Alejandro S. Mendoza, MD, Cytopathology Fellow
Alaa Afify, MD, Professor and Director of Cytopathology
Lydia Howell, MD, Professor and Chair of the Department of Pathology and Laboratory Medicine
Successful pathologic diagnosis after an image-guided fine needle aspiration (FNA) depends on several factors including nature of the lesion, skill of the aspirator, and availability of rapid on-site evaluation (ROSE)1. ROSE has proven to improve the sensitivity and diagnostic yield of FNA according to several published articles2-5. Pathologists, cytotechnologist, and sometimes pathology residents/fellows provide ROSE service to check the cellular content and adequacy of fine-needle aspiration smears and biopsy touch imprints. ROSE can inform the operator of the need to obtain additional samples and in some instances allows for preliminary diagnosis6.
Preparation starts with a detailed clinical and radiologic information about the patient and the site of the specimen. Prior history and current clinical findings are helpful to know if primary or a metastatic tumor is to be expected, as well as whether an infectious process is in the differential. Knowledge of the site is helpful in trimming down possible differential diagnoses. Abnormal serum markers (CA19.9, CA125, AFP, etc.) also provide useful clues for primary cancer diagnosis1.
Laboratory Best Practice: How to Perform ROSE
The entire ROSE procedure can be done in few minutes. There are numerous studies and articles describing ROSE methodology7-10. The actual on-site evaluation involves 3 rapid common steps: smearing, staining, and microscopic evaluation.
Smearing: The purpose of making a smear is to fix the FNA aspirate onto the slide and to prevent the sample from being lost during a staining procedure. A small drop of the sample (about 4-5mm in diameter) is placed in a pre-cleaned, labeled slide, near its frosted end. Using another slide as a spreader (held at a 30-40 degrees angle), the droplet is quickly smeared to the opposite unfrosted end for a good feathered edge. If the aspirate is bloody, it is better to just submit the entire specimen in a preservative for cell block. Blood can make a smear thick leading to an obscuring artifact. A cell block preparation is better in preserving the cellular material present in the bloody aspirate.
Staining: The staining method used for ROSE is important in influencing the rapidity of the procedure and the quality of the slide for microscope evaluation. Though the Papanicolaou stain is commonly used for routine interpretation, it is impractical for ROSE since the staining time and many reagents make it impractical for use outside of the laboratory at the site of collection. There are two commonly used methods for rapid staining of smeared-slides during ROSE:
- Modified Wright-Giemsa, also known commercially as Diff-Quik (DQ): DQ reduces a standard 4–minute process of Wright-Giemsa stain into a simplified 15-second procedure. It uses 3 different solutions; a fixative reagent (e.g. methanol), an eosinophilic solution (e.g. Xanthene dye), and a basophilic solution (e.g. thiazide dye) 11. Another advantage of DQ is the quality of the cytoplasmic and extra cellular material details in eosinophilic and basophilic hue. Microbiologic agents also appear easily in DQ12. A major disadvantage of DQ is that it requires an air–dry sample prior to staining which can extend the time necessary to ROSE. Some samples are either mucin-rich or bloody and therefore require extra time for drying. Mucinous or bloody specimens can also be obscured by the stain preventing full evaluation by ROSE. Another disadvantage is the quality of the eosinophilic and basophilic color, since this is dependent on the time the slide is left in the staining solutions. More time is required for a crisp detail of the cells. Since time is gold, other staining methods are being explored and tried in ROSE.
- Tolonium chloride, aka Toluidine blue (TB): The Cytopathology Laboratory at UC Davis prefers TB for our in-house ROSE. This has been our preferred staining method for over 30 years. Our cytotechnologists primarily perform ROSE and all are well trained and very experienced with the procedure and with interpretation. TB is a cationic (basic) thiazine metachromatic dye which has a high affinity for acidic tissue components and turns nucleic acid blue and polysaccharides purple11. The use of TB in ROSE is efficient, economical, and practical. The use of TB saves time as it does not require air-drying of the smeared specimen and only involves one stain solution in the process. A major advantage is that the slide can later be re-stained with the Pap stain which provides a better nuclear detail for final diagnosis. A disadvantage is that TB does not provide a two-tone eosinophilic-basophilic contrast of the tissue components. It provides a monotone appearance of the tissue sample which therefore requires some degree of experience for interpretation. TB may not adequately penetrate areas of the smear with obscuring artifacts such as blood and thick mucus, limiting evaluation during ROSE.
