Veterinary Clinical Pathology ISSN 0275-6382

Use of destained cytology slides for the application of routine special stains
o, F. Ferreira, R.A.F. Monteiro, E. Rocha R. Marcos, M. Santos, N. Santos, F. Malha
Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS - Institute of Biomedical Sciences Abel Salazar, UP, University of Porto, Porto, Portugal

Key Words Cytochemistry, cytology, destaining, dog, special stains Correspondence Ricardo Marcos, Cytology Diagnostic Services, Institute of Biomedical Sciences Abel Salazar ICBAS, Lg. Prof Abel Salazar, 2, 4099-003 Porto, Portugal E-mail: rmarcos@icbas.up.pt DOI:10.1111/j.1939-165X.2008.00098.x

Background: Special stains to demonstrate microorganisms or intra- and extracellular substances have not been evaluated in detail regarding their applicability and usefulness in destained cytologic specimens. Objectives: The aim of this study is to compare the results of routine special stains on destained slides previously stained with Hemacolor and on fresh (unstained) specimens. Methods: Archival Hemacolor-stained ne needle aspirate specimens of inammation with infectious agents (bacterial, mycobacterial, and fungal infections), neoplasia (melanoma, myxosarcoma, and mammary adenocarcinoma), and hemorrhage (pericardial effusion) from 14 dogs and 7 cats were selected. Cells in a minimum of 4 elds were photographed and 5 slides from each case were then destained by different methods (alcohol acid or microwave). Seven special stains were applied selectively to the destained slides, depending on the cytologic ndings: periodic acid Schiff, GrocottGomori methenamine silver, Grams, ZiehlNeelsen, Alcian blue, FontanaMasson, and Prussian blue. The same elds were rephotographed and 2 observers evaluated the slides qualitatively, with comparison to fresh cytologic specimens from similar lesions. Results: Special stains applied to destained slides demonstrated the expected cellular and extracellular material or organisms independent of the destaining method. Staining intensity, nonspecic staining (background), cell morphology, and nuclear counterstaining results were similar to those of special stains applied to fresh unstained slides. Conclusions: Destaining does not appear to affect the results of routine special staining for cytologic specimens. Destaining before special stains may be a valuable diagnostic strategy when few slides are present or only stained slides are available.

Introduction
Almost 150 years have passed since Bernard demonstrated ferric iron in tissues with the Prussian blue reaction, introducing histochemical methods in pathology.1 Since then, different reactions and stains have been described for demonstrating carbohydrates, proteins, lipids, pigments, and microorganisms, including bacteria and fungi.2 Histochemical methods play a major role in diagnostic pathology for conrming or reaching a diagnosis, especially in atypical infections or undifferentiated neoplasia.2 Histochemical stains have long been applied to cytologic specimens in medical

and veterinary pathology3,4; several recent reports have been published that highlight the use and importance of special stains in veterinary cytology.511 One of the major drawbacks for the use of special stains in cytology practice is the highly variable sample quality among slides. Smears often differ in cellularity, contaminants, individual cell populations, and morphology.12,13 Sometimes, consecutive aspirations of the same mass yield widely different samples, and acellular samples and blood contamination frequently occur. Typically, the use of special stains is done after the review of a routine Romanowsky-stained smear, and thus requires an additional unstained sample.14 If

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Table 1. Special stains and counterstains applied to destained cytologic specimens in this study. Lesion Bacterial infection (septic purulent inammation) Mycobacterial infection Fungal infection Melanoma Myxosarcoma Mammary adenocarcinoma Hemorrhagic effusion Structure/Cell/Material Stained Bacteria Mycobacteria Fungal cell wall Melanin granules Mucin Mucin (secretory material) Iron (ferric) Special Stain Grams and Tworts Gram ZiehlNeelsen PAS, GMS MassonFontana Alcian blue PAS Prussian blue Color of a Positive Reaction Blue (gram-positive) or red (gram-negative) Red Purple, black Black Sky blue Purple Blue Nuclear Counterstain Basic fuchsin and neutral red1 fast green Methylene blue Gills hematoxylin, light green Light green or neutral red Gills hematoxylin Gills hematoxylin Neutral red

