Doing more with less: multiple uses of a single slide in veterinary cytology. A practical approach

Abstract

Veterinary cytology faced a remarkable evolution in the last 15 years, in part due to increase recognition of the advantages of the cytology by veterinary clinicians. Simultaneously, there has been a growing awareness by the owners about the importance of a complete diagnostic workup aimed at defining a proper treatment protocol. With the extended use of cytology, challenging diagnostic cases are more frequent, and more clinically useful answers are requested. In this scenario, the use of cytology specimens to perform ancillary techniques is a valid approach. Rather than being simply archived, cytology slides can be a valuable source and a good platform to carry out cytochemistry, immunocytochemistry, and molecular techniques. Therefore, several diagnostic techniques can be applied in tiny samples, thus following the "doing more with less" principle. The aim of this approach is to refine the cytologic diagnosis and provide additional prognostic and therapeutic information. Herein, we detailed this principle in veterinary cytology and reviewed the use of cytology specimens for ancillary techniques as a single procedure, i.e., using the whole slide, or multiple procedures, i.e., multiple procedures applied in the same slide.

Keywords: Cat; Cytology; Diagnosis; Dog; Immunocytochemistry; Molecular biology.

Fig. 1

Schematic overview of the multiple uses of a cytologic smear. Single procedure refers to the full use of the smear (meaning that no Romanowsky-stain is retained) for special cytochemical staining (A), immunocytochemistry (B) or for molecular biology (C). In contrast, multiple procedures refer to the simultaneous application of different techniques, while retaining Romanowsky-stained material in part of the slide, which can be covered (D) or split and transferred to other slides (E). Multiple uses can also be achieved in manual immunocytochemistry (F), by applying several antibodies (AB1 and AB2) on the same slide, retaining the Romanowsky-stained area

Fig. 2

Pyogranulamatous inflammation in the skin of a dog. Various fungal organisms (later diagnosed as Microsporum spp.) can be seen among neutrophils and macrophages in aqueous Romanowsky (Hemacolor, Merck) (A). After restaining with periodic-acid Schiff (PAS) more organisms could be detected (B); a second restaining with Grocott–Gomori methenamine silver stain (applied over the PAS) highlighted more fungal organisms, especially when cells and debris overlapped (arrows) (C). After each staining procedure, the slides were scanned using the Olympus VS110 Virtual Slide Scanning System (Olympus, Japan) and the same areas of slide were compared

Fig. 3

Pleural effusions from one dog with a benign effusion (A, B) and a second dog with a malignant effusion (C, D) stained with a double immunocytochemistry protocol against pan-cytokeratins (AE1/AE3) and Wilms-tumor 1 (WT1), as a screening test for carcinomatous cells. (A) Top panel shows a cytocentrifuged preparation with isolated cells or small clusters Hemacolor stained (aqueous Romanowsky). (B) After the double immunocytochemistry, all cells have a strong brown nuclear positivity (WT1 antibody, Diaminobenzidine chromogen) and weak cytoplasmic red staining (AE1/AE3 antibody, Aminoethylcarbazole chromogen), which is consistent with mesothelial cells and a benign effusion; this was later confirmed by cell block immunohistochemistry and follow-up. (C) Bottom panel shows macrophages (asterisks), cell clusters of mesothelial cells (arrows), intermingled with neoplastic carcinomatous cells (arrowheads) Hemacolor stained. (D) After the double immunocytochemistry, macrophages appear negative for both markers (asterisks), mesothelial cells have double positivity (arrows) (i.e., nuclear positivity with WT1 antibody, Diaminobenzidine chromogen, and cytoplasmic staining with AE1/AE3 antibody, Aminoethylcarbazole chromogen), whereas neoplastic carcinomatous cells have single positivity (arrowheads) (i.e., red cytoplasmic staining with AE1/AE3 antibody, Aminoethylcarbazole chromogen). In this double immunocytochemistry protocol, slides were first incubated with WT1, followed by an acidic glycine buffer rinse to elute these antibodies, and then incubated with pan-cytokeratin antibody

Fig. 4

Fine needle aspiration of an enlarged lymph node submitted for molecular testing (PCR for antigen receptor rearrangement, PARR). After removing the coverslip, the smear was scraped (A) into a vial (arrow in B). After Proteinase K digestion (B), the DNA is extracted using commercial kits. After ensuring that sufficient DNA of good quality is present, PCR can be performed (C). The DNA products can be either sequenced or sorted in agarose gels or by capillary electrophoresis (D), in which the peaks of the sample are compared with control samples of T and B lymphomas. In this case, the sample was consistent with a T-cell lymphoma

Fig. 5

Ascitic fluid of a dog, in which brown to greenish extracellular material was observed in Romanowsky staining (detailed in E). In order to elucidate the identity of this material, the right part of the slide was covered with mounting medium (cover 1 in A) and the Hall’s staining was applied in the uncovered area (B, D). After immersing in xylene to remove the previous cover, a second application followed. The left part of the slide was now covered with mounting medium (cover 2 in A) and the Perl’s Prussian Blue applied over the uncovered area (C). Note that besides biliary material (arrows in B, D and E, the latter two correspond to the same area), there were also hemosiderin that appeared blue in Perl’s Prussian Blue (arrowheads in C). In this case, the sample was consistent with biliary peritonitis

Fig. 6

Fine needle aspiration of a dog with enlarged lymph nodes submitted for tissue transfer (A). After heating the mounting medium (in a water bath at 60ºC for 1 h), the medium is lifted from the slide (B), then clipped in pieces (C) and transferred to new slides (D). The receptor slides should be moistened and firm pressure should be applied after the transfer (for instance, the slides can be left overnight with even pressure applied under a heavy book)

Fig. 7

Fine needle aspiration of an enlarged lymph node submitted for panel immunocytochemistry against CD3, a marker of T lymphocytes and PAX5, a marker of B lymphocytes. The Romanowsky-stained slides were previously scanned using the Olympus VS110 Virtual Slide Scanning System and the same areas were compared before (A) and after the immunostaining (B). By comparing the same regions (C, D and E, F pairs) it is possible to disclose a positive nuclear immunoreactivity to PAX5 (D) meaning that a proliferation of neoplastic B lymphocytes was present, whereas only few scattered lymphocytes were positive for CD3 (arrows) (F)