Histologic® Celebrates Its Reviewed Predictions That Had Been Have Not Changed Much; Microtomy 30Th Anniversary! Over the Years It Made for the 1970-1980 Decade

HISTOLOGIC ® T echnical Bulletin for Histotechnology Vol. XXXIV, No. 2, Nov. 2001

it has made a difference in spread- web site at www.sakuraus.com. 30 Years and Still ing valuable information to histo- It is interesting to realize that some Going Strong technologists and pathologists of the same conclusions made then around the world. also apply today. Faster, more Vinnie Della Speranza automated processing systems are Scientific Editor In 1981 for this publication’s 10th not yet here; automated stainers are anniversary, Lee G. Luna, editor, now widely used, but their principles This year HistoLogic® celebrates its reviewed predictions that had been have not changed much; microtomy 30th anniversary! Over the years it made for the 1970-1980 decade. has not evolved significantly except has been read by close to 1,000,000 The original article can be retrieved for the more widespread use of people worldwide. We believe that from the Sakura Finetek U.S.A. motorized units.

21 There have been advances, how- times are critical in advancing ever, with automated coverslippers. The Golden Age technology and histologic technique. For example, by using the Tissue- Barry R. J. Rittman, PhD Tek ® SCA™ Film Coverslipper, Much of the work being carried out slides can be viewed sooner under My perspective is largely colored today is based on the discoveries the microscope, thanks to the fast by my training as a medical labora- made in the 18th and 19th centuries. drying time. The fact that it cover- tory technologist and histologist in It was not until the 1820s that slips one slide every 3 seconds also England and the United States, microscope lenses which corrected helps! Slide labeling now can be including my training in electron for chromatic aberration were being done without using paper labels manufactured, usually on a custom microscopy and image analysis, and 1 and practical bar-coding is on the my teaching in histology and basis. Slides used in the 18th and horizon. Other areas of rapid histologic technique. This is a early 19th centuries were made of growth are immunohistochemistry somewhat different perspective ivory with a central hole; the and in situ hybridization. than for most histotechnologists. specimen was covered on both surfaces with small pieces of mica. What progress will be made over By the 1830s, glass plates were in the next 10 years? From the devel- use. These were often known as slides "1 ן "opment of the very first microscope, sliders. The standard 3 histopathology became a discipline still in use today were recommended largely of visual images. (Be sure to by the Microscopical Society of read “ The Golden Age” in this London around 1839. Cover glasses issue.) Diagnosis continues to rest were starting to be made thinner by upon interpretation of those very the mid-1800s. In 1855, Chance images, in their spectacular color, manufactured cover glasses, but it which is the hallmark of what we was left to Beale in 1880 to produce do in the laboratory. With the ever cover glasses that were thin enough increasing sophistication of com- to take advantage of the high dry puters, and as imaging devices objectives then being manufactured.1 become easier to operate with better quality and cheaper price By 1840, the major cell structures tags (see “ Histology and Digital such as mitochondria and chromo- Imaging — Present and Future Most technologies have a golden somes had been discovered and both Technologies” in this issue), the age— an era when advances are mitosis and meiosis described. The days of pulling (and refiling) glass continually being made which activity of cells in embryology, ferti- slides for conferences, tumor board, significantly move that technology lization, pathology, and physiology or teaching may well be behind us. forward. What do we consider to be was the subject of investigation at How long will it be before the glass the golden age of histologic tech- about this time. The concept of fixa- slide becomes relegated to the nique? Depending on outlook, tion was not yet developed but museum display case? some might believe it to be the tissues were “ hardened” in alcohol, 19th century when independent chromic acid, and formalin. There are strong indications that work by “ gentlemen scientists” Bracegirdle cites the following dates automation of the embedding developed early methods for for the introduction of substances process will become reality in the examining tissues which still form for either hardening or fixing tissues: near future. Faster, more efficient the basis of much of our current alcohol 1743; mercuric chloride 1846; processing technologies are also at histology work. Others might look potassium dichromate 1860; picric the testing stage. This could lead to acid 1879; Bouin’s fixative 1897; and at the developments within the past 2 drastic changes to the workflow of two decades and suggest that the formaldehyde 1893. routine anatomic pathology explosive technological develop- laboratories. I can only wonder if ment that has taken place now puts Despite the scarcity of stains prior to today’s sophisticated motorized us in the middle of an exciting the 1850s, there are several publica- microtomes are paving the way for golden age. Still others, in trying to tions with very detailed histology a day when instruments will be able look ahead, may think that the which came from meticulous to section tissue at high speed golden age is yet to come— that we observations of unstained tissues. without human intervention. are merely the pathfinders for van Leeuwenhoek is credited with ensuring the advance of histologic using the first stain, where he showed Since HistoLogic is here to stay for contrasting muscle fibers with technology.All are correct in their 2 many years to come, it will be fun own way and only vary in percep- saffron around 1714-1719. In the to revisit these predictions in 2010. tion. It is certain that specific key early years of microscopy, carmine events and discoveries at optimal and indigo were the most commonly

22 used stains, starting around 1850. I began working in an oral histo- sections for frozen section diagnosis. Aniline dyes were not readily pathology laboratory in London, While freezing microtomes using available until 1856 and many of England, in 1957, transferred to the liquid carbon dioxide was in com- these were adapted from the dyeing University of Iowa in 1968, and then mon use, the cryostat allowed for of fabrics. However, stains provided a to the University of Texas in 1989. both the preparation of sequential technological leap forward allowing While in London, pathologic speci- sections that remained frozen and distinctions between tissue com- mens had to be produced in a timely the comparison of histochemistry on ponents whose existence up to that manner but there was ample time unfixed sections with those fixed by time had been controversial. 3-5 for study, reflection, experimentation various other methods. Some interesting observations can with new and old methods, and be found in documents from the late personal training. Technicians Image analysis and three- 19th century.The book by Miller carried out all the histology, histo- dimensional reconstruction were (1887) opines, “ nine-tenths of the chemistry, electron microscopy, carried out routinely using a microtomes are purchased because museum technique, and tea making. camera lucida and a pencil, pro- of failure in free hand work with a jecting images on flat sheets of wax. dull knife.” 6 Miller used free-hand This was an exciting time, when new In this way individual features sectioning and hardening agents discoveries about the structure and could be traced, and by cutting such as alcohol and chromic acid. physiology of the cell were being these out and sticking sequential Teeth were decalcified in chromic made on a regular basis. The sections together, a three- acid. Sections were produced by electron microscope explored anew dimensional reconstruction was infiltrating from alcohol into the structure of cells and tissue, and prepared. Some reconstructions Bayberry tallow or in celloidin. in many ways its application of individual enamel spindles Wax sections were collected in paralleled the use of the optical produced in this manner are still benzol and treated as free-floated microscope in the 19th century. regarded as classic examples. The sections. Staining was done with Along with the use of the electron amount of work in tracing hundreds hematoxylin and eosin, borax car- microscope came the need for or thousands of features in serial mine, or carmine plus picric acid, improved techniques to visualize sections can only be imagined. and sections were dehydrated and tissue, as well as the development of Stereology by manual analysis of permanently mounted. The per- ultramicrotomes, glass knives, new individual images provided valuable manence of some of these tech- processing techniques, electron information but again was niques is seen in a section (that I dense stains, and histochemical exceedingly time-consuming. possess) from 1861 of cerebellum techniques that were applicable stained with borax carmine in which specifically for electron microscopy. Tissue had always been processed the details are still crystal clear. A point of interest here is that manually.An automatic processing Weigert’s method for myelin and Gustav Mann recommended the use machine, known as the Auto- Gram’s stain are also from this of glass knives to prevent contam- technicon, was described in 19092; period (1884). Metallic impreg- ination of sections that would be 8 nations using silver and gold salts tested for the presence of iron. Managing Editor, Gilles Lefebvre were used by some investigators A useful reference in the early Scientific Editor, Vinnie Della Speranza, during this era. development of electron microscopy MS, HTL (ASCP) HT, MT instrumentation and techniques is By 1890, optical microscopes had Hall.9 The general belief was that reached the level of theoretical the improved resolution of the IN THIS ISSUE resolution using transmitted light. electron microscope would revolu- 30 Years and Still Going Strong ……………21 Pearse cited the iodine reaction for tionize routine diagnosis. In most The Golden Age ……………………………22 starch by Colin and de Claubry in cases this has not been realized. A Field of Poppies ……………………………25 1815 as one of the first histochemical Histology and Digital Imaging — 7 methods. In the 1930s and 1940s, In light microscopy, the emphasis Present and Future Technologies……………27 there was a revival of interest in was on the quality of sections. A Simple Method to Demonstrate understanding the basis of Results needed immediately in the H pylori and Mast Cells ……………………32 histochemical reactions and operating room used tissue fixed Concepts in Epitope Retrieval developing new techniques. Staining in hot formal-saline for 1 minute, for Immunohistochemistry …………………33 techniques didn’t progress rapidly followed by frozen sections and Marine Histopathology at until after the Second World War— rapid staining to obtain a section Cooperative Oxford Laboratory……………37 before and during the war, aniline within 4 minutes of receipt of the Ups and Downs of a Histology Student …………………………40 dyes were unavailable from tissue. The introduction of the Latex Allergy…………………………………41 Germany.The first transmission cryostat and its popularity due to Reprocessing of Tissue Blocks ……………42 electron microscope was introduced the work of Everson Pearse revolu- Mark Your Calendar…………………………43 by Knoll and Ruska (1932). tionized the production of uniform

23 it offered automatic transfer of antibodies were also developed. The understands the technology but is specimens to a new solution and historical development of histo- able to make it work and then then to baths of molten paraffin. chemistry is summarized in Pearse.7 provide that information in a However, the Autotechnicon only reproducible and reliable manner. gained popularity in the United Monoclonal antibodies were intro- States, and was later replaced in the duced in immunohistochemistry by There are usually rapid steps forward late 1970s with closed processors Cuello et al.10 The use of lectins to in instrumentation followed by a offering the capability of several demonstrate carbohydrate moieties much longer period during which different programs. Microtomy started to become popular in histo- application of that technology occurs. utilized rotary machines such as the chemistry in the 1970s and 1980s I believe that single cell analysis such MSE and the AO 820— the latter when it was recognized that their as laser scan microdissection is one regarded by many as the ultimate specificity was similar to antigen- technique that will be in common microtome of its day. Cambridge antibody reactions.11 PCR and in situ use. Antibodies will be used to Rocking microtomes were still used hybridization are commonplace localize most substances and their by many and a modified sturdier today and allow us to visualize not concentrations within blocks of vital model formed the basis of the initial only where substances are located tissue. Images will be scanned, commercial cryostats. (The Rocking but also whether they are being analyzed, and compared with normal microtome was introduced in 1881 synthesized. Automated machines locations and values, all in automated and the rotary microtome in 1883.) for staining and also for immuno- fashion. The histotechnologist will histochemical reactions are now in still remain a vital link in this common use. Antigen retrieval process. While many things can be techniques have, in many instances, automated, the ability of a good circumvented many of the limita- histotechnologist to solve problems tions imposed by fixation. is not yet matched by a machine.