PAS indicates periodic acid-Schiff; GMS, GrocottGomori methanamine silver.

additional samples lack representative material, false negative or inconclusive results may occur. The objective of this study was to evaluate the use of special stains relevant in the diagnosis of pathologic lesions in cytologic samples from dogs and cats, specically to determine whether destaining slides after Romanowsky staining was a reliable option. We also evaluated 2 different destaining methods. The results using destained slides were compared with the results of special stains applied to fresh (unstained) samples from similar lesions.

Materials and Methods

Slides were retrieved from the archives of the Cytology Diagnostic Services at ICBAS, University of Porto, from September 2002 to June 2007. Twenty-one cases (from 14 dogs and 7 cats) with the following lesions or diagnoses were selected: inammation with infectious agents (bacterial, mycobacterial, and fungal infections), neoplasia (melanoma, myxosarcoma, and mammary adenocarcinoma), and hemorrhage (pericardial effusion). Cases were included only if a minimum of 5 representative slides was present and if the diagnosis was later conrmed by histopathology or microbiological methods (except for the pericardial effusion). The slides had been stained with Hemacolor (Merck, Darmstadt, Germany) with extended postxation and staining times, as described by Jorundson et al.15 The slides were mounted with DPX mounting medium (Merck). All slides were photographed with a digital camera (Camedia 4050, Olympus, Tokyo, Japan) tted to a microscope (BX-50, Olympus), in recorded positions on the x and y axes of the microscope stage (minimum of 4 elds per slide, using 10, 40, and 100 objectives). Afterwards, to remove the coverslip, the slides were immersed in xylene for 13 days. After coverslips were removed, the slides were left for

1 additional day in xylene to ensure complete removal of the mounting medium. The 5 representative slides of each case were destained by one of 2 main methods: microwave at 600 W, placing the slides either in citrate buffer (pH = 6.0) or in distilled water; and alcohol acid, in which we tested 1%, 5%, and 10% solutions (ie, ethanol 70% with 1, 5, and 10 mL of concentrated HCl). Afterwards, the slides were hydrated in a decreasing series of alcohols, tap water, and distilled water (2 minutes in each solution) followed by cytochemical staining using conventional techniques and appropriate counterstains (Table 1).1,16 The following special stains were applied to the destained slides, depending on the type of lesion (Table 1): periodic acid Schiff (PAS), GrocottGomori methenamine-silver (GMS), Grams (including the Tworts variant), ZiehlNeelsen, Alcian blue, Fontana Masson, and Prussian blue. Positive and negative controls (parafn tissue sections) were run for all the special stains. A minimum of 15 slides from 3 different cases were evaluated per special stain. The slides were rephotographed in the same positions as for the Hemacolor-stained specimens and both specimens were evaluated qualitatively by 2 observers (RM, MS) blinded to the diagnosis. The observers assessed staining intensity, the presence of nonspecic (background) staining, cell morphology, and nuclear counterstaining. In addition, ZiehlNeelsen, Alcian blue, and PAS stains were applied directly to 3 extra Hemacolor-stained slides from the mycobacterial and fungal lesions and the myxosarcoma (which were hydrated in a decreasing series of alcohols and distilled water, but not destained). In the case of fungal specimens, we also tested the consecutive use of PAS and GMS stains. Fresh material from one case of each inammation with infectious agents (bacterial, mycobacterial, fungal), neoplasia (melanoma, myxosarcoma, mammary adenocarcinoma), and hemorrhage (pericardial effusion) was

