HISTOLOGIC ® Vol. XXXVIII, No. 1 May 2005 Managing Editor, Gilles Lefebvre Scientific Editor, Vinnie Della Speranza, MS, HTL(ASCP) HT, MT silver (GMS) stain is probably the most widely used fungal stain, however, the somewhat capricious nature of silver staining may challenge even the most experienced of histology practitioners. Insufficient silver impregnation, overstaining that results in high background staining, or undesirable precipitate may all contribute to difficulties in finding stained fungal organisms. Proper determination of staining endpoint with a microscope, in many instances, is essential to a successful outcome. Fig. 1. Positive fungus control. PAS stain, 400X Fig. 2. Positive fungus control. Chromic acid-Schiff stain, 400X As a result, some pathologists will routinely order a periodic acid- A Common Mistake Schiff (PAS) stain in the hope of attaining a rapid and more fool- When Staining for Fungi proof stain. This method is among the most frequently employed Vinnie Della Speranza, MS, HTL(ASCP) techniques in histology labora- Rena Fail, HT(ASCP) tories. It is relatively simple to Medical University of South Carolina perform and reliable, even in Charleston, SC inexperienced hands. The PAS [email protected] stain will reliably demonstrate the polysaccharide-laden wall of Fungal stains remain an important tool in the histology laboratory’s most fungal organisms, with the diagnostic arsenal for identifying infectious microorganisms. exception of Histoplasma Dermatomycoses, or superficial fungal infections involving the skin, are capsulatum.2 rarely a serious health problem in man; however, systemic mycoses with such organisms as Cryptococcus neoformans and Candida albicans can cause life-threatening infections, especially among those who are immune IN THIS ISSUE suppressed. Therefore, fungal stains are often ordered with some urgency A Common Mistake When Staining by the surgical pathologist when hints of fungal infection are observed for Fungi…………………………………… 1 Fatty Tissue Fixation Using Microwave with the H&E stain. Technology………………………………… 3 Methods for Assessment of Proliferation Most fungi are fairly large and their walls are rich in polysaccharides in an in vitro Tissue Model (1,2-glycol groups), which can be demonstrated with Schiff’s reagent or of Oral Epithelia…………………………… 6 1 Rapid Detetion of Lipid in Livers hexamine (methenamine) silver solutions. Both techniques rely on the for Transplantation…………………………11 use of an oxidizer (periodic acid or chromic acid) to create aldehyde To B-5 or Not To B-5 ………………………15 binding sites for the Schiff or silver ions. The Grocott’s methenamine Letter From the Editor………………………18 1 One drawback to employing the fungi. In fact, if we were to substitute the further oxidation of aldehyde PAS for fungal staining, however, periodic acid for chromic acid in the by chromic acid, less density of is that it will stain polysaccharides GMS stain, essentially we will have aldehyde reaction is achieved and wherever they appear in tissues. performed the Jones’ method, which structures with fewer glycol groups This method will stain many intra- specifically stains basement are not demonstrated, such as cellular components in various membranes and reticular fibers. basement membranes and types of normal cells, from a wide collagenous and reticular fibers.”5 variety of animal species. Some of Yes, the Jones’ technique can This statement by Lillie tells us these intracellular components are demonstrate fungi; however, it will why chromic acid is the better listed below3: also stain many other structures, oxidizer when staining fungi, since making the discovery of fungal these organisms have a greater • glycogen organisms much more difficult. density of sugar (polysaccharides) • neutral mucosubstances Whether we are using Schiff or in their cell wall. silver stain, the weaker periodic acid • some epithethial sulfomucins oxidizer creates aldehyde binding While chromic acid continues and sialomucins sites in many more tissue structures. further oxidation of all In this example, we can see how the polysaccharides, complete • colloid of the thyroid selection of an oxidizing agent can conversion in the areas of heaviest • basement membranes significantly determine which concentration takes longer, structures will be stained. This allowing these to remain reactive • reticular fibers choice will maximize the probability with Schiff (or silver).2 Since the of achieving the desired results. walls of fungi are particularly rich • paneth cell granules in 1,2-glycol groups, they stain • adrenal chromaffin cells These observations were actually more intensely than most other quite well known a generation or reactive components in the tissue • neuronal glycolipids two ago. However, this