Today, confocal microscopy allows In looking back at previous work in us to look at structures and localize the 19th century, we can only be some materials in intact blocks of awed by the wealth of information tissue. The confocal microscope gleaned by simple instrumentation (originally known as the tandem and meticulous observation, but at scanning reflected light microscope) the same time amused by some of was introduced by Egger and Petran the erroneous deductions by in 1967 using a Nipkow disk system.12 investigators. I wonder how future Digital photography has largely histologists will view our attempts eliminated the need for with what we now regard as photographic film of gross speci- sophisticated instrumentation, mens and sections. New antibodies techniques, and training. are being produced almost on a daily basis. Automatic tissue References 1. Bradbury S. The Evolution of the Microscope. processors and automatic stainers Pergamon Press. London; 1967. are commonplace. 2. Bracegirdle B. 1978. A History of Microtechnique. Cornell University Press; 1978. 3. Conn HJ. 1948. History of Staining. Commission on During the 1950s and 1960s, paraffin In 1902, Gustav Mann suggested Standardization of Biologic Staining. 2nd ed. 1948; New York, NY. wax sections were routinely cut at that “ we had learnt almost all we 4. Gierke H. 1884. Färberei zu mikroskopischen 6-7 microns. Double embedding was could of the organelles in the cell.” 8 Zwecken. Zts. Wis. Mikr.1884;1:62-100. 5. Lewis FT.The introduction of biological stains: used routinely for hard specimens During the 1970s when image employment of saffron by Vieussens and and celloidin for large specimens. analysis was rapidly developing, the Leeuwenhoek. Anat Rec. 1942;83:229-253. 6. Miller MN. A course of normal histology for students Histochemical reactions that were suggestion was that automatic image and practitioners of medicine. Practical Microscopy. used included von Kossa technique, analysis would allow rapid and New York, NY:William Wood and Co; 1887. 7. Pearse AGE. Histochemistry Theoretical and Perls’ Prussian blue, Periodic Acid reproducible diagnosis of conditions Applied. Vol 1. Preparative and Optical Technology. Schiff, and the Feulgen reaction. such as with the Pap smear. This New York, NY: Churchill Livingstone; 1980. 8. Mann G. Physiological Histology. Methods and Immunochemistry and immuno- has only partially materialized. Theory. Oxford: Clarendon Press. 1902. cytochemistry came into existence Automated systems such as image 9. Hall CE. Introduction to Electron Microscopy. New York, NY: McGraw Hill; 1953. during this time. In 1967, Gibb et al analysis and receptor data can 10. Cuello AC. 1993. Immunohistochemistry II. IBRO. produced antibodies to dopamine simplify the tasks and provide more (handbook series Vol. 14). Wiley Publishing; 1993. 11. Liener IE, Sharon N, Goldstein IJ.The Lectins. ß-hydroxylase, and in 1969, information for us to use in diag- Properties, Functions and Applications in Biology Geffen et al used indirect immuno- nosis, however, they cannot replace and Medicine. New York, NY:Academic Press; 1986. 7 12. Egger MD, Petran M. New reflected light microscope fluorescence on adrenal glands. the final arbiter— the pathologist. for viewing unstained brain and ganglion cells. During this period, enzyme-labeled The histotechnologist not only Science. 1967;157:305.

24 green separates: a wool blazer, a and hope for the best. In 1905, A Field of Poppies cotton turtleneck, and a silk scarf. Curtis recommended ponceau S for Richard W. Dapson, PhD All have to be the same color, and trichromes in a paper written in Anatech, Ltd. each must be treated with a dif- French, but the chemical identity of ferent dye to achieve that color that dye is unknown. Since then, Adapted with permission from because each fiber reacts with dyes dozens of red dyes have been Mikro-Graf, vol. 26, no. 4, Michigan uniquely. Eddie Bauer couldn’t suggested as Curtis’ ponceau S. Society of Histotechnologists care less about the names of the A strikingly parallel story began dyes used to achieve that end, and a few years later when Masson Recently, a histotech reported the manufacturers pull names (also French) reported on the use having difficulty with her seemingly out of a hat. The same of ponceau de xylidine in tri- trichrome, a procedure that had dye may be sold under any of chromes, another mystery dye that “ always worked before.” Only one dozens of names, and different dyes has come to be equated (somewhat change had occurred that coincided are given the same or nearly arbitrarily) with ponceau 2R. with the disaster: her original identical names. supply of red dye, which had been With that as background, is it any around for many years, had been Many dyes have letters appended wonder that we have trouble consumed. A newly purchased dye to their names, e.g., ponceau S. obtaining the same product year of the same name had been used, Sometimes these have real after year? We can achieve some with very unsatisfactory results. meaning, designating a shade measure of consistency by relying The red dye in question is ponceau (eosin Y for yellowish, as opposed on Colour Index numbers (C.I.#), S, one of the most confused dyes in to eosin B for bluish). Occasionally, numerical designations assigned to histology. Ponceau is French for the letter denotes a chemical nearly all dye molecules by the poppy, an allusion to the poppy- characteristic. Alizarin contains no Society of Dyers and Colourists. red color of the aqueous solution. ionic groups and is insoluble in These numbers provide a stan- water. Add a sulfonic acid group to dardized identification for each dye the molecule and you have alizarin that transcends language, culture, red S (S for sulfonic), the familiar, and whim. If your old bottle has a water-soluble dye used to stain C.I.# on it, order a new bottle with calcium deposits. Sometimes the the same number. Really old letter is doubled (ponceau SS) or bottles will show a three-digit a combination of letters and number from the 1st edition of the Conn’s Biological Stains (9th ed., numbers is used (crystal ponceau Colour Index; current numbers 1977) lists 17 dyes whose name 6R, used in Lendrum’s MSB stain from the third edition have five starts with the word ponceau, but for fibrin); in many of these digits. Conn’s Biological Stains has doesn’t include three others that are instances, a deeper, richer color is a table matching the two systems. available currently.Among all of implied. Here is where a good bit those, there are two called ponceau of the problem lies. So what did our friend have S, a ponceau SS, and two given the that was labeled ponceau S? designation ponceau SX. To com- To match Eddie Bauer’s woolen Here is a list of likely candidates: pound the problem further, there is color swatch, the dye manufacturer a crystal ponceau 6R, a brilliant may make an extra pure batch to C.I.# Name ponceau G, and an acid ponceau. heighten the color, or might add 14700 ponceau SX How is a person ever going to inert ingredients to tone down 15635 ponceau S replace an old dye with an equiva- the color. Chemically, it could 16140 ponceau scarlet lent one? There are ways, better be the same dye molecule. 27190 ponceau SS today than a few years ago, but still Meanwhile, another manufacturer 27195 ponceau S they are not foolproof. Here’s why. could take a different dye, fiddle with its concentration to match These differ from one another in The names of dyes are assigned the cotton swatch, and give it a several ways, as the molecular arbitrarily by manufacturers, most name similar or identical to that models show (Figs. 1-5). I have of whom are in business to serve used to dye wool. designed these models so that the textile trade. Their customers important features, such as ionic do not care which dye is used, only Biological supply houses then buy groups, are prominent. Sulfur is that it produces a given shade on a small quantities of these dyes, yellow, oxygen is green, nitrogen is particular fiber or fabric. sometimes keeping the name intact blue, carbon is gray, and hydrogen or changing it to one of their own. is black. Each molecule has one For example, Eddie Bauer wants Years ago, histologists had little hydroxyl group (green oxygen with to assemble a collection of mint choice but to buy what was offered one black hydrogen atom) but

25 Compare C.I. 27190 with C.I. 27195 (Fig. 6). The former has two sulfonic acid groups stuck on one end, leaving a large, uncharged portion dangling free of the tissue. This is a less stable configuration because of the loose end. The latter has four sulfonic groups spread all over the molecule, providing a much more reliable fastening to cytoplasm.

Fig. 1. Ponceau SX, C.I. 14700. Two rings, each Fig. 5. Ponceau S, C.I. 27195. Three rings with a sulfonic acid group. and four sulfonic acid groups, spread all over the molecule.

these probably play no role in our story. Sulfonic acids (yellow sulfur with three green oxygen atoms) are important because they bear a negative charge that could bind to cytoplasmic proteins.

See how the dyes vary? The first three molecules each have two sets of rings, while the rest have three sets. The number of sulfonic acid Fig. 2. Ponceau S, C.I. 15635. Two rings, but only groups ranges from one to four. one sulfonic acid group. The number and placement of these groups directly affect how they interact with the tissue in a Fig. 6. Comparison of C.I. 27190 with C.I. 27195. trichrome stain. Now look at the remaining dyes. Trichromes work by competitive With only one opportunity for ionic displacement of red dye by attachment, C.I. 15635 (Fig. 2) blue or green dye from select tissue probably will not provide as clean elements (e.g., collagen). The red a trichrome as C.I. 14700 (Fig. 1) or dye must be bound tightly to C.I. 16140 (Fig. 3). cytoplasm to resist displacement. Collagen is weakly ionic and My guess is that our friend had attracts the blue or green dye by C.I. 14700, 16140, or 27195 in the nonionic forces (probably some original bottle (if in fact the dye Fig. 3. Ponceau scarlet, C.I. 16140. Two rings and type of van der Waal’s force). two sulfonic acid groups, but both are on was one of those listed above). the same ring. Cytoplasm is more strongly She probably purchased C.I. 15635 charged and attaches to dyes via as the replacement, as this is ionic bonds. Red acid dyes that are commonly sold as ponceau S. well charged should bond strongly If I were to use one of these in a to cytoplasm, especially if their trichrome, I would choose 27195 negative charges are spaced evenly because of its uniform distribution around the molecule. Those with of many ionic groups. fewer charges, or with several charges concentrated at one end of the molecule, will be more easily Acknowledgment dislodged by the blue or green dye. Peggy Wenk of William Beaumont Hospital, Royal Oak, Mich, first told me about the histotech who reported the This will produce muddy cyto- problem to her following one of Peggy’s workshops. plasmic colors.