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acid solutions compared with 1% alcohol acid solution and the microwave. The use of citrate buffer vs distilled water resulted in no difference in destaining time or cell morphology in the microwave method. In destained slides of bacterial infections, rodshaped and lamentous bacteria were stained red with Grams stain (gram-negative), whereas cocci stained blue (gram-positive) (Figure 1). Grams stain and Tworts stain rendered comparable results, although the distinction of bacteria tended to be slightly easier with the latter, because of the greenish background. ZiehlNeelsen clearly stained acid-fast bacilli of Mycobacterium lepraemurium in destained slides of a subcutaneous mass in a cat. Direct application of this stain to Hemacolor-stained slides tended to increase the staining intensity (Figure 2). PAS stained fungal elements positively in destained slides containing Microsporum and Trichosporum

Figure 1. Bacterial infection in a cutaneous nodule from a dog. (A) Moderately karyolytic neutrophils with intra- and extracellullar coccoid bacteria (Staphylococcus sp.). Hemacolor. (B) Tworts Gram stain applied after destaining the Hemacolor slide with 5% alcohol acid. The bacteria are blue (gram-positive) and the erythrocytes are light green. Bar = 22.5 mm.

obtained from 4 dogs and 3 cats by ne-needle aspiration at the Veterinary Clinic at ICBAS - University of Porto. A minimum of 2 unstained slides were obtained per case. Cytochemical stains were applied directly (without the destaining step) and the staining results were compared, by the same observers, with those of the same special stains on destained samples.

Results
Destaining was achieved using both acid alcohol and microwave techniques; no major differences in cell morphology were noted between the destaining methods. The time needed to completely destain the slides ranged from 1 to 15 minutes, depending on the thickness of the smear. Nevertheless, destaining was faster (taking half the time) with the 5% and 10% alcohol

Figure 2. Mycobacterial infection (Mycobacterium lepraemurium) in a subcutaneous nodule from a cat. (A) Large numbers of macrophages lled with negative linear rod-shaped bacteria. Hemacolor. (B) ZiehlNeelsen staining of the Hemacolor-stained slide. In A note that in some cells (arrows) and in the background, the acid-fast bacteria that can be seen in B are not apparent. Bar = 22.5 mm.

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Figure 3. Organisms in a lymph node aspirate from a dog. (A) Numerous round to oval budding yeasts with a thin halo (later diagnosed as Trichosporum sp.) can be seen among the lymphocytes. Hemacolor. (B) Periodic-acid Schiff (PAS) stain applied to the Hemacolor slide in A after destaining with 5% alcohol acid. (C) GrocottGomori methenamine silver stain applied to the destained PAS slide in B. Bar = 22.5 mm.

(Figure 3). Like ZiehlNeelsen, PAS applied directly to Hemacolor-stained slides (ie, without destaining) tended to increase the staining intensity. GMS stained hyphae and fungal spores positively in 2 cases of dermatophytosis (Figure 4); however, staining intensity was variable between slides. Despite the use of light green counterstain, it was difcult to recognize the nuclei of inammatory cells. Good results were obtained with the consecutive use of PAS and GMS (Figure 3); slides appeared similar when stained with PAS, destained a second time, and restained with GMS. In contrast, the silver precipitate in GMS-stained slides could not be removed by destaining (data not shown). In destained melanoma specimens, Fontana Masson stained melanin granules black within macrophages, melanocytes, and in the background (Figure 5). Counterstaining with light green provided better contrast than with neutral red, making recognition of cells easier. Alcian blue stained the background blue and the cytoplasm of neoplastic cells in myxosarcomas blue (Figure 6). In these lesions, Alcian blue applied directly to Hemacolor-stained slides (without destaining) also gave acceptable results (data not shown). PAS stained the cytoplasm of neoplastic cells (isolated and in clusters) in destained specimens of mammary gland tumors (Figure 7). In the hemorrhagic pericardial effusion, Prussian blue stained the cytoplasm of macrophages that con-

tained hemosiderin and exhibited erythrophagocytosis, whereas the mesothelial cells were not stained (Figure 8). Results of cytochemical stains in destained slides were similar to those in fresh material. The only difference was in nuclear detail (membrane outline and chromatin pattern), which was slightly poorer in the destained samples. However, in the cases of neoplasia, this did not compromise the ability to diagnose malignancy.