Microscopic evaluation: The cytotechnologist or pathologist makes a quick scan of the stained slide. Presence of neoplastic cells, artifacts, etc. are reported immediately to the physician performing the FNA.
Final tips for ROSE
- Do not expect a definitive diagnosis. The main purpose of the procedure is to evaluate specimen adequacy- in other words, to obtain the sufficient cellular material for definitive diagnosis and for ancillary studies. The collecting physician should expect to be told whether or not diagnostic material is present or no diagnostic material is present during the procedure.
- Communicate clearly the sites, special requests, and possible differential diagnosis. Different potential diagnoses may need special collection and orders for ancillary tests. Some tests require special container such as RPMI for lymphoma which the cytotechnologists may not otherwise have readily prepared.
- Expect to collect 3-4 FNA passes for smear preparation/ROSE. After the first adequate sample is obtained, 3-4 extra passes for cell block should be collected, if the patient can tolerate the additional collections. If needle core biopsies are planned, these should include 2-3 needle core samples with at least 2 cm length.
- Expect 3-4 days for the final result and additional more days if IHC stains are ordered.
- Equivocal ROSE evaluations may occur: In ROSE, no technique, stains, or procedure is perfect. Sometimes, the nature of the specimen may limit the ability of ROSE to fully see and evaluate the cells obtained.
- If the final diagnosis is unsatisfactory or not representative of the lesion after ROSE, know that the procedure is imperfect. Know that it is a standard practice in our laboratory to correlate ROSE findings with final adequacy evaluations. Know that these are monitored and tracked to ensure that we stay within the expected range of practice for ROSE.
- Gong Y. Metastatic Neoplasms in Fine-Needle Aspiration Cytology. DOI 10.1007/978-3-319-23621.
- Diacon AH, Schuurmans MM, Theron J, et al. Utility of rapid on-site evaluation of transbronchial needle aspirates. Respiration. 2005;72:182-188.
- Diette GB, White P Jr, Terry P, Jenckes M, Rosenthal D, Rubin HR. Utility of on-site cytopathology assessment for bronchoscopic evaluation of lung masses and adenopathy. Chest. 2000;117:1186-1190.
- Klapman JB, Logrono R, Dye CE, Waxman I. Clinical impact of on-site cytopathology interpretation on endoscopic ultrasoundguided fine needle aspiration. Am J Gastroenterol. 2003;98:1289-1294.
- Pellise Urquiza M, Fernandez-Esparrach G, Sole M, et al. Endoscopic ultrasound-guided fine needle aspiration: predictive factors of accurate diagnosis and cost-minimization analysis of on-site pathologist. Gastroenterol Hepatol. 2007;30:319-324.
- Tambouret, et. Al., Cytopathology and More | FNA cytology: Rapid on-site evaluation—how practice varies. CAP TODAY, May 18 , 2016.
- Howell LP, Gandour-Edwards R, Folkins K, Davis R, Yasmeen S, Afify A: Adequacy evaluation of fine-needle aspiration biopsy in the breast health clinic setting. Cancer 2004; 102: 295–301.
- Gosey LL, Howard RM, Witebsky FG, et al: Advantages of a modified Toluidine Blue stain and bronchoalveolar lavage for the diagnosis of Pneumocystis carinii J Clin Microbiol 1985; 22: 803–807.
- Canil M, Meir K, Jevon G, Sturby T, Moerike S, Gomez A. Toluidine Blue staining is superior to H&E staining for the identification of ganglion cells in frozen rectal biopsies. Histologic 2007; 40: 1–3.
- Layfield LJ, Bentz JS, Gopez EV. Immediate on-site interpretation of fine-needle aspiration smears: a cost and compensation analysis. Cancer (Cancer Cytopathol). 2001;93:319-322.
- Sridharan, G; Shankar, AA (2012). ” Toluidine Blue: A review of its chemistry and clinical utility”. J Oral Maxillofac Pathol. 16: 251–5. doi:10.4103/0973-029X.99081.
- da Cunha Santos G, Ko HM, Saieg MA, Geddie WR. “The petals and thorns” of ROSE (rapid on-site evaluation). Cancer Cytopathol. 2013 Jan;121(1):4-8. doi: 10.