Fig. 4. Ponceau SS, C.I. 27190. Three rings and two sulfonic acid groups, but both are on the same ring, leaving a long floppy tail that will not bond to cytoplasm. 26 into a standard JPEG image file state of the art. These examples are Histology and Digital or a low-resolution streaming offered for illustrative clarity and Imaging – Present and video output. Useful for simple comparison. However, it is low-resolution telepathology3 and very important to be aware that Future Technologies conferencing, standard video there are other comparable cameras do not produce an image of competitive models available and James H. Nicholson high enough quality to satisfy most new ones will be forthcoming. Research Image Core Facility diagnostic pathologists. Department of Pathology and Single Chip (One-pass) Laboratory Medicine Digital Cameras Color Camera Medical University of When the A/D converter is incor- The most common method used to South Carolina porated in the camera itself, it is generate color is to incorporate considered a digital camera because color filters directly on the sensor Introduction the output is in a computer readable so that different pixels are sensitive The practice of histotechnology is image file format. At the least to red, green, or blue light (RGB). devoted to the creation of visual expensive level, the imaging sensor The actual array of filters is usually information. A change in image differs very little from a standard by rows of pixels, most often in a recording equipment as well as video camera. To produce image pattern of Green-Red-Green-Blue. This means that one-half of the ,480 ן image storage, retrieval, and dis- resolutions greater than 640 semination technologies funda- larger nonvideo-standard sensors pixels are sensitive to green light mentally affect the usability and are used with resolutions that range and only one-fourth each to red value of the histology lab’s output. into the millions of pixels (picture and blue. The rest of the image data Few technologies have developed as elements). Besides pixel count is filled in by digital interpolation. rapidly as digital imaging. That in (image resolution), the cameras are A1 million pixel count one-pass turn has created profound change in further distinguished by the manner color camera produces a digital all imaging applications, from home in which they form color images. image file with 3 million data points pictures to magazine publication. These design differences are im- (3 MB), but only one-third are Histology is certainly no exception. portant to the cost, ease of use, and derived from actual resolved image The advent of relatively inexpensive types of application, so they will be information. This has contributed and easy-to-use digital microscope discussed individually. For each to giving first generation digital cameras has opened new possi- camera type, a specific commercial cameras a reputation for inadequate bilities for the pathologist and other example will be offered from resolution, especially for low- consumers of histologic material. models that represent the current power microscopy. Cameras A discussion of digital imaging must first focus on the often confusing and rapidly expanding range of available cameras.1 They all share the use of a solid-state sensor, most often a CCD (Charge Coupled Device).2 In fact, a CCD produces an analog signal, so the creation of a digital computer image file requires the use of an A/D (analog to digital) converter.

Standard Video and Image Capture When this conversion takes place outside of the camera, it is usually called image capture. Inexpensive “ frame-grabbers” now cost less than one hundred dollars. They achieve simplicity of use through “ plug-and-play” USB (Universal Serial Bus) interfaces and very user-friendly software. The output Fig. 1. The Nikon Coolpix 995 is a high-end prosumer-grade fixed-lens digital camera that can be of any standard video source or mounted on a microscope with an optical adapter. The half of the camera containing the LCD camera can be captured quickly display can be freely rotated for the optimal viewing angle.

27 One solution is to increase the total number of pixels until the resolution becomes acceptable, despite the interpolated nature of some of the image data. Current models of prosumer-grade digital cameras have pixels 2000 ן resolutions above 1500 (3 megapixel). With the addition of an inexpensive microscope adapter, they are converted to a surprisingly capable microscopic camera, even at low magnifications. The Olympus C-3030,4 the Kodak MDS 290,5 and the Nikon Coolpix 9956 (illustrated) are all examples priced below $3000. The Nikon Coolpix 995 has the advantage in that the center pivot allows the microscope user to orient the view screen for the most convenient angle. The addition of a USB interface and simple control image of placenta (Frazier-Landrum fibrin stain) taken with a (ןFig. 2. (A) A low power (4 software allows the capture of relatively inexpensive Nikon Coolpix 995. (B) An enlargement of a small area of the image showing images directly to the user’s the surprisingly credible low power performance of the high pixel count (3.34 million) sensor of the computer. These cameras do type being used for top-quality mass-market digital cameras today. a reasonably good job of automatic the small portable LCD screen. reports and records, teaching, white balancing for a neutral back- The SPOT INSIGHT 7 color camera research, and publication. ground, but they lack some of the pixel mosaic 1200 ן uses a 1600 image processing features of more color CCD that can operate at a rate Network Microscope Camera specialized microscope cameras. up to 15 frames a second for near The increasing popularity of One frequent complaint is the real-time focusing. At a price tag of telepathology8 and image exchange difficulty of focusing on the small some $4000, it provides a practicing with remote collaborators has LCD screen, which is not optimally pathologist with an easy-to-use, high- created a need to integrate digital designed for microscope imagery, quality microscopic instrument which cameras directly into networks. especially at low power. These can produce images suitable for The Nikon DN100 Digital Network cameras are a good choice for a lab on a tight budget or one that needs a camera for occasional microscope use. One advantage is the camera can be easily removed from the adapter and used for other imaging chores around the lab.

Dedicated Single Chip Color Microscope Camera Another group of cameras uses sensors of similiar size and performance to the prosumer cameras, but they differ in that they are designed for dedicated microscope use. These cameras use custom software to perform specialized operations like shading correction (subtracting an image of the microscope light field to ensure a flat uniform background). Often, shading correction, exposure time, and white balance can be stored for each microscope objective ensuring optimal quality at all magnifications. Full monitor screen Fig. 3. The SPOT INSIGHT is a dedicated microscope camera. The computer interface and software focusing overcomes the handicap of provide full computer monitor screen focusing and advanced image processing features especially designed for microscope imagery. 28 (at a reduced resolution), and downloaded at full resolution. Operation is nearly as simple as a home web-camera, but with an image quality that will support serious histological diagnosis.

High Resolution Multi-pass Cooled Color Camera There are two methods that can overcome the inherent resolution limitations of the single-sensor color camera. One method is to have three separate sensors with an assembly of prisms and filters to deliver the correct wavelength to each CCD. This allows for the simultaneous acquisition of all three colors. This is a very popular and successful strategy in the design of high-end professional video cameras. The expense and bulk of three high- Fig. 4. The Nikon DN100 is a dedicated network camera. A remote user can connect with it through a resolution sensors make this option standard web browser to view and download images. less practical in digital cameras. A less expensive option is to have a Camera9 is an example of a new class of a host computer. It has a built-in single high-resolution monochrome of instruments that will undoubtedly network connection that allows the sensor and a tricolor filter selector .pixel operation of the camera from a that acquire each color separately 960 ן grow rapidly.A 1280 camera, not dissimilar to instruments remote location via a standard This does not allow the camera to be already described, features a Internet link. Essentially, the remote used for moving objects because of specially designed control box that user calls up the camera as if it were the inherent time lag between color allows the acquisition and transfer a web site. Images can be viewed at a channels, but it is quite workable for of images with or without the use frame rate of one every few seconds static histology specimens. A popular example of this class of camera is the pixel 1200 ן SPOT RT10 with a 1600 count CCD that produces a 5.76 MB image, similar in size to many of the single-chip cameras.

The difference is that all of the pixels represent actual image data rather than interpolated data, producing a substantially higher quality image even though the file size is the same. In this class of research-grade cameras, the CCD is often electronically cooled, providing digital noise suppression for the long exposures required in fluorescent techniques such as FISH.11

Another adaptation to achieve higher resolution is to combine some of the characteristics of a digital camera and a scanner to offset or step the sensor to achieve higher sample sizes. Examples of this class of “ pixel-shifting” Fig. 5. The SPOT RT uses the same software interface as the SPOT INSIGHT but offers higher 12 resolution images through three-pass color capture. In addition, it is electronically cooled for long cameras include the Zeiss Axiocam exposures to support extreme sensitivity for weak fluorescent signals. This model has the optional and the Nikon DXM 1200.13 These filter slider that allows the camera to operate in a true monochrome mode, bypassing the color filters.

29 cameras can generate extremely objective to scan an entire micro- negative, slide, and print file cabinets large image files that give excellent scope slide at resolutions up to of an earlier era. In addition, a well- results even at very low magnifi- 54,000 pixels per inch. For compari- designed image database can support cations. Coupled with powerful son, a typical high-resolution 35mm recordkeeping, reports, research, and computer image processing and slide scanner operates at a teaching18 with different software deconvolution software, these resolution of 2000 pixels per inch. packages drawing on the same image instruments are stretching the The resulting slide contains all of files. This can end the need for theoretical limits of optical light the image information from the separate sets of transparencies for resolution.14 Prices for this class of original tissue slide as if each field teaching, prints to file with reports, and negatives to archive. To achieve ןcameras vary from $7000 to $15000, were photographed with a 20 making them more suitable for objective. The resulting file is 3 GB this level of functionality requires research and publication purposes in size and even with image com- designing an integrated camera/ than routine histological records. pression, only about three images database/network system19 for the can be stored on a CD-ROM. archiving and communication of This description of camera types is Despite its exciting potential in medical images.20 hardly exhaustive but does reflect research and teaching, the huge the great range of pricing and capa- volume of data will require the For example, consider a surgical bility that the modern histologist can development of cheaper network pathologist working in a teaching choose from for digital imaging. A storage, writeable DVD-ROM disks, hospital environment. In this case, recent informal survey of practicing and higher bandwidth networks to the system needs to be capable of histologists and pathologists at the support widespread adoption. acquiring and storing microscopic Medical University of South Carolina and private labs in the area indicated that approximately 79% of images in their work were currently digital as opposed to 21% film. Eighty percent of the respondents expected to abandon film images completely within five years, indicating continued growth for these instruments.