Discussion
The special stains used in this study have been applied successfully for decades in human and veterinary cytology, where they have been applied directly to fresh air-dried8,10,11 or alcohol-xed6 smears. Destaining, however, is not commonly used and only isolated reports recommend destaining slides when special stains are required, namely when no unstained smears remain or when a particular feature has been observed in a single slide.9,17 For the stains tested here, we conrmed that destaining could be a useful procedure when limited material or only previously stained material is available. Thus, the recommendation of leaving unstained smears for special stains may be

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eas. Our use of these stains directly on Hemacolorstained cytologic samples yielded good results. The special stains used in this study have multiple applications and are used routinely in veterinary cytology. The Grams staining characteristics of bacteria are important for selecting initial antibiotic therapy.16 Although often used in histopathology, this method has been applied less frequently in cytology samples10,14,1820 and, to the best of our knowledge, it has not been evaluated in destained cytology samples. Gram staining of destained cytologic preparations can be particularly useful when it is difcult to distinguish bacteria from granular precipitate or from a granular proteinaceous background.18,21 Because both grampositive and gram-negative bacteria stain equally well

Figure 4. Dermatophytosis (later diagnosed as Microsporum canis) in a dog. Degenerated neutrophils and fungal spores (arrows) localized around a hair shaft (asterisk). (A) Hemacolor. (B) GrocottGomori methenamine silver stain applied to the Hemacolor-stained slide in A after destaining with 5% alcohol acid. Bar = 10 mm.

unwarranted because it does not guarantee substantially better quality staining and because false negative results may occur if representative material is absent in the unstained slides.12 In fact, using special stains in unstained slides may be risky if we consider that it is not unusual for cytologists to evaluate multiple slides from a lesion, collected by the hands of experienced clinicians, in which only a single slide is representative and diagnostic.13 Although both destaining methods worked equally as well in our hands, we recommend destaining with 5% alcohol acid solution because it is faster, especially in thick specimens. Based on the evaluation of limited Hemacolor-stained samples that were directly stained with PAS, ZiehlNeelsen and Alcian blue, this destaining step can be obviated with good results. With PAS, the periodic acid partially destains the slides, whereas with ZiehlNeelsen and Alcian blue, the special stains actually highlight previously unstained ar-

Figure 5. Cutaneous melanoma from a dog. (A) A fusiform melanocyte, numerous erythrocytes, a lymphocyte, and a neutrophil are seen. Hemacolor, bar = 22.5 mm. Inset : round melanocyte with eccentric nucleus and black-green cytoplasmic granules that also appear in the background. Hemacolor, bar = 45 mm. (B) FontanaMasson stain with light green counterstain applied to the Hemacolor-stained slide after destaining with 5% alcohol acid. The melanin granules are positive (black) and the erythrocytes appear green. Bar = 22.5 mm. Inset : Fontana-Masson counterstained with neutral red. Bar = 45 mm.

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PAS stains a myriad of carbohydrate-containing substances like glycogen, brin, amyloid, certain epithelial sialo- and sulphomucins, and fungal cell walls,1,3 and is useful for demonstrating yeast-forming fungi.16,18,27 PAS has been used previously in destained cytologic samples from humans, where it is recommended for the diagnosis of cryptococcosis, when few or thin-walled organisms are present.28 To our knowledge, PAS has not been evaluated previously in destained samples from animals, although based on our results, it should also be useful. As we showed, slides stained with PAS can even be destained and restained with GMS, a stain recommended for the identication of fungi.17 This practice may be useful for avoiding misinterpretation of PAS-positive material

Figure 6. Myxosarcoma from a dog. (A) Population of fusiform and stellate cells with mild anisokaryosis and slightly eosinophilic cytoplasm. Amorphous, faintly eosinophilic material is visible in the background. Hemacolor. (B) Alcian blue staining of the Hemacolor-stained slide destained with 5% alcohol acid. The background and cytoplasm of some neoplastic cells are stained blue, positive for mucin. Bar = 65 mm.

