Use of Red O stain in the cytologic diagnosis of canine liposarcoma

Carlo Masserdotti, Ugo Bonfanti, Davide De Lorenzi, Nicola Ottolini

Background: , a stain commonly used to demonstrate in frozen tissue, also may be used to stain air-dried cytologic specimens. Objective: The purpose of this study was to prospectively evaluate the value of Oil Red O in identifying lipid to aid in the differentiation of liposarcomas from other types of sarcoma. Methods: Twelve tumor specimens from dogs were evaluated. The tumors were included in the study if initial cytologic evaluation indicated a sarcoma, and if histologic confirmation was available. Oil Red O was applied to all cytologic specimens. Results: Tumor specimens were diagnosed histologically as liposarcoma (3 well-differentiated, 1 pleomorphic), hemangiopericytoma (n 5 3), fibrosarcoma (n 5 3), malignant fibrous histiocytoma (n 5 1), and undifferentiated sarcoma (n 5 1). Cytologic specimens from all liposarcomas showed strong positive of cytoplasmic vacuoles for lipid. Specimens from other sarcomas stained negative for Oil Red O, with the exception of weak, irregular positive staining in 1 hemangiopericytoma. Conclusions: Our results suggest that Oil Red O staining may be an easy, inexpensive, and useful diagnostic tool for the differentiation of liposarcoma from other mesenchymal neoplasms. (Vet Clin Pathol. 2006;35:37–41) 2006 American Society for Veterinary Clinical Pathology

Key Words: Dog, liposarcoma, mesenchymal tumors, special stains, Oil Red O

u

Liposarcoma is an uncommon tumor in animals and develops sarcomas, and to compare the results with the histopatho- predominantly in adult dogs.1 The neoplasm arises spontane- logic diagnosis. ously and is not derived from either of its benign counterparts, namely, lipoma or infiltrative lipoma. Diagnosis is based on the results of light microscopy of histologic sections2–4 and on Materials and Methods ultrastructural features.5,6 Cytologic features of liposarcoma have been reported as ovoid to spindle cells with small to large Subcutaneous masses from 12 dogs during the period lipid globules in the cytoplasm.6–9 In some cases, variable September 2001–April 2004 were evaluated. The tumors were morphologic characteristics may be found in different areas prospectively selected if the cytologic results were indicative of the same tumor, similar to what is frequently found in of sarcoma. Only masses for which a definitive histopatho- other sarcomas7; this is especially true for less differentiated logic diagnosis subsequently was obtained were included in neoplasms,8 and hampers definitive cytologic diagnosis. the study. The masses were aspirated by use of a fine-gauge To the author’s knowledge, only Sudan III, Sudan IV, needle, and at least 4 cytologic smears were prepared from Sudan Black, and Oil Red O stains are available to demon- each lesion. Air-dried smears were stained with May- strate lipid in a neoplasm originating from adipose tissue. Gru¨ nwald Giemsa (Carlo Erba Reagenti, Milan, Italy) and These stains can be used only on fresh or frozen tissue since with Oil Red O (Diapath Microstain, Martinengo, Italy). are removed by standard tissue-processing procedures. Briefly, without fixation in alcohol, the smears were covered This may limit their use, as most surgically removed tissue with a few drops of Oil Red O stain (that had been diluted specimens routinely are placed into formalin and embedded 1:0.6 with distilled water) for 5 minutes, according to the in paraffin for histologic examination. Cytologic smears, method of Johnson.10 The slides were then rinsed in running which typically are air-dried rather than fixed in alcoholic tap water and counterstained by immersion for 5 minutes in preservatives, represent a possible source of cells for use in Mayer’s hematoxylin (Diapath Microstain). After rinsing special staining for the detection of lipids. Evaluation of again in running tap water and air drying, the slides were cytologic specimens using Oil Red O could allow for the covered overnight with a drop of aqueous mounting fluid morphologic differentiation between liposarcoma and other (Crystal Mount, Biomeda, Foster City, CA, USA), then covered mesenchymal tumors, and would permit the prior establish- with balsam (Eukitt, Electron Microscopy Sciences, Hatfield, ment of staining protocols or fixation procedures for surgically PA, USA) and a coverslip. After cytologic examination and removed tissue specimens. The purpose of this study was to diagnosis, all neoplasms were surgically removed and tissue use Oil Red O as an adjunct stain in the cytologic diagnosis of specimens were submitted for histologic examination. Tissue