The Virtual Microscope It is a safe prediction that digital cameras will continue to evolve with higher resolving power, greater ease of use, and lower prices. In addition, a new class of instruments is emerging— the virtual microscope.15 The concept is to digitally capture and store all of the visual information contained in a histological sample and allow the field to be scrolled and the magnification varied in a manner similar to a microscope. Earlier versions have used a combination of low-power Fig. 6. A well-designed image database such as ImageQUEST is an essential component of any image archiving system. It maintains good organization and security for large and diverse image collections scanning with a transparency while making them instantly accessible through accession coding, demographic criteria, or Boolean scanner16 and indexed higher mag- search. nification images. Other systems use software to “ tile” or “ stitch” indi- Picture Archiving and and gross images that will be used vidual high magnification images in Communications System (PACS) for reports, records, consultation, a manner similar to the software The efficiency and ease of use of clinical/pathological conferences, that stitches together individual digital cameras encourage their use quality assurance, teaching, and snapshots to form a panoramic for routine documentation. This possibly research. The acquisition view. An interesting new instru- quickly results in the rapid accum- process must be streamlined to ment currently in prototype is the ulation of large numbers of images. reflect the time and case flow Aperio ScanScope17 microscope Fortunately, digital files have the pressures in the modern diagnostic slide scanner. The ScanScope uses potential for more compact and well- lab. The image database must be an interchangeable microscope organized storage compared to the integrated into, or at least 30 compatible with, the Laboratory ImageQUEST,26 which allows the Histological Digital Imaging Information System (LIS) and pathologist to operate his camera in the Future conform to the security and directly in the database using a How might these technologies affect network protocols of the TWAIN interface similar to that the laboratory of the future? With institution.21 The system must also used to control a desktop scanner the rapid pace of camera and be compliant with applicable within a word processing program. software development, it is difficult HIPAA22 patient privacy regulations The program supports the mapping to accurately predict change over and DICOM23 medical image of fields of information from other time. Still, long-term planning interoperability standards. In databases, as well as the dictated or requires that the wise lab manager addition, there will be oversight and typed entry of diagnostic dictation anticipates at least the general regulation by academic at the microscope. These data fields directions of growth. It is a reason- accreditation bodies and various are then searchable by accession able assumption that a continued state and federal agencies. In brief, number, patient name, diagnosis, shortage of qualified histo- the system must provide a high level or any other attached data. technologists,28 managed care cost of security from unauthorized By simple point-and-click, the containment, and competitive access24 and other safeguards such pathologist can produce an pressures will make the histology lab as fail-safe image tracking and illustrated report using premade of the future more automated. This, image-change logging to ensure the templates with selected image and along with a growing concern for integrity and accuracy25 of the data information. This report can error reduction and patient security, images and the attached data. be forwarded to the referring will make embedded positive sample identification such as bar-coding necessary. Presuming that the histologic slide carries such identification, a suitable reader on the microscope will automatically access the Laboratory Information System and call up all relevant data that the pathologist needs to evaluate the specimen. The fully automatic digital camera will make all necessary adjustments for exposure, color balance, and focus in real time on a full screen display. Images will be snapped at the click of a foot pedal or voice command, allowing the pathologist’s hands to always remain on the microscope. Each image will be inserted automatically into the database along with the other patient data and notes the pathologist dictates through voice recogni- tion software. If required, the camera can also be used for real-time tele- pathology29 or remote consultation. A fully illustrated surgical pathology Fig. 7. A schematic diagram showing the theoretical flow of images and image data based on a possible pathology departmental system at a teaching hospital. report is automatically generated and filed. When performing final sign-out, the pathologist cross-checks Fortunately, manufacturers of physician by either encrypted image the images with the report to reduce Laboratory Information Systems email or accessed through a pass- error and improve quality assurance. are finally recognizing the word-protected web site. With a A notification is then automatically importance of integrating images networked conference room and generated and sent to the referring with other clinical and demographic a computer-driven classroom physician by encrypted email. That patient information. Other projector, the stored images can physician can then use a preassigned companies are producing stand- be presented directly from the password to access a secure web site alone image database programs database for a weekly clinical to view and download the report. that can supplement and interface conference or downloaded to When the pathologist attends tumor with the Laboratory Information presentation software such as board or a weekly clinical confer- System. A good example is PowerPoint for teaching.27 ence, he can call up the database from

31 the conference room computer and 17. Aperio Technologies. Available at: a solution containing the cultured http://www.aperiotech.com. display the images and information as 18. Harrison M, Koh J,Tran S, Mongkolwat P,Channin Hpylori.Within a week, Marshall required in real time without spending DS. Research and teaching access to a large clinical was ill with gastritis. They published picture archiving and communication system. J Digit additional preparation time gener- Imaging. 2001;14(2 suppl 1):121-124. their experiment in 1985 under the ating a formal slide presentation. 19. vanEssen J, Hough T.An overview of a picture title “ Attempt to fulfill Koch’s pos- archiving and communications system procurement. 3,4 Later, when preparing for a lecture, J Digit Imaging. 2001;14(2 suppl 1):34-39. tulates for pyloric Campylobacter.” professional conference, or publica- 20. Blado ME. Management of the picture archiving and Not all of the postulates were communications system archive at Texas Children’s tion, he can search the database by Hospital. J Digit Imaging. 2001;14(2 suppl 1):84-88. fulfilled because neither one of diagnosis or other criteria to find 21. Gaytos B. Developing policies and procedures for a them had developed ulcers.1 The picture archiving and communication system. J Digit suitable images. Imaging. 2001;14(2 suppl 1):44-47. infection is found in 60% to 80% 22. HIPAA Standards for Privacy of Individually of patients with stomach ulcers and Identifiable Health Information. Available at: How far off in the future is such a http://www.hcfa.gov/hipaa/hipaahm.htm. 90% of patients with duodenal system? Almost all of the equip- 23. Digital Imaging and Communications in ulcers. It is prevalent throughout Medicine (DICOM). Available at: ment and software mentioned exists http://medical.nema.org/dicom.html. most of the world and it is estimated today, at least in a prototype form. 24. Harding DB Jr, Gac RJ Jr, Reynolds CT 3rd, that approximately 10% of the US Romlein J, Chacko AK. Security model for picture Commercial vendors and individual archiving and communication systems. J Digit population will become infected labs are even now putting together Imaging. 2000;13(2 suppl 1):202-203. with this intestinal pathogen. 25. Stauch G, Schweppe KW, Kayser K. Diagnostic errors systems with many of these in interactive telepathology. Ann Cell Pathol. features. As these systems demon- 2000;21(3,4):201-206. 26. ImageQUEST Image Database. Available at: strate cost-effectiveness, improved http://www.visual-med.com. quality control, and competitive 27. Frank MS, Schultz T, Dreyer K. Integrating digital teaching-file systems with off-the-shelf presentation advantage, other labs will rapidly software to facilitate speaker-led conferences. J Digit adopt them. As usual in medicine, Imaging. 2001;14(2 suppl 1):98-101. 28. Medical Laboratory Personnel Shortage Act of 2001. scientific curiosity will open the HR 1948 IH. Available at: http://thomas.loc.gov/cgi- door, technology will create new bin/query/z?c107:H.R.1948. 29. Gombas P.Informational aspects of telepathology in opportunities, but in the end, it is routine surgical pathology. Ann Cell Pathol. always cost-savings that topples 2000;21(3,4):141-147. empires and creates new worlds.

References A number of methods to detect 1. Introduction to Cameras. Available at: http://www.theimagingsource.com/prod/cam/ Hpylori have been reported in the camintro_2.htm. literature, yet individuals continue 2. Spring KR. Scientific imaging with digital cameras. BioTechniques. 2000;29(1):71-73. A Simple Method to to inquire about what is the best 3. Kayser K, Beyer M, Blum S, Kayser G. Recent method to demonstrate this developments and present status of telepathology. Ann Cell Pathol. 2000;21(3,4):101-106. Demonstrate Hpylori organism. Few offer the simplicity, 4. Olympus C-3030. Available at: speed, and low cost of the method http://www.olympusamerica.com/innards/ and Mast Cells h_microscopes.asp?s=11. that follows. While many regard the 5. Kodak MDS 290. Available at: silver stains as offering the best http://www.kodak.com/US/en/health/scientific/ Rena Fail, AS, HT(ASCP) products/mds290/. Medical University of South Carolina contrast, they are costly and capri- 6. Coolpix 995. Available at: cious, making consistent and repro- http://www.nikonusa.com/usa_group/group.jsp?cat=5 &grp=26. Discovered in 1983 and classified as ducible results problematic. With 7. SPOT INSIGHT. Available at: those methods the presence of any http://www.diaginc.com/insight/indexinsight.htm. Campylobacter, Helicobacter pylori 8. Mairinger T.Acceptance of telepathology in daily (H pylori) is a gram-negative spiral- precipitate can obscure the bacteria, practice. Ann Cell Pathol. 2000;21(3,4):135-140. requiring repeat staining which can -micro 3.0 ן Nikon DN100. Available at: shaped bacteria 0.5 .9 http://www.nikonusa.com/usa_group/group.jsp?cat=5 meters with 4 to 6 sheathed flagella delay turn-around time and increase &grp=26. the cost to the lab. 10. SPOT RT Available at: attached to one pole. The spiral http://www.diaginc.com/spotrt.htm. shape aids the bacteria in making its 11. Tibiletti MG, Bernasconi B, Dionigi A, Riva C. What follows is an inexpensive and The applications of FISH in tumor pathology. way into the mucous lining of the Adv Clin Pathol. 1999;3(4):111-118. stomach and duodenum. It is the rapid method for the detection of 12. Zeiss AxioCam. Available at: Hpylori that offers good contrast http://www.zeiss.de/us/micro/home.nsf/. only bacteria able to colonize in the 13. Nikon DXM 1200. Available at: gastric mucosa.1,2 In Perth, Australia, for organism identification and is http://www.nikonusa.com/usa_group/group.jsp?cat=5 simple to perform. Using this &grp=26. after months of trying, the “ new” 14. Malkusch W, Bauch H, Schafer L. Digital light organism was finally cultured by method, the contrast is sharp microscopy: prerequisite for optimum contrast enough for the bacteria to be seen ן enhancement and increase of resolution. Exp Dr. Robin Warren and Dr. Barry Gerontol. 2001;36(7):1199-1217. Marshall because of a staff shortage at 40 .Itis also quite useful for the 15. Felten CL, Strauss JS, Okada DH, Marchevsky demonstration of mast cells. The use AM.Virtual microscopy: high resolution digital over a holiday weekend. The two photomicrography as a tool for light microscopy were frustrated in their efforts to of the microwave to heat the stain simulation. Hum Pathol. 1999;30(4):477-483. reduces the staining time in the 16. Ventura L, Chiominto A, Colimberti P,Sarra M, Dal prove H pylori responsible for many Mas A, Leocata P.Creating low-power ulcers. To prove their theory, working Giemsa from 20 minutes photomicrographs by digital scanning of histological in a 60° C oven to 2 minutes. sections. Pathologica. 2000;92(1):9-12. Marshall and a volunteer swallowed 32 6. Rinse quickly in distilled water. represented a new era for the demonstration of antigens, allowing 7. Rinse in three changes of pathologists to correlate immuno- methanol. logical findings with cytological and 8. Clear in xylene, and mount. histological criteria.1 In the decades Results: that followed, there were various Red blood cells ………………pink attempts to improve the sensitivity Nuclei …………………………blue and specificity of the IHC method. H pylori, fungi, and These included the development of other bacteria …………………blue primary antibodies optimized for antigen binding in formalin-fixed Method: Microwave Giemsa Stain Mast cells ……………………purple Rickettsia ……………………purple tissues, enhanced detection strate- Fixation: 10% neutral buffered gies employing various enzyme- formalin In our hands, the use of methanol to chromogen systems, and techniques dehydrate stained sections prior to for signal and chromogen amplifi- Quality Control: Tissue known to cation. Today, despite tremendous be positive for H pylori mounting seems to provide lighter staining of surrounding tissue, efforts by instrument manufacturers Solutions: offering better contrast with stained to bring this technology within 1. Methanol, absolute organisms. reach of any laboratory, irrespective of staff expertise, IHC staining has 2. 1% Acetic Acid become more complex, fraught with Glacial Acetic Acid … … … 1.0 ml References 1. Helicobacter pylori in peptic ulcer disease. NIH pitfalls for both the uninitiated and Distilled water …………99.0 ml Consens Statement Online. January 1994;12(1):1-23. 2. Scoarughi GL, Cimmino C, Domini P. Helicobacter experienced alike. Thus, the ability 3. Stock Giemsa Solution 25 pylori: a eubacterium lacking the stringent response. to produce accurate, reliable, and J Bacteriol. 1999;181(2):552-555. Giemsa powder ……………1.0 g 3. Sweet M. The Sidnet Morning Herald.August 2, 1997. reproducible staining with the vast Glycerin …………………66.0 ml 4. Marshall BJ,Armstrong JA, McGechie DB, Glancy RJ. array of antibodies to tissue and Attempt to fulfill Koch’s postulates for pyloric Methanol…………………66.0 ml Campylobacter. Med J Aust. 1985;142:436-439. cellular antigens is tenuous at best. 5. Luna L. AFIP. 3rd ed. McGraw Hill. 236. Mix glycerin and giemsa powder, Experts have long lamented the microwave* on high for 1 minute, need for standardization of the IHC stir, and cool. When the solution has method to allow for comparable cooled to room temperature, add testing from one laboratory to the methanol. If a microwave oven Concepts in Epitope another, but the many variables that is not available, the solution may be Retrieval for can affect staining have stymied the prepared using a conventional Biological Stain Commission’s laboratory oven by placing the Immunohistochemistry efforts at standardization over the glycerin-giemsa mixture at 60° C for past decade.2 The failure of all 2 hours before stirring. Store the Vinnie Della Speranza laboratories to use a standard fixa- stock solution in the refrigerator. MS, HTL(ASCP) HT tion protocol for various tissues is * Sharp carousel model R-410DK, 1200 watts. Medical University of South Carolina but one illustration of the daunting 4. Working Giemsa Solution challenge facing proponents of IHC A newcomer to the world of immuno- standardization. Stock Giemsa ……………7.0 ml histochemical (IHC) staining will Methanol …………………5.0 ml quite likely become quickly Distilled water …………38.0 ml The widespread appeal of discouraged and overwhelmed. formaldehyde as a routine fixative Staining procedure: Attaining clean, specific immuno- during the past century has been 1. Deparaffinize and hydrate to staining of all appropriate antigens largely due to its superior ability to distilled water. with no distracting or misleading preserve morphological detail. nonspecific staining is no small feat, Although the exact mechanism of 2. Rinse slides in methanol for even for the most experienced 2 minutes. formalin fixation is still unclear, the among us. The discussion that follows chemical process leading to protein 3. Preheat the working Giemsa summarizes the relevant information fixation by formalin appears to solution in the microwave on needed to allow the reader to involve multiple chemical reactions high for 45 seconds. Filter the develop a coherent yet reliable among formaldehyde, various hot solution into a coplin jar method for antigen unmasking. amino acid residues, and even containing the slides. Stain for peptide bonds. An additive fixative, 2 minutes. First appearing on the scene in this small molecule may offer an diagnostic histopathology in the 4. Rinse quickly in distilled water. efficient means, through cross- early 1970s, the application of linking, of retaining localized 5. Dip slides once in 1% acetic acid. IHC to routinely processed tissues antigen in tissue. This is exemplified 33 LOOKING GOOD. WORKING SMART. YEAR AFTER YEAR.