with Romanowsky-stains, Grams staining of cytologic preparations is needed to determine gram-staining characteristics. In contrast with other bacteria, acid-fast bacilli do not stain with Hemacolor; rather, negative staining occurs, presumably because of hydrophobic interactions between the water-based stain and the lipid cell wall of the bacilli.22 Because these negative images may be difcult to recognize, ZiehlNeelsen has been recommended to effectively diagnose mycobacterial infections in dogs and cats18; this stain has been mostly used in unstained cytologic specimens.2326 Based on the results of this study, the application of ZiehlNeelsen stain to destained Hemacolor slides is a viable method for the conrmation or diagnosis of mycobacterial infections. Recently, Freeman described the use of ZiehlNeelsen superimposed on a WrightGiemsastained smear,18 a strategy that we also tested and recommend for this special stain.

Figure 7. Mammary adenocarcinoma from a dog. (A) Acinar structure of neoplastic cells with moderate anisokaryosis, vacuoles in the cytoplasm (arrowheads), and basophilic material (black arrow), presumably secretory. Additional peripheral material (white arrow) is present. Hemacolor. (B) Periodic-acid Schiff stain of the Hemacolor-stained slide in A, after destaining with 5% alcohol acid. The cytoplasm, cytoplasmic vacuoles (arrowheads), and peripheral material (black arrow) are positive for neutral glycoprotein, in contrast with the cell debris (white arrow in A), which is negatively stained. Bar = 22.5 mm.

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Figure 8. Pericardial effusion from a dog. (A) Cluster of mesothelial cells and macrophages with erythrophagocytosis (arrowheads). Hemacolor. (B) Prussian blue stain of the same Hemacolor-stained slide after destaining with 5% alcohol acid. The macrophages are positive for ferric iron, in contrast with the mesothelial cells. Bar = 22.5 mm.

in macrophages as fungi. In addition, predigestion with diastase before PAS can reduce nonspecic staining of nonfungal material or cells (eg, neutrophils).1 GMS stains different types of fungi in cytologic samples,17,29 but the conventional technique is timeconsuming, taking about 2 hours.16 This time can probably be reduced substantially, to minutes, by using a 80 1C water-bath for the oxidation and reduction step30 instead of using only a 58 1C oven for the latter.16 Even with technical renements, however, GMS is still laborious, expensive, and requires well-trained personnel. For that reason, and considering our results, we would recommend PAS as a conrmatory stain for fungi, instead of GMS. FontanaMasson can also be used for the differential staining of fungi,27 but is most commonly used for the detection of melanin. In our study, we found FontanaMasson useful in the detection of coarse melanin granules within macrophages, in which it is often dif-

cult to distinguish melanin from nuclear debris or from other dark pigments like hemosiderin.31 To our knowledge, FontanaMasson has not been evaluated previously for use in destained cytologic specimens from dogs and cats. Staining with FontanaMasson is laborious, requires great care to prepare the staining solution in order to avoid background, and is timeconsuming.1,16 However, our results indicate it can be used in destained slides, and the technique we followed can probably also be optimized to reduce the time needed.32,33 Acid and neutral glycoproteins in cells and in extracellular matrix are stained by Alcian blue and PAS, respectively.1 The diagnostic use of Alcian blue has been noted in a few reports in animals using fresh cytologic samples from abdominal uid,11 a myxosarcoma,34 and a liposarcoma.7 We demonstrated in this study that Alcian blue can also be used in destained ne-needle aspirate smears to highlight the mucinous nature of a myxosarcoma. PAS has also been used in unstained smears, for evaluating abdominal uid,11 conjunctival impressions (goblet cells),6 and in aspirates of cutaneous nodules in the dog.8 In the latter, PAS was used to demonstrate mucus secretion, thus helping in the diagnosis of metastatic adenocarcinoma. In this study, we also applied PAS to destained smears of neoplastic lesions in which mucinous cellular products could be demonstrated. Prussian blue stain has been described previously for use with destained human cytologic samples.3 Prussian blue stains ferric iron blue, and is helpful in the differentiation between anthracotic pigment, melanin, and hemosiderin.1,31 In samples from animals, Prussian blue is used often in the detection of hemosiderophages.18,35,36 In our study, we veried the use of Prussian blue for detecting iron-laden macrophages in destained specimens from pericardial effusions. Although we did not test other specimens, the stain also can likely be used to demonstrate iron in other types of destained samples. In conclusion, we demonstrated that good results can be obtained for routinely-used special stains applied to destained Hemacolor specimens from dogs and cats. Staining quality was comparable to the quality seen in unstained smears. Limited results in our laboratory with destaining MayGrunwaldGiemsastained slides, using the destaining strategy presented here, suggest that these results can probably be generalized to other Romanowsky-type stains. Based in our results, and depending on the available cytologic material, there is no strong support for leaving slides unstained for later use or for not using special stains when limited material or only previously stained slides are available.