older, yet section. The contrast between fungi still important information can and background is often more • intranuclear inclusions sometimes be overlooked. Modern distinct than what is seen in in vas deferens histology practitioners can use this duplicate sections stained by the • afibrillar cells of arterioles information today to guide surgical PAS reaction3 (see Figs. 1 and 2). of kidney pathologists in selecting the most This discussion suggests that if appropriate staining method fungi are present in your tissue • granules within available. section, they are less likely to be reticuloendothelial cells missed when a stronger oxidizer Bauer (1933) is credited with the like chromic acid is used. • salivary glands earliest report that the oxidation • gastric and Brunner’s glands of sugars (1,2-glycols) with chromic Work in our laboratory has acid creates aldehydes that will convinced us that using chromic • mucin from respiratory tract bind the Schiff molecule.4 Many acid as oxidizer, followed by Schiff, others, including Hotchkiss, yields results superior to PAS for • acinar and endocrine glands, McManus (1960), Spicer, Pearse, fungal staining, as background including the prostate in man and Lillie continued to study the staining is greatly decreased. This aldehyde-Schiff reaction using approach is as simple as doing a Staining of these elements in various oxidizers. It became well typical PAS, yet avoids the need to some tissues may be distracting understood that the strength of the perform a diastase digestion, which when trying to find PAS-stained oxidizer, the duration of exposure, adds unnecessary work and fungal organisms. As a result, and the density of sugars in the expense to fungal staining. The pathologists who utilize the PAS tissue structures of interest method reported by Casella3 for fungal staining will often determine the location where the utilizing potassium permanganate request the stain with diastase Schiff molecule will bind. as oxidizer is another suitable digestion in an attempt to alternative to periodic acid and minimize staining structures Both chromic acid and potassium yields similar results to chromic of little or no interest. permanganate, which are stronger acid when staining fungi. Interestingly, few histology oxidizers than periodic acid, also practitioners question the use of produce aldehydes in Interestingly, chromic acid-Schiff the oxidizer periodic acid in the polysaccharides, but they further (CAS) is remarkably similar to the PAS stain, yet we use the oxidizer attack and destroy those aldehydes method by Gridley6 that can be chromic acid in the GMS stain for if given sufficient time. “Because of 2 found in many histology texts well during routine tissue except that Gridley’s technique Fatty Tissue Fixation processing.2 Tissue thickness and also requires the use of aldehyde Using Microwave fat content are two main factors fuchsin following Schiff. This is causing the problem of under- done to achieve more intense Technology processed tissue. staining.7 The aldehyde fuchsin acts as an aldehyde and occupies Introduction uninvolved linkages of the Schiff Mary Faith Abbuhl, MS Diagnostically, the most critical fatty reagent, thus reinforcing the depth HTL(ASCP)QIHC tissues are breast and lymph node of the stain.2 While we have Andrea Williams, HT(ASCP) specimens. Any delay in processing confirmed that aldehyde fuchsin University Hospitals of Cleveland these specimens may affect patient does in fact intensify the stained Cleveland, OH care. We are continually faced with organisms, we speculate that the [email protected] inadequate processing of fatty Gridley method is not widely used tissues, most often due to both the today because the aldehyde fuchsin Abstract thickness of the sections when stain requires the use of The microwave oven has been in grossed in and their fat content. paraldehyde, a controlled substance use in the histology laboratory for The use of alcoholic formalin in that may be problematic to obtain many years. In recent years, preprocessing and processing, often in some laboratories. laboratory-grade microwave recommended as a solution to this instruments have become more problem, is not an option for some In conclusion, the next time your widely adopted as standard immunohistochemical procedures, pathologist requests a PAS for equipment in many laboratories.1 especially those typically used for fungus, we recommend that you These microwave units may be breast samples. We have found that suggest the CAS as a much better used to accelerate a number of formalin fixation done in a alternative. It makes little sense to laboratory procedures, including microwave oven offers a suitable utilize an oxidizer that will create fixation, processing, decalcification,