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©2001 Sakura Finetek U.S.A., Inc. Visit our web site at www.sakuraus.com by the reported observation that The past decade has witnessed a alkaline solutions may have a cytologic samples fixed in alcohol proliferation of reports in the decidedly negative effect upon will demonstrate improved IHC scientific literature describing section adherence to glass, and more staining when post-fixation with strategies and methods for restoring importantly may be deleterious to formalin is used, followed by antigen tissue antigenicity which will undo tissue morphology. retrieval.3 the denaturing effects of various fixatives. Attempts by industry to Proteolytic Enzymes The chemical actions of the formal- identify a fixative that does not Treatment of formalin-fixed, dehyde molecule in tissues are denature antigens while providing paraffin-embedded tissue sections largely time dependent. Cross- acceptable morphology has thus far with proteases has been linking is believed to require at least been unsuccessful. This voluminous demonstrated to effectively unmask two distinct chemical reactions body of literature, combined with a some antigens. Studies have shown involving amino acid NH2 groups, as number of competing proprietary that the required time of exposure well as their side chains which vary methods to achieve epitope to enzyme is dependent upon the among the amino acids. Thus, the unmasking, can all serve to confuse duration of formalin exposure combined results of these binding the user who wants to develop a which, unfortunately, is not known actions upon varied chemical simple but reliable method for in many instances, especially for constituents make it difficult to routine application. Disparate archival tissues. Overexposure to completely predict formaldehyde’s reports in the literature recommend proteases can destroy tissue effects on tissue antigens. It is the use of either proteolytic antigenicity, creating a false generally accepted that, as a enzymes, chelating agents (EDTA), negative result. Optimal exposure consequence of formaldehyde- or high heat (Heat Induced Epitope times must be determined and induced cross-linking of proteins, a Retrieval – HIER)— with the tailored to each laboratory’s highly dense protein scaffold results, proponents of each method touting fixation and processing protocol. which can sterically hinder the theirs as best. accessibility of antigens.4 The time- Incubate hydrated tissue sections to sensitive nature of these combined The early work of Fraenkel-Conrat6 trypsin or pepsin for 20-30 minutes reactions, especially when suggested that the denaturing effects (as a starting point) at 37oC. Prepare considering the variable fixation of formaldehyde fixation, ie, the enzyme as follows: times for surgical tissues in the cross-linkages, could be disrupted at 40 mg pepsin in 100 ml 0.01N HCl clinical laboratory, adds to the high temperatures or with strong 10 mg trypsin in 100 ml 0.1 M Tris- challenges of antigen demonstration alkalis. This observation laid the buffered saline (pH 7.8). with IHC. This contributes to the foundation for the current approaches Following enzyme incubation, rinse difficulties often encountered when to antigen unmasking. While the sections in three changes of Tris- working with archival tissues. development of one standard buffered saline (pH 7.4). retrieval method that is effective for In order to fully appreciate the all antigens has not been forth- Heat Induced Epitope Retrieval impact of cross-linking fixation on coming thus far, sufficient data have (HIER): antigen staining, it is essential to emerged to enable the reader to Heat induced antigen retrieval has consider the three-dimensional develop a sensible strategy for been purported to offer the best impact of the attachment of the attaining unmasking at the bench. means for a standardized approach formaldehyde molecule to protein. to antigen unmasking. A number of The protein scaffolding continues to Epitope retrieval methods may be reported observations regarding the build upon itself over time which broadly characterized as those that use of heat for epitope retrieval may, in effect, throw a “ net” over the require heat and those that do not. warrant emphasizing. antigens of interest. This can result 1. The relationship between tem- in steric hindrance and lead to a Epitope retrieval without heat: perature (T) and time (t) is inverse. conformational (shape) change Alkaline solutions That is, the higher the temperature, (denaturation) of the epitope you The exposure of celloidin-embedded the shorter the time needed to are trying to visualize, which might tissues to a strong alkaline solution achieve beneficial results. Tem- prevent binding of the antibody (NaOH/methanol ratio 1:5 for peratures below 80oC are not molecule being used for staining. 30 minutes) resulted in improved significantly beneficial. A temper- Despite these shortcomings, immunostaining.7 This demonstrated ature of 100oC is optimal. One formalin-induced cross-linking of that there is an inverse relationship recent report indicated that intense protein appears to serve as an between concentration of alkali (C) IHC staining can be achieved efficient means of in situ and time (t), ie, the greater the with the following conditions: preservation of antigen in most concentration of sodium hydroxide, ן minutes; 90oC 20 ן 100oC circumstances, despite the simul- the less time required to achieve the ;minutes 50 ן minutes; 80oC 30 taneous masking of antigenicity.5 desired result. However, strongly hours.3 10 ן 70oC