From Laboratorio Biodiversity–Divisione Veterinaria (Masserdotti), via Corfu`, Brescia; Clinica Veterinaria Gran Sasso (Bonfanti), Milan; Clinica Veterinaria S. Marco (De Lorenzi, Ottolini), Padova, Italy. Corresponding author: Carlo Masserdotti ([email protected]). ª2006 American Society for Veterinary Clinical Pathology

Vol. 35 / No. 1 / 2006 Veterinary Clinical Pathology Page 37 Oil Red O Staining of Canine Liposarcoma

Table 1. Cytologic and histologic diagnoses and Oil Red O staining results for 12 tumors from dogs.

Case Cytologic Diagnosis Histologic Diagnosis Oil Red No. O Results 1 Liposarcoma Well-differentiated liposarcoma Positive 2 Hemangioperycytoma Hemangiopericytoma Negative 3 Liposarcoma Well-differentiated liposarcoma Positive 4 Sarcoma Fibrosarcoma Negative 5 Poorly-differentiated sarcoma Undifferentiated sarcoma Negative 6 Liposarcoma Well-differentiated liposarcoma Positive 7 Sarcoma with Malignant fibrous histiocytoma Negative multinucleated giant cells 8 Hemangiopericytoma Hemangiopericytoma Focally positive 9 Sarcoma Fibrosarcoma Negative 10 Sarcoma Fibrosarcoma Negative Figure 1. Cytologic smear of a liposarcoma. The neoplastic cells have 11 Poorly-differentiated Pleomorphic liposarcoma Positive indistinct cytoplasm filled with round, sharply demarcated vacuoles. May- liposarcoma Gru¨nwald Giemsa, 3100 objective. 12 Hemangiopericytoma Hemangiopericytoma Negative

In cytologic samples from all 4 liposarcomas, Oil Red O specimens were fixed, processed, and stained routinely with staining resulted in red-orange coloration of both the intra- H&E. The cytologic diagnosis and results of Oil Red O staining cytoplasmic and extracellular vacuoles (Figure 3), demon- were compared with the final histopathologic diagnosis. strating their lipid content. The intensity of staining was highly variable, ranging from complete to partial or peripheral staining of the large globules; cells in 1 sample had fewer Results smaller vacuoles (Figure 4). Hematoxylin counterstaining provided good contrast for visualization of the overall Four cases of liposarcoma were diagnosed based on both architectural arrangement, cytoplasmic features, and nuclear cytologic and histologic specimens (Table 1). The cytologic structures, and permitted excellent general evaluation of the samples had good to excellent cellularity. The cells were smears. The cytologic and histologic features, including the arranged in a perivascular pattern, with the neoplastic cells positive results of Oil Red O staining, led to a final diagnosis organized around a vascular axis. Generally, the neoplastic of well-differentiated liposarcoma for 3 of the 4 tumors and cells had an indistinct spindle shape; cells in 1 case had an of pleomorphic liposarcoma for the fourth. ovoid shape. The cells had abundant, slightly eosinophilic cytoplasm that often was filled with round, sharply de- marcated vacuoles (Figure 1). High numbers of large vacuoles were seen in cells from 3 tumors, whereas cells in 1 tumor had smaller, fewer vacuoles. Abundant lipid-like material also was evident in the background of the smears. In samples from 3 of the 4 tumors, large ovoid cells were observed in which the cytoplasmic vacuolation deformed the profile of the nucleus, consistent with lipoblasts. In samples from 3 tumors, nuclei were round to oval, with finely clumped chromatin. In samples from the remaining case, anisokaryosis was moderate and nuclei had clumped chromatin with macronucleoli. Binucleated cells were found in specimens from all 4 cases. The histologic features in specimens from 3 of the 4 cases of liposarcoma were consistent with the proliferation of ovoid or spindle cells with abundant, macrovacuolated cytoplasm and round to oval nuclei with finely stippled chromatin. Cells were arranged in large lobules supported by a stromal com- ponent (Figure 2). Vascular capillary structures were present in all tissue sections. One tumor had less differentiated lobular structures, in which large round cells with variable amounts of Figure 2. Histologic section of a well-differentiated liposarcoma, in microvacuolated cytoplasm, anisokaryosis, and large, round which vacuolation of neoplastic cells and fibrous septa are evident. H&E, nuclei containing clumped chromatin were evident. 340 objective.