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Acknowledgment
` The authors deeply thank Prof. Mario Caniatti (Universita degli Studi di Milano) for his generosity in supplying material from 1 cat used in the evaluation of ZiehlNeelsen stain.

14. Lumsden JH, Baker R. Cytopathology techniques and interpretation. In: Baker R, Lumsden JH, eds. Color Atlas of Cytology of the Dog and Cat. St. Louis, MO: Mosby; 2000:720. 15. Jorundsson E, Lumdsen JH, Jacobs R. Rapid staining techniques in cytopathology: a review and comparison of modied protocols for hematoxylin and eosin, Papanicolaou and Romanowsky stains. Vet Clin Pathol. 1999;28:100108. 16. Stevens A, Francis RJ. Micro-organisms. In: Bancroft JD, Stevens A, eds. Theory and Practice of Histological Techniques. 4th ed. New York: Churchill Livingstone; 1996:291308. 17. Shelly SM. Cutaneous lesions. Vet Clin North Am Small Anim Pract. 2003;33:146. 18. Freeman KP. Veterinary Cytology. Dog, Cat, Horse and Cow. London: Manson Publishing; 2007:125170. 19. Tater KC, Scott DW, Miller WH, Erb HN. The cytology of the external ear canal in the normal dog and cat. J Vet Med A. 2003;50:370374. 20. Lake AM, Scott DW, Miller WH, Erb HN. Gross and cytological characteristics of normal canine anal-sac secretion. J Vet Med A. 2004;51:249253. 21. Parry BW. Synovial uid. In: Cowell RL, Tyler RD, Meinkoth JH, eds. Diagnostic Cytology and Hematology of the Dog and Cat. 2nd ed. St. Louis: Mosby; 1999:104119. 22. Maygarden SJ, Flanders EL. Mycobacteria can be seen as negative images in cytology smears from patients with acquired immunodeciency syndrome. Mod Pathol. 1989;2:239243. 23. Carpenter JL, Myers AM, Conner MW, et al. Tuberculosis in ve Basset Hounds. J Am Vet Med Assoc. 1988;192:15631568. 24. Malik R, Hughes MS, James G, et al. Feline leprosy: two different clinical syndromes. J Feline Med Surg. 2002;4:4359. 25. Turinelli V, Ledieu D, Guilbaud L, et al. Mycobacterium tuberculosis in a dog from Africa. Vet Clin Pathol. 2004;33:177181. 26. Courtin F, Huerre M, Fyfe J, Dumas P, Boschiroli ML. A case of feline leprosy caused by Mycobacterium lepraemurium originating from the island of Kythira (Greece): diagnosis and treatment. J Feline Med Surg. 2007;9:238241. 27. Veerappan R, Miller LE, Sosinski C, Younberg GA. Narrow-spectrum staining pattern of Pityrosporum. J Cutan Pathol. 2006;33:731734. 28. Kumar P, Saran RK, Gondal R, Malhotra V. Smear morphology of cryptococcosis presenting as a subcutaneous swelling in healthy adults: a report of three cases. Cytopathology. 2005;16:143146.