35 2. Retrieval solution pH is important. (to avoid the boiling over of 4oC overnight. Sections that did not A number of markers appear to retrieval solutions) are necessary receive exposure to calcium stained stain well regardless of retrieval for optimal IHC staining. One intensely positive for these markers, solution pH. These include L26, report indicates that microwave yet those exposed to calcium PCNA, AE1, EMA, and NSE.3 antigen retrieval may yield chloride did not. Furthermore, Other antigens, including MIB-1 unanticipated artifact staining when sections receiving calcium and ER, exhibit diminished patterns.8 chloride pretreatment were subse- staining at neutral pH but strong quently treated with EDTA, staining at very acidic or very If you are using a domestic pressure a calcium chelator, intense immuno- alkaline pH. A last group of anti- cooker with 103 kPa/15 psi, keep in staining was restored, suggesting gens, such as HMB-45, shows weak mind that you are at temperature that calcium may induce conforma- focal positivity at low pH but when pressure is achieved. Excessive tion changes in antigen that are yields intense staining at high pH. incubation of slides at high heat and reversible. These data suggest that, with few pressure can result in compromised exceptions, a retrieval buffer (Tris- histology, particularly in skin where Conclusion HCl or sodium acetate buffer) of collagen and elastin can appear frag- Immunohistochemical staining pH 8.0-9.0 may be suitable for mented and disrupted. Pressurized techniques continue to be an most antigens. Low pH buffers boiling for 1-2 minutes is sufficient evolving science. While attempts are appear better for some nuclear for epitope unmasking. under way to identify a means for antigens, such as retinoblastoma standardization of this technique, protein, estrogen receptor, and With a household microwave oven, the complexity of tissue fixative androgen receptor. In this instance, it is important that you determine interactions, lack of standard acetate buffer at pH 1.0-2.0 or (calibrate) the time needed to fixation protocols from laboratory glycine buffer (pH 3.6) appear achieve temperature with your to laboratory, and the wide array of superior to citrate buffer. particular oven. Repeated boiling clinically relevant antigenic markers cycles have been reported to be and detection strategies make 3. The significance of retrieval more effective than extending the standardization unlikely in the short solution composition is unclear. boiling time of a single cycle. term. It is left to each of us to deter- High heat using distilled water Heat a plastic coplin jar or other mine a rational and reproducible alone will achieve improved IHC microwave-safe vessel until the protocol for antigen unmasking that staining, suggesting that the solution begins to boil, taking care is tailored to the fixation and chemical composition of the not to boil the solution out of the processing schemes employed in our retrieval solution is not important vessel. If your device takes respective laboratories. in many cases. When heat induced 5 minutes to boil, allow 1-2 minutes epitope retrieval was first reported, in between heating cycles to References buffer solutions containing zinc maintain temperature without 1. Taylor CR. Immunologic studies of lymphoma: past, (sulfate) or lead (citrate) salts were present and future. J Histochem Cytochem. boiling over. Repeat again for a 1980;28:777-787. recommended. However, it is now third cycle of heating. 2. Taylor CR, Shi S-R, Cote RJ.Antigen retrieval for generally agreed that the use of immunohistochemistry: status and need for greater standardization. Appl Immunohistochem. solutions containing such toxic salts The tendency among some vendors 1996;4:144-166. offers no advantage over simple 3. Chaiwan B, Shi S-R, Cote R, Taylor CR. Major factors to give their retrieval solutions pro- influencing the effectiveness of antigen retrieval buffer solutions and should prietary names may prevent the user immunohistochemistry. In: Antigen Retrieval therefore be avoided. While many Techniques. Natick, Mass: Eaton Publishing; 2000:42. from knowing what buffer has been 4. Roth J, Ziak M, Guhl B. Nonheating antigen retrieval laboratories have adopted the use techniques for light and electron microscopic purchased, which will put you at a of citrate buffer, in some instances immunolabeling. In: Antigen Retrieval Techniques. disadvantage. You haven’t purchased Natick Mass: Eaton Publishing; 2000:276. this solution has appeared less 5. Shi S-R, Gu J,Turrens JF, Cote RJ,Taylor CR. some special magic in their effective than acetate buffer, Development of the antigen retrieval technique: proprietary concoction, but simply philosophical and theoretical bases. In: Antigen Tris-HCl, or glycine-HCl. Retrieval Techniques. Natick, Mass: Eaton Publishing; the convenience of having the buffer 2000:28. 4. The heating source is not solution premade for you. Insist on 6. Fraenkel-Conrat H, Olcott HS. The reaction of formaldehyde with proteins. Cross-linking between significant to outcome. Despite knowing what you are using. amino and primary amide or guanidyl groups. J Biol what others may tell you, the Chem. 1948;174:2673-2684. 7. Shi S-R, Cote RJ,Taylor CR.Antigen retrieval manner in which required tem- EDTA: immunohistochemistry used for routinely processed perature is achieved and main- A recent report by Morgan et al9 celloidin-embedded human temporal bone sections. In: Antigen Retrieval Techniques. Natick, Mass: Eaton tained (vegetable steamer, suggests that calcium ions bound to Publishing; 2000:290. microwave, or pressure cooker) proteins inhibit IHC staining of 8. Margiotta M. Atypical cytokeratin staining of sentinel node biopsies for breast cancer due to microwave is more a matter of convenience. those proteins. This observation has pretreatment. HistoLogic. 1999;31(2):22. Microwave users may maintain been confirmed by others.5 Parallel 9. Morgan JM, Navabi H, Jasani B. Role of calcium in high temperature antigen retrieval at different pH that they can complete retrieval frozen sections were stained for values. J Pathol. 1997;182:233-237. faster, but keep in mind that 2 to MIB-1 and thrombospondin after

3 cycles of heating and cooling prior incubation in 50 mM CaCl2 at 36 The US Bureau of Commercial The Diagnostics & Histology Lab is Fisheries established the Oxford a subproject of the Oyster Disease Laboratory in 1960 for the primary Research Project (ODRP) of the purpose of investigating oyster Fisheries Division within MD-DNR. diseases that struck Chesapeake ODRP actually encompasses Bay and Delaware Bay in the late investigations into the health of 1950s. It became the Cooperative any and all species of aquatic Oxford Laboratory in 1987, resources, as required. There are through an agreement between four state of Maryland staff Maryland Department of Natural members: Charlie Gieseker, MS, Marine Resources (MD-DNR) and the and I are pathologists; Sue Tyler, HT, NOAA National Marine Fisheries and Jud Blazek are histotechs. Histopathology at Service to share the facility and NOAA also has a histotech on staff, Cooperative Oxford cooperate in research. Currently, Dorothy Howard, HT.The state staff administration of federal programs provides technical support to both Laboratory at COL is carried out by NOAA’s state and federal projects. The National Ocean Service Center for Immunoassay Laboratory is also Carol B. McCollough Biotechnology. Scientists at COL part of ODRP.This lab is primarily HT/HTL(ASCP) investigate health problems of concerned with the development finfish, shellfish (including oysters, and adaptation of clinical immuno- It’s 4 PM on a crisp October afternoon when the white pickup various species of clams, scallops, logical techniques, molecular truck rolls into the driveway at and blue crabs), and other aquatic biology, and shellfish pathology. Cooperative Oxford Laboratory life in the Chesapeake Bay and Lee Hamilton is the immunology (COL). Stacked in the bed are along the Atlantic Coast. They also technician. Chris Dungan, MS, is 10 onion sacks filled with dirty collaborate with scientists the research scientist who develops brown lumps— oysters. Roy Scott nationally and internationally studies for and administers the comes to my office and announces, to improve understanding of ODRP. Federal scientists include “I’ve got some samples for you,” aquatic animal health and to Gretchen Messick, MS, HT, so I head out to the lot to help haul develop management strategies crustacean pathologist; Shawn the sacks into the Wet Lab to store to prevent and mitigate diseases. McLaughlin, PhD, shellfish and them in tanks with flowing seawater The laboratory participates in the coral pathologist; Jay Lewis, MS, from the Tred Avon River. This is National Marine Mammal and shellfish pathologist; and Fred the true beginning of a day in the Sea Turtle Stranding Network, Kern, Fishery research biologist. life of the Diagnostics & Histology investigating strandings of these Laboratory at COL. Next morning, rare and endangered animals in my staff and I begin the annual Maryland, and sharing informa- process of determining the health tion, samples, and expertise with of Maryland’s oyster population. other institutions and coastal By the time we are finished in mid- states. New techniques for December, we will have examined classifying and mapping critical 1330 individual animals for the reef habitats in Chesapeake Bay routine samples, plus another have been developed recently at 360 ± 60, all using routine paraffin the laboratory. These methods are histology and microbiological assays now being applied in Maryland’s for oyster diseases. oyster restoration efforts. Fig. 3. Jud Blazek embeds razor clams in paraffin.

Work on oysters is focused primarily on two protozoan pathogens, Perkinsus marinus (dermo disease) and Haplosporidium nelsoni (MSX disease). A pair of us necropsy each 30-animal sample, measuring and shucking each oyster carefully, noting macroscopic anomalies, and collecting two tissue samples. The oyster’s rectum is excised and cultured for P marinus, since it is Fig. 1. Charlie Gieseker trims paraffin blocks Fig. 2. Carol McCollough sections paraffin blocks after embedding crab tissue. of oyster spat. believed that the route of infection 37 is through the gut epithelium. Work in the immunology laboratory stains is being used to characterize A cross-section through the mantle, has focused on the detection of the organism, including PAS, gills, and digestive diverticulum is P marinus, both in oyster tissue and Protargol (Bodian), Feulgen, and collected and fixed for histology. in the water column. A polyclonal alcian blue. The histology staff also After embedding and sectioning, antibody was developed to works in the field collecting and these tissues are all stained P marinus and has been used for fixing lesioned fish for EM. manually with H&E. Charlie flow cytometry studies of parasite We recently collaborated with and I then examine them for the load in natural water, an ELISA scientists from the Maryland presence of H nelsoni and any assay, and DAB streptavidin-biotin Department of Agriculture in other microscopic pathology. and FITC immunostains useful on identifying a new species of After incubation, the rectal biopsy paraffin-embedded tissues. This Mycobacterium in wild striped bass, is fished out of the culture tube, antibody has also been useful in performing hundreds of AFB stains macerated on a glass slide, and shedding light on the taxonomy of in the process. These fish also stained with Lugol’s iodine. the Apicomplexa and their possible exhibited severe dermal ulcerations. The starchy cell wall of P marinus relationship to the dinoflagellates. Occasional fish kills occur in wild stains black and is easily seen at Efforts are now under way to or aquaculture populations of Some animals are infected so develop a monoclonal antibody, finfish, and our staff prepares and .ן40 heavily that we can see the black and to isolate and propagate examines the histological slides to color develop with the naked eye. Perkinsus sp. that infect other shell- determine causes of mortality. In these cases, the host oyster is in fish hosts to determine if these hosts Planned studies of fish health and the terminal stage of the disease and harbor the same parasite species as population status are also part of not likely to survive longer than two oysters, or others. our repertoire. One recent study to weeks. P marinus is difficult to characterize American eel popula- visualize with an H&E, but tions in Maryland’s Chesapeake H nelsoni is readily identifiable. Bay had us preparing hundreds of Its plasmodium looks like a small H&E slides of eel gonads. Great purple potato and has multiple cocktail party conversation! nuclei. The name “ MSX” stands for multinucleate sphere, unknown. Work with crustaceans at COL Typically it first invades the gills, emphasizes the health of blue crabs, and once past the basement those feisty, tasty morsels that are membrane of the gill epithelium the mainstay of summer seafood in it becomes systemic. Sporulation the mid-Atlantic. Crabs have been occurs infrequently, typically in Fig. 5. Cyst containing spores of Kudoa species of interest at COL for many years, young hosts, and the eosinophilic infecting muscle of Atlantic menhaden (Bevoortia and a definitive treatise entitled tyrannus). Gram’s stain; note gram-positive polar :Histology of the Blue Crab “ ןand slightly refractile spores are bodies. 40 found in the epithelium of the A Model for the Decapoda” was digestive diverticula. These spores Lest you think that oysters occupy authored by now-retired Phyllis are also acid-fast; this is a charac- all of our interest, be assured that T. Johnson. Recent research has teristic of Haplosporidia. In animals other aquatic species also receive focused on Hematodinium sp., with heavy infections, the H&E our scrutiny. During the recent a parasitic dinoflagellate found in cross-sections appear homogeneous Pfeisteria piscicida scare in Mary- both the hemolymph and tissues of to the naked eye, while normal land, the histology staff necropsied sick crabs. Watermen from Maryland and less heavily infected animals and fixed, embedded, sectioned, coastal bays have reported crabs show a great deal of tissue detail stained, and examined hundreds of dying in crab pots. Hematodinium sp. and contrast. Atlantic menhaden exhibiting infections were found in adult and dermal lesions. No histological juvenile crabs from the coastal bays evidence linking P piscicida with of Maryland, Delaware, and these lesions was found, however, Virginia. Other crustaceans affected spores of Kudoa sp., a myxosporean by Hematodinium spp. include parasite, were found in the muscu- amphipods and green crabs, plus lature of all specimens with lesions. Tanner crabs and other commer- In addition, a novel plasmodium cially important species. These was detected in many of the parasites are easily visualized in lesioned fish, and work to identify both cytology smears and tissue this organism is presently under way sections using an H&E stain. They in collaboration with senior scientists exhibit a characteristic clumped Fig. 4. Perkinsus marinus in oyster (Crassotrea at US-FDA and The Johns Hopkins chromatin that stains dark blue .University.A broad suite of special with hematoxylin ןvirginica). Wet mount with Lugol’s iodine. 40