Page 38 Veterinary Clinical Pathology Vol. 35 / No. 1 / 2006 Masserdotti, Bonfanti, De Lorenzi, Ottolini

Figure 3. Cytologic smear of a well-differentiated liposarcoma. Note the Figure 5. Cytologic smear of a hemangiopericytoma. Note the positive red-orange color of the cytoplasmic vacuoles, indicative of lipid. Oil Red O staining of small, irregular vacuoles in the cytoplasm. Oil Red O with with hematoxylin counterstain, 3100 objective. hematoxylin counterstain, 3100 objective.

Three tumors were definitively diagnosed as heman- of light or deep blue cytoplasm that frequently was micro- giopericytomas based on both cytologic and histologic vacuolated. Large nuclei with clumped chromatin, anisokar- examination (Table 1). In cytologic specimens, the cells had yosis, and macronucleoli were observed. All of the smears exfoliated in 2-dimensional, noncohesive sheets, and in 1 contained bi- or trinucleated cells, and 1 sample contained sample a focal perivascular arrangement of cells was seen. multinucleated giant cells. Histologically, 1 tumor was di- Cytologic specimens contained spindle or stellate cells with agnosed as a poorly differentiated sarcoma, with large, plump slightly blue cytoplasm and oval to round nuclei with finely ovoid or caudate, deeply cells with a few micro- irregular chromatin. All smears contained binucleated and vacuoles, irregularly shaped nuclei with clumped chromatin, multinucleated cells; in the latter, nuclei were arranged around and frequent atypical mitoses. Three of these tumors were the cytoplasmic border leading to their designation as ‘‘crown diagnosed histologically as fibrosarcomas. The sarcoma with cells.’’11 In 2 of the tumors the cytoplasm of the cells contained multinucleated giant cells in cytologic specimens was classified small vacuoles; in 1 of these tumors, the vacuoles were histologically as malignant fibrous histiocytoma. The tumor partially and weakly positive to Oil Red O staining (Figure 5). diagnosed as poorly differentiated sarcoma by cytologic exam- Five cases were diagnosed as unclassified sarcomas based ination was classified histologically as undifferentiated sarcoma. on cytologic examination. The cytologic specimens contained Of the 5 tumors with a cytologic diagnosis of unclassified either single or aggregated spindle cells, with variable amounts sarcoma, none reacted positively with Oil Red O stain (Figure

Figure 4. Cytologic smear of a pleomorphic liposarcoma. Note the Figure 6. Cytologic smear of a fibrosarcoma. The spindle cells are positive staining of cytoplasmic microvacuoles. Oil Red O with hema- unstained, but the background is focally positive for lipid. Oil Red O with toxylin counterstain, 3100 objective. hematoxylin counterstain, 3100 objective.

Vol. 35 / No. 1 / 2006 Veterinary Clinical Pathology Page 39 Oil Red O Staining of Canine Liposarcoma