References
1. Bancroft JD. An Introduction to Histochemical Technique. London: Butterworth & Co; 1967:62175. 2. Grahn B, Peiffer R, Cullen C, Haines D. Classication of feline intraocular neoplasms based on morphology, histochemical staining, and immunohistochemical labelling. Vet Ophthalmol. 2006;9:395403. 3. Sachdeva R, Kline TS. Aspiration biopsy cytology and special stains. Acta Cytol. 1981;25:678683. 4. Andrews JM, Malarkey DE. Advanced diagnostic techniques. In: Raskin RE, Meyer DJ, eds. Atlas of Canine and Feline Cytology. Philadelphia, PA: Saunders; 2001:401410. 5. Blauvelt M, Weiss D, McVey A, Bender J, Aird E. Spaceoccupying lesion within the calvarium of a cat. Vet Clin Pathol. 2001;31:1921. 6. Bolzan A, Brunelli A, Castro M, et al. Conjunctival impression cytology in dogs. Vet Ophthalmol. 2005;8:401405. 7. Boyd S, Taugner F, Serrano S, Gregory S, Tennant K. Matrix blues: clue to a cranial thoracic mass in a dog. Vet Clin Pathol. 2005;34:271274. 8. DellOrco M, Bertazollo W, Vergine M, et al. Gastric mucinous adenocarcinoma with cutaneous metastases in a dog: diagnosis by ne-needle aspiration cytology. J Small Anim Pract. 2005;46:449453. 9. Marcos R, Santos M, Oliveira J, et al. Cytochemical detection of calcium in a case of calcinosis circumscripta in a dog. Vet Clin Pathol. 2006;35:239242. 10. Peeters DE, McKiernan BC, Weisiger RM, Schaeffer DJ, Clercx C. Quantitative bacterial cultures and cytological examination of bronchoalveolar lavage specimens in dogs. J Vet Intern Med. 2000;14:534541. 11. Owens S, Gossett R, McElhaney R, Christopher M, Shelly S. Three cases of canine bile peritonitis with mucinous material in abdominal uid as the prominent cytologic nding. Vet Clin Pathol. 2003;32:114120. 12. Tyler RD, Cowell RL, Baldwin CJ, Morton RJ. Introduction. In: Cowell RL, Tyler RD, Meinkoth JH, eds. Diagnostic Cytology and Hematology of the Dog and Cat. 2nd ed. St. Louis, MO: Mosby; 1999:119. 13. Meinkoth JH, Cowell RL. Sample collection and preparation in cytology: increasing diagnostic yield. Vet Clin North Am Small Anim Pract. 2002;32:11871207.

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29. Nassar A, Zapata M, Little JV, Siddiqui MT. Utility of reex Gomori methenamine silver staining for Pneumocystis jirovecii on bronchoalveolar lavage cytologic specimens: a review. Diagn Cytopathol. 2006;34:719723. 30. Pintozzi RL. Modied Grocotts methenamine silver nitrate method for quick staining of Pneumocystis carinii. J Clin Pathol. 1978;31:803805. 31. De Lorenzi D, Bonfanti U, Masserdotti C, et al. Nasal melanosis in three dogs. J Small Anim Pract. 2006;47:682685. 32. Barbosa A, Castro L, Nogueira AM. A simple and economical modication of the MassonFontana method for staining melanin granules and enterochromafn cells. Stain Technol. 1984;59:193196.

33. Leong A, Gilham P. A new, rapid, microwavestimulated method of staining melanocytic lesions. Stain Technol. 1989;64:8185. 34. Raskin RE. Skin and subcutaneous tissues. In: Raskin RE, Meyer DJ, eds. Atlas of Canine and Feline Cytology. Philadelphia, PA: Saunders; 2001:3592. 35. Sprague WS, Hackett TB, Johnson JS, SwardsonOliver CJ. Hemochromatosis secondary to repeated blood transfusions in a dog. Vet Pathol. 2003;40:334337. 36. DeHeer HL, McManus P. Frequency and severity of tracheal wash hemosiderosis and association with underlying disease in 96 cats: 20022003. Vet Clin Pathol. 2005;34:1722.

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