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©2001 Sakura Finetek U.S.A., Inc. Visit our web site at www.sakuraus.com Work in the Diagnostics & explained our physical makeup and Now that I had the “ perfect sec- Histology Lab at COL is varied and why our bodies function the way tions,” I was ready to stain them interesting. We work on a diverse they do. The knowledge that I was before turning them in to the pro- group of species exhibiting a variety gaining kept me thinking about my fessor for grading. All was going of diseases and pathologies. While future career in histology, even when well, and then I noticed that my most of the work consists of tinc- I wasn’t at school. I would meet old tissue was no longer on the slide torial stains on FFPE tissues, we are friends and begin explaining what but floating its way to the bottom expected to execute necropsies, I had learned and how it was a great of the coplin jar. I persevered and immunostains, and microbiological change in my life. And with this stained another slide. This time assays. No day is ever the same, even positive attitude I made it through the tissue remained on the slide, when working on the same project, the first year with confidence that my but the differentiation was terrible. because each sample is different, change in careers was a good choice. Who ever said a Masson’s was easy? and one never knows what fasci- nating discovery lies in the next In the fall of 1995, I began the After many attempts, I finally began animal or on the next slide. Or what semester with a renewed to get a grasp on the staining. unique situation or question might exuberance. Then the test began! The next glitch was coverslipping. have to be addressed. The question I started my second year and It looked easy and I thought I did this morning was from a field biolo- encountered the histology course. a good job. Then I noticed the gist who had just finished surveying I had no idea that the professor professor looking at one of my a pond and had discovered the expected me to know ALL of the slides, so I decided to ask her how it skeletal remains of several young cells and tissues in the human body. looked. She said that my staining sturgeon— “ How should I preserve Now the thoughts of whether or not was all right and that she wanted me the skulls to use as show-and-tell I had made the best choice by to look and tell her what I thought material?” What fun!! entering the field of histology of the slide. As I started to look in began to surface. But I wanted this the scope I heard her chuckle and new career and I buckled down to soon saw why.There was this small, work. Ultimately, the semester went perfectly round circle surrounded by Ups and Downs of a quite well and I mastered the a dark line right in the center of the Histology Student material. And then, during the final tissue. Slowly I was learning to be semester came the part of the humbled. It really was an art and I Richard Sparling, AS, HT(ASCP) training that I had long awaited— needed to learn to become the artist. Microtechniques, the hands-on In 1994 I decided to make a major portion of my training. Learning the It was only after leaving college and career change in my life. As a father proper way to preserve, process, starting work in a histology lab that of two, working full-time on the cut, and stain tissues was so I truly realized why the professor night shift to help defray expenses, exciting. The professor began had required that students learn I entered the Histology Program at showing us how to set up the so many details in her courses. State University of New York’s processor and cut the proper size I worked at that job for a year and College at Cobleskill. It was at this tissue for cassettes. None of this then had an opportunity to return to time that I began my experience in seemed to look all that difficult. SUNY Cobleskill as a lab technician the exciting world of histology. As the weeks went on and we were for the biology courses, including all I started out feeling interested, taught the way to section, stain, and of the histology courses. My favorite curious, excited, and most of all, coverslip, through both lecture and course is Microtechniques— the one a bit scared. As the first semester demonstration in lab, I felt that I struggled through years ago. began, I realized that the path I had these also looked quite easy. Well, Now my job allows me to be in a chosen to take in life was going to reality set in quickly! How could position to help students with the be a greater change than anything something that the professor made same problems I used to have. It is else I had ever done. look so easy, be so very difficult for very gratifying and I feel that my me to do? I thought that there must presence is an asset to the training In my two years at SUNY Cobleskill, be something physically wrong with program. I have the expertise of a I experienced many ups and downs. me. Try as I might, the sections kept certified histotechnician, but I am I would like to share some of these sticking, folding, and tearing, until still on a similar peer level as the with you and I’m sure that you will I finally got the section I had long students. I watch students going be able to relate to them. Starting awaited. I carefully moved it toward through the same ups and downs this training at the age of 44 was the waterbath, only to have the air that I had. When they are exper- different, to say the least, but it was currents whip the ribbon against iencing the downs, I am now able to also a slight advantage. I was here to my arm! After many frustrating ease their anxieties and help them to learn a career that would allow me attempts, I finally managed to get a persevere by providing technical to help others even if it was behind ribbon of wrinkle- and scratch-free advice and sharing my experiences the scenes. My emotions ran high as sections onto the waterbath and with them. I explain that they aren’t I began my biology courses, which then onto the slide. having any problems that thousands 40 of histotechs before them have not difficult. In a hospital setting, most donned a pair of latex gloves. This experienced. It is beneficial to all devices have a latex component reaction generally increases in students to hear similar explana- somewhere in them. Gloves are severity with each exposure. It is tions not only from the professor, obvious culprits, but syringes have also called delayed hypersensitivity, but also directly from a former rubber stoppers, stethoscopes have or Type IV allergy. Symptoms may student. Sometimes it is an advan- rubber tubing, tourniquets are often not appear for 1 to 3 days after tage to me as the technician to not latex strips, and even the floor mats exposure with this type of reaction.1 be in a position of authority. I feel in entryways have rubber backs. that being a student, working in the Thus, prevention of latex allergies The most severe reaction is Type I, field, and now returning to the can be a daunting task. Fortunately or immediate hypersensitivity.This college to work with the students for healthcare workers, many reaction can manifest as hives, is one of the most rewarding hospitals have adopted a latex-safe asthma, or a respiratory allergic experiences I have ever had. policy and have switched to latex- reaction similar to hay fever. The free devices wherever possible. patient can experience itchy, watery These hospitals have outlawed eyes, runny nose, and excessive latex balloons in their facilities, and sneezing. Anaphylaxis is the most have switched to vinyl and nitrile severe of the Type I reactions, where gloves in place of latex for many a rapid drop in blood pressure is the Latex Allergy purposes, such as food handling hallmark symptom. Immediate and patient exam room use. medical intervention is required Wanda Shotsberger, HT(ASCP) with this reaction, as severe shock Prevention efforts are noble in or death can occur.1 Medical University of South Carolina intent, but what should you do if you suspect they have not helped Once diagnosed, the second step In 1987, the Centers for Disease you? If you suspect you have a latex for the latex allergic patient Control issued its recommendations allergy, there are several steps you obviously is to limit exposure to for latex glove use as part of should take. The first step is to have latex, but this is not as easy as it universal precautions against HIV your allergy diagnosed. From there may seem. Latex gloves are the infection.1 This action, coupled with the steps you take will depend on most obvious threat to the allergic OSHA’s Bloodborne Pathogen the severity of your reaction. healthcare worker, but the list of Standards, increased latex glove use 1 products, both medical and by 17-fold, and thus, dramatically Diagnosing a latex allergy is not household, containing latex is increased healthcare workers’ easy.There are only two accepted extensive. Blood pressure cuffs, exposure to latex allergens. The ways to diagnose a latex allergy: Band-Aids, catheters, condoms, result has been an alarming increase blood work, which is the allergen- ACE bandages, some balloons, in the reports of latex allergy among specific IgE test, and skin prick T-shirt iron-ons, the handles on healthcare workers. Although allergy testing. IgE testing has a high false “ comfort grip” tools— all of these to latex is considered uncommon negative rate, so skin prick testing and many, many more products among the general population with 2 has been shown to be the more contain latex, which may cause a less than 1% prevalence, among reliable test,2 however, it carries a life-threatening reaction. Consider healthcare workers and others with risk of anaphylactic shock and the following suggestions: high rates of exposure to latex the 1 should only be administered by an rate is as high as 17%. experienced professional. 1. Switch from powdered latex gloves to nitrile gloves. Vinyl Latex is a plant derivative. It is Reactions to latex vary from sub- gloves are permeable to many produced from the sap of the tree acute reactions to anaphylaxis. Sub- of the chemicals we use in the Hevea brasiliensis.1 Thus, latex con- acute reactions are rashes that are histology lab, so they are not an tains plant proteins, many of which usually limited to areas of skin option for laboratory use. are now believed to be plant directly exposed to latex. An defense proteins. The proteins 2. When eating out, ask your wait- example would be dry, itchy skin protect plants by causing unpleasant person if latex gloves are used in on the hands limited to the area reactions, which keep herbivores the kitchen. Food handling leaves covered by gloves. These reactions from returning to a particular plant.2 latex proteins on food, which can are referred to as “ irritant contact These proteins are also thought to cause an allergic reaction. dermatitis.” 