6), with the exception of a few globules in the background dog.29 To the best of our knowledge, this is the first report of all smears that stained a typical red-orange color. about use of Oil Red O in staining cytologic samples from canine neoplasms. Further studies will be necessary to validate the method for making a final cytologic diagnosis of Discussion liposarcoma, including criteria to avoid false-positive results and for correctly interpreting the presence of fatty material. In this series of cases, Oil Red O was used to accurately In conclusion, Oil Red O is a simple, inexpensive, and diagnose liposarcomas in cytologic specimens based on the useful stain for demonstrating the presence of fat in cytologic identification of cytoplasmic lipid. Cells in 1 of 8 other samples, which might be ideal material for confirming sarcomas also were positive for lipid, however, a diagnosis a morphologic suspicion of neoplasia originating in adipose of hemangiopericytoma was made in that case based on tissue. It should be mentioned that we used air-dried specimens other cytologic features. that were not treated with alcohol-based solvents. Our results Liposarcoma is a rare tumor in dogs and cats, representing strongly suggest that Oil Red O can assist in the diagnosis of only a small percentage of all animal neoplasms. The tumor liposarcoma and, in particular, might aid in correct interpreta- may arise in almost any body site and has been found in tion of the lipid content of the vacuolation observed in cytologic spleen,12,13 bone,14–16 abdominal organs,17 extradural spinal samples. Moreover, a suspicion of liposarcoma in cytologic location,18 liver,19 heart,20 and bone marrow21,22; however, the specimens that is supported by positive Oil Red O staining subcutis of extremities and the trunk is the most common might be useful preliminary information for selecting the best site.22,23 In humans, liposarcomas often are localized to deeper fixation procedures to be adopted after surgical removal of soft tissues, the gluteal region, thigh, lower portion of the masses to obtain a more rapid, definitive, histologic diagnosis. extremities, and retroperitoneum. Histologic diagnosis of liposarcoma is made on the basis of light microscopic results, and the neoplasm is subdivided into well-differentiated, myxoid, and pleomorphic types.5 In well-differentiated var- References iants, the neoplasm is characterized by round, spindle, or 1. Goldschmidt MH, Hendrick MJ. In: Meuten, DJ, ed. Tumors in Domestic stellate cells, and vacuolated cytoplasm. Frequent coalescence Animals. 4th ed. Ames, IA: Iowa State Press; 2002:97–99. of the vacuoles gives rise to a single large vacuole that displaces 2. Garvin CH, Frey DC. Liposarcoma in a dog. J Am Vet Med Assoc. 1962;140:1073–1075. the nucleus at the periphery of the cytoplasm. In pleomorphic 3. Jabara AG. Three cases of liposarcoma in dogs. J Comp Pathol. 1964;74: or anaplastic types, only a small percentage of cells contain 188–191. cytoplasmic vacuolation, but the characteristic morphology 4. Hendrick MJ, Mahaffey EA, Moore FM, Vos JH, Walder EJ. Histological of deformed nuclear profiles is often observed. The myxoid Classification of Mesenchymal Tumors of Skin and Soft Tissues of Domestic variant is identified by neoplastic cells that are loosely ar- Animals, Washington, DC: Armed Forces Institute of Pathology; ranged in mucoid, Alcian blue-positive stroma.1 In the present 1998:20–21. series of cases, 3 tumors were definitively diagnosed as well- 5. Doster AR, Tomlinson MJ, Mahaffey EA, Jordan CW. Canine lip- osarcoma. Vet Pathol. 1986;23:84–87. differentiated liposarcoma, and 1 was the pleomorphic type; myxoid variants were not observed. Ultrastructural studies 6. Messick JB, Radin MJ. Cytologic, histologic and ultrastructural characteristics of a canine myxoid liposarcoma. Vet Pathol. 1989;26: and special stains for neutral often are helpful in estab- 520–522. 5,23 lishing the lipid content of cytoplasmic vacuoles. 7. Cowell RL, Tyler RD, Meinkoth JH. Cytology of the Dog and Cat. The cytologic diagnosis of liposarcoma in 3 of 4 cases was St. Louis, MO: Mosby; 1999:230–239. suspected on the basis of previously published criteria such as 8. Baker R, Lumsden JH. Color Atlas of Cytology of the Dog and Cat. large, round, cytoplasmic vacuoles,6–8 presence of lipoblasts,6,8 St. Louis, MO: Mosby; 2001:47–48. peripheralization of nuclei,6,8 and perivascular arrangement of 9. Raskin, RE, Meyer DJ. Atlas of Canine and Feline Cytology. Philadelphia cells.6,24–26 Cells in 1 of the 4 cases had minimal evidence for PA: WB Saunders; 2001:306–309. differentiation, with ovoid neoplastic cells, variable numbers 10. Johnson F.B. Lipids. In Prophet EB, Harrington JB, Sobin LH, eds. Laboratory Methods in Histotechnology. Washington, DC: American of small to medium-sized vacuoles, and nuclear pleomor- Registry of Pathology; 1992:177–178. phism. Our observations based on the cytologic examination 11. Caniatti M, Ghisleni G, Ceruti R, Roccabianca P, Scanziani, E. of samples with morphologic features suggestive of lip- Cytological features of canine haemangiopericytoma in fine needle osarcoma were supported by clear evidence of positivity of aspiration biopsy. Vet Rec. 2001;149:242–244. the cytoplasmic vacuolation to Oil Red O, identifying a crucial 12. Spangler WL, Culbertson MR, Kass PH. Primary mesenchymal feature of this type of neoplasm, compared with that of other (nonangiomatous/ nonlymphomatous) neoplasms occurring in the canine spleen: anatomic classification, immunohistochemistry and mesenchymal tumors, which appeared to be weakly positive mitotic activity correlated with patient survival. Vet Pathol. 1994;31: in only 1 case of hemangiopericytoma. 37–47. 27 Oil Red O stain has been used in human cytopathology. 13. Weinstein MJ, Carpenter JL, Schunk CJ. Nonangiogenic and non- Kim and Goldblatt describe the use of Oil Red O staining of air- lymphomatous sarcomas of the canine spleen: 57 cases (1975–1987). J Am Vet Med Assoc. 1989;195:784–788. dried cytologic smears to differentiate malignant fibrous histiocytoma from pleomorphic variants of liposarcoma and 14. Misdorp W, Van Der Heul R.O. An osteo-(chondro)liposarcoma 28 (‘‘malignant mesenchymoma’’) of the radius in a dog, with two types rhabdomyosarcoma in humans. Hoenerhoff documented of metastases. Zentralbl Veterinarmed A. 1975;22:187–192. Oil Red O-positive staining of liposarcomatous regions in 15. Brodey RS, Sauer RM, Medway W. Canine bone neoplasms. JAmVet histologic sections of a multipotential osteosarcoma in a Med Assoc. 1963;143:471–495.

