanent mounts Formaldehyde, as 10% neutral buffered formalin (NBF) is the most common fixative used in diagnostic pathology. Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced.

Zenker's fixatives are recommended for reticuloendothelial tissues including lymph nodes, spleen, thymus, and bone marrow. Zenker's fixes nuclei very well and gives good detail. Bouin's solution is sometimes recommended for fixation of testis, GI tract, and endocrine tissue. Glutaraldehyde is recommended for fixation of tissues for electron microscopy. Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap.

Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned, there is no problem from induced brittleness. For fixing frozen sections, you can use just about anything-- though methanol and ethanol are the best Formic acid in a 10% concentration is the best all-around decalcifier. The presence of a fine black precipitate on the slides, often with no relationship to the tissue (i.e., the precipitate appears adjacent to tissues or within interstices or vessels) suggests formalin-heme pigment has formed. This can be confirmed by polarized light microscopy, because this pigment will polarize a bright white (and the slide will look like many stars in the sky). Tissues such as spleen and lymph node are particularly prone to this artefact. SPECIAL STAINSMucin stains There are a variety of mucin stains, all attempting to demonstrate one or more types of mucopolysaccharide substances in tissues.

The types of mucopolysaccharides are as follows:

Type of mucin	PAS	Alcian blue
Neutral	Positive	Negative
Acid simple- epithelial	Positive	Positive at pH 2.5
Acid simple- mesenchymal	Negative	Positive at pH 2.5
Acid complex	Positive	Positive at pH 1

Melanin stains Melanin is normally found in the skin, eye, and substantia nigra. It may also be found in melanomas. The commonly used Fontana-Masson ("melanin stain") method relies upon the melanin granules to reduce ammoniacal silver nitrate Schmorl's method uses the reducing properties of melanin to stain granules blue-green Lipochrome stains These are the breakdown products within cells from oxidation of lipids and lipoproteins. They are the wear-and-tear pigments found most commonly in heart, liver, CNS, and adrenal cortex (zona reticularis). The less highly oxidized "ceroid" pigment of testis interstitium and seminal vesicle is another form of lipochrome. Lipochrome can be stained by Sudan black B Iron (hemosiderin) Hemosiderin (storage iron granules) may be present in areas of old hemorrhage or be deposited in tissues with iron overload Perl's iron stain 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 an insoluble blue compound (the Prussian blue reaction) Calcium stains Only calcium that is bound to an anion (such as PO4 or CO3) can be demonstrated. Calcium forms a blue-black lake with hematoxylin to give a blue color on H&E stain, usually with sharp edges. VonKossa stain is a silver reduction method that demonstrates phosphates and carbonates, but these are usually present along with calcium. This stain is most useful when large amounts are present, as in bone. Alizarin red S forms an orange-red lake with calcium at a pH of 4.2.

It works best with small amounts of calcium (such as in Michaelis-Gutman bodies) Copper stain The rubeanic acid and rhodanine stains are utilized to detect the cytoplasmic accumulation of copper in the liver Fat stains The oil red O (ORO) stain can identify neutral lipids and fatty acids in smears and tissues. Fresh smears or cryostat sections of tissue are necessary because fixatives containing alcohols, or routine tissue processing with clearing, will remove lipids. It can be useful in identifying fat emboli in lung tissue or clot sections of peripheral blood. ANTICOAGULANTS USED IN HEMATOLOGY EDTA and sodium citrate remove calcium which is essential for coagulation. Calcium is either precipitated as insoluble oxalate or bound in non ionized form Heparin binds to anti thrombin thus inhibiting interaction of several clotting factors EDTA is used for blood counts Sodium citrate is used for coagulation testing and ESR (westergren method) Any anticoagulant can be used for collecting blood for flow cytometry EDTA sodium and potassium salts are powerful anticoagulants - dipotassium salt is the most preferable excess of EDTA can cause shrinkage and degenerative changes of both red cells and leukocytes platelets can swell and disintegrate leading to an artificially high platelet count leucoagglutination can occur (naturally occurring anti platelet antiplatelet antibody- if seen, blood collected again in citrate) used for ESR with Wintrobe method (double oxalate may also be used for the same)

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