1 be the cause of latex allergies, as 3. Avoid toys (balloons, Koosh™ they are present in latex gloves and Next in severity is “ allergic contact balls, tennis balls) that contain other products made from latex. dermatitis,” characterized by latex. redness, swelling, and weeping sores. As with any medical condition, This reaction is not limited to the 4. Avoid other household products prevention is preferable to treating area directly exposed to latex and that contain latex such as a condition or attempting to cure can cover the arms and chest of an stretchy Band-Aids, rubber tub one, but preventing latex allergy is allergic person who has simply mats, rubber dish draining 41 products, Playtex™ gloves, and Latex allergy is a difficult condition. To reprocess the specimen, the even pantyhose. Obviously, the The difficulties in diagnosing the paraffin block surrounding it list of products containing latex condition are problematic, and pre- must first be removed. To do so, is extensive, and eliminating vention and treatment are quite place the block in a base mold exposure is impossible. a daunting task. The statistics will that is sized to accept the solid Exposure often comes from continue to show a rise in latex block. Place the base mold onto the unlikely sources, and patients allergy symptoms among those warming plate of a tissue embed- do not always know what has people continually exposed unless ding console. The block will take caused a reaction. latex is greatly reduced in hospital approximately 20 minutes to melt. and industrial settings. Considering Once the paraffin is melted, transfer Many patients also experience the difficulties this presents, it is not the specimen in a properly identi- cross-reactions with different foods. likely to happen in the foreseeable fied cassette. Place the cassette in These reactions are thought to future. the tissue processor basket. result from the plant proteins discussed earlier. These foods include, References With some commercial tissue 1. Larson LE. Latex allergy: how a protective material but are not limited to: banana, kiwi, threatens some of its users. Laboratory Medicine. processors, the operator can set up avocado, peanuts, and fruits known 1998:29. a program reversing the processing 2. Advice from Your Allergist… Latex Allergies. as the stone fruits, which include American College of Allergy,Asthma, and steps. However, this reversed peaches and cherries.3 These fruits Immunology web site [brochure]. 1998. Available at: process does cause reagent http://www.medem.com/search/article_display.cfm? ® and other foods that cross-react are path=n:&mstra-/ZZZ1C571A9C.html&soc= contamination. On the Tissue-Tek best avoided. ACAA1&srch_typ=NAV_SERCH.Accessed VIP™ Series tissue processors, the September 3, 2001. 3. Weiss SJ, Halsey JF.A nurse with anaphylaxis to stone use of the built-in cleaning cycle Latex exposure is not limited to fruits and latex sensitivity: potential diagnostic makes the procedure fast and difficulties to consider. Annals of Allergy, Asthma and direct exposure. People with latex Immunology. 1996;77:504. economical since the processing allergies are at risk from airborne reagents are not used. The Tissue- exposure as well. Latex particles Tek ® VIP™ will automatically attach to the powders in gloves and prompt the operator at the end of become airborne with donning and the processing cycle to start a removal of gloves, especially if cleaning cycle. A cleaning cycle can gloves are “ snapped” off.1 also be done on demand. Simply Exposures of this type are not place the basket containing the limited to the work environment, Reprocessing of specimens that need to be however. Walking into an auto parts reprocessed into the processor and store where hoses, floor mats, tires, Tissue Blocks start a cleaning cycle. Automatically, and windshield wiper blades are the Tissue-Tek® VIP™ system will Gilles Lefebvre, Managing Editor unpacked, transferred, and stored is first heat the retort for 10 minutes also a hazard. If you have a latex to remove any residual paraffin. At times, a tissue block may need allergy, consider carefully what is Then, nine changes of xylene will reprocessing. Many factors can lead inside a place of business before you take place, followed by five changes to blocks unsuitable for diagnosis, even open the door. of alcohol. On the latest model, the including inadequate fixation time, Tissue-Tek® VIP™ 5 system, the user a specimen that is too thick, and The final step in treating a latex can program the number of changes improper reagent rotation schedule. allergy is necessary only for those for each of the cleaning stations. Some of the well-known results of with a Type I hypersensitivity. These improper processing are: patients should wear a Medic Once the cleaning cycle is Alert™ -style bracelet engraved with • Depression in the block once it completed, the specimens can then “ Latex Allergy,” and should carry has cooled. Sometimes the be processed again starting with a an epinephrine syringe in case of a depression may take 24 hours lower grade alcohol, such as 70%, reaction. These patients should also or more to develop. to avoid buffer salt precipitation call the fire rescue unit nearest within the tissue. The reprocessing • The specimen is “ mushy.” them and report their allergy. This should not be performed with the will allow that station to stock non- • The color of the block is not standard overnight process typically latex gloves for any calls to the uniform, appearing more opaque used with fresh tissue. The extent of home of the allergic individual. where the tissue is located. chemical exposure renders the These may seem like extreme specimens dangerously over- • Sections are difficult to obtain measures, but for those with a dehydrated, to the point that they because there are holes where Type I hypersensitivity, treatment may become unsuitable for diag- the tissue should be. by a latex glove-wearing paramedic nosis. A shorter program should be gives a whole new meaning to a life • Sections “ explode” when floated used, with immersion times at least and death situation. in the water bath. half the original ones. 42 √ Mark Your Calendar! Educational Opportunities in 2002 January 16 NATIONAL SOCIETY FOR HISTOTECHNOLOGY May 17-18 GEORGIA SOCIETY FOR HISTOTECHNOLOGY TELECONFERENCE Site: Holiday Inn, Macon, GA Title: “ Temporal Artery Biopsy” Contact: Connie Waurin (404) 686-2385 Speaker: Peggy Wenk, HTL(ASCP) Email: [email protected] Contact: NSH office (301) 262-6221 June 6-8 NSH REGION III/ARIZONA SOCIETY FOR January 18 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ HISTOTECHNOLOGY SAN ANTONIO Site: Sheraton Phoenix Airport Hotel Teleconference: 12:00 pm Central Time 1-800-982-8868 Contact: Andi Grantham (520) 626-4415 Title: “ Mohs Histographic Surgery” Email: [email protected] Speaker: Clifford M. Chapman, MS, HTL(ASCP)QIHC June 15 ALABAMA SOCIETY FOR HISTOTECHNOLOGY February 15 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ Site: Birmingham, AL SAN ANTONIO Contact: Rita Humphrey (205) 939-9639 Teleconference: 12:00 pm Central Time 1-800-982-8868 Title: “ Handling Chemical Hazards in Histology” June 19 NATIONAL SOCIETY FOR HISTOTECHNOLOGY Speaker: Maureen Doran, BA, HTL(ASCP) TELECONFERENCE Title: “ Basic Troubleshooting of the H&E Stain” February 20 NATIONAL SOCIETY FOR HISTOTECHNOLOGY Speaker: Joan Vesey, HT(ASCP) TELECONFERENCE Contact: NSH office (301) 262-6221 Title: “ Laboratory Microwave Ovens: Theory and Non-staining Applications in Histology” June 21 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ Speaker: Jan Minshew, HTL(ASCP)HT SAN ANTONIO Contact: NSH office (301) 262-6221 Teleconference: 12:00 pm Central Time 1-800-982-8868 Title: “ Paraffin or Plastic: Concepts for Optimal March 15 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ Management of Bone Specimens” SAN ANTONIO Speaker: Robert A. Skinner, HTL(ASCP) and Teleconference: 12:00 pm Central Time 1-800-982-8868 Frances Swain, HT(ASCP) Title: “ Breast Cancer: Immunohistochemistry and Prognostic Factors” July 17 NATIONAL SOCIETY FOR HISTOTECHNOLOGY Speaker: David Tacha, HT(ASCP)HTL TELECONFERENCE Title: “ Competency Assessment for the Histotech” March 15-16 KENTUCKY SOCIETY FOR HISTOTECHNOLOGY Speaker: Daniel Huan Site: Holiday Inn, Hurstbourne Contact: NSH office (301) 262-6221 Louisville, KY Contact: Cynthia Long (859) 323-6114 July 19 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ Email: [email protected] SAN ANTONIO Teleconference: 12:00 pm Central Time 1-800-982-8868 March 20 NATIONAL SOCIETY FOR HISTOTECHNOLOGY Title: “ Quality Asurance in Anatomic Pathology– TELECONFERENCE It’s Everybody’s Job” Title: “ Ocular Histology” Speaker: Joseph W. Galbraith, MBA, HTL(ASCP)MT Speaker: Pat Lewis, HT(ASCP) Contact: NSH office (301) 262-6221 August 16 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ SAN ANTONIO March 21-24 NSH Region III hosted by TENNESSEE SOCIETY Teleconference: 12:00 pm Central Time 1-800-982-8868 FOR HISTOTECHNOLOGY Title: “ Space– the Final Frontier” Site: Clarion Hotel, Chattanooga, TN Speaker: Elizabeth Sheppard, BA, HT(ASCP) Contact: Michael LaFriniere (423) 495-6117 Email: [email protected] August 21 NATIONAL SOCIETY FOR HISTOTECHNOLOGY March 22-23 IOWA SOCIETY OF HISTOTECHNOLOGY TELECONFERENCE Annual Spring Symposium Title: “ Immunohistochemistry” Site: Best Inn & Suites, Des Moines, IA Speaker: David Tacha, HT(ASCP)HTL Contact: Debra Foster (563) 244-5665 Contact: NSH office (301) 262-6221 Email: [email protected] September 20 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ April 11-13 NSH REGION II & NEW JERSEY SOCIETY FOR SAN ANTONIO HISTOTECHNOLOGY Teleconference: 12:00 pm Central Time 1-800-982-8868 Site: Holiday Inn, Atlantic City, NJ Title: “ Tissue Culture” Contact: [email protected] Speaker: Gillian Rittman, ONC [email protected] September 28- NSH SYMPOSIUM/CONVENTION April 17 NATIONAL SOCIETY FOR HISTOTECHNOLOGY October 3 Site: Long Beach, CA TELECONFERENCE Contact: NSH Office Title: “ The Highs and Lows of pH” 4201 Northview Drive, Suite 502 Speaker: Betty Tallardy, HT(ASCP)QIHC Bowie, MD 20716-2604 Contact: NSH office (301) 262-6221 (301) 262-6221 Fax: (301) 262-9188 April 19 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ Email: [email protected] SAN ANTONIO Web page: www.nsh.org Teleconference: 12:00 pm Central Time 1-800-982-8868 Title: “ Histopathology of the Prostate” October 18 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ Speaker: Dean Troyer, MD SAN ANTONIO Teleconference: 12:00 pm Central Time 1-800-982-8868 April 26-27 WASHINGTON STATE SOCIETY FOR Title: “ The Conversion of Special Stain Procedures HISTOTECHNOLOGY to Immunohistochemistry” Site: Seattle area Speaker: Nancy L. Shellhorn, BA, MT(ASCP) Contact: Linda Cherepow (206) 587-0430 ext. 5044 Email: [email protected] November 3 CONNECTICUT SOCIETY FOR HISTOTECHNOLOGY Site: Mid State Hospital, Meriden, CT May 4-6 FLORIDA SOCIETY OF HISTOTECHNOLOGY Contact: Cindy DeRiso (203) 694-8587 ext. 3017 Site: Deerfield Beach Resort, Deerfield Beach, FL Contact: Tonia Breckenridge (850) 937-9995 November 15 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ Email: [email protected] SAN ANTONIO Teleconference: 12:00 pm Central Time 1-800-982-8868 May 15 NATIONAL SOCIETY FOR HISTOTECHNOLOGY TELECONFERENCE Title: “ Breast Cancer– The Role of the Histology Lab” Title: “ Cytochemical Detection of Apoptotic Cell Death” Speaker: I-Tien Yeh, MD Speaker: Mark C. Willingham, MD December 18 NATIONAL SOCIETY FOR HISTOTECHNOLOGY Contact: NSH office (301) 262-6221 TELECONFERENCE May 17 UNIVERSITY OF TEXAS HEALTH SCIENCES CTR/ Title: “ Cytoprep for the Histotech” SAN ANTONIO Speaker: Beth Cox, SCT(ASCP) Teleconference: 12:00 pm Central Time 1-800-982-8868 Contact: NSH office (301) 262-6221 Title: “ Special Stains and the Liver Biopsy” Speaker: Michael Lovell, MD

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