Page 40 Veterinary Clinical Pathology Vol. 35 / No. 1 / 2006 Masserdotti, Bonfanti, De Lorenzi, Ottolini

16. Nielsen SW. Comparative pathology of bone tumours in animals, with 23. Yager JA, Scott DW. The skin and appendages. In: Jubb KVF, Kennedy particular emphasis on the dog. Recent Results Cancer Res. 1976;54:3–16. PC, Palmer N, eds. Pathology of Domestic Animals. San Diego, CA: Academic Press; 1993:725–726. 17. Ackerman LJ, Silver JN. Abdominal liposarcoma in a dog. Mod Vet Pract. 1984;65:470–471. 24. DeMay RM. The Art and Science of Cytopathology. Chicago, IL: American Society for Clinical Pathology; 1996:575–576. 18. Lewis DD, Kim DY, Paulsen DB, Kerwin SC. Extradural spinal liposarcoma in a dog. J Am Vet Med Assoc. 1989;195:1580–1583. 25. Geisinger KR, Naylor B, Beals TF, Novak PM. Cytopathology including transmission and scanning electron microscopy of 19. Sundararaj A, Thanikachalam M, Murali Manohar B, Thilakarajan N, pleomorphic liposarcoma in pleural fluids. Acta Cytol. 1980;24: Ramakrishnan R. A case of liposarcoma in the liver of a dog. Indian 435–441. J Cancer. 1984;21:85–87. 26. Hajdu SI. Pathology of Soft Tissue Tumours. Philadelphia, PA: Lea & 20. Machida N, Kobayashi M, Tanaka R, Katsuda S, Mitsumori K. Primary Febiger; 1979:321–323. malignant mixed mesenchymal tumour of the heart in a dog. J Comp Pathol. 2003;128:71–74. 27. Sachdeva R, Kline TS. Aspiration biopsy cytology and special stains. Acta Cytol. 1981;25:678–683. 21. Davis PE, Dixon RT, Johnson JA, Paris R. Multiple liposarcoma of bone marrow origin in a Greyhound. J Small Anim Pract. 1974;15: 28. Kim K, Goldblatt PJ. Malignant fibrous histiocytoma. Cytologic, light 445–456. microscopic and ultrastructural studies. Acta Cytol. 1982;26:507–511. 22. Baez JL, Hendrick MJ, Shofer FS, Goldkamp C, Sorenmo KU. 29. Hoenerhoff MJ, Kiupel M, Rosenstein D, Pool RR. Multipotential Liposarcomas in dogs: 56 cases (1989–2000). J Am Vet Med Assoc. 2004; osteosarcoma with various mesenchymal differentiations in a young 224:887–891. dog. Vet Pathol. 2004;41:264–268.

Vol. 35 / No. 1 / 2006 Veterinary Clinical Pathology Page 41