Yinong Wang, Robert A. Goulart, L. Pantanowitz
Apr 1, 2011
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Diagnostic Cytopathology
Abstract
Oil red O (Solvent Red 27 or Sudan Red 5B) is a lysochrome (fat-soluble) diazo-dye used for staining neutral triglycerides and lipids in frozen tissue sections or unfixed (air-dried) cytologic slides (smears and/or touch preparations). Histochemical staining must be performed on fresh samples, as both alcohol fixation and the paraffin embedding process remove lipids. Oil red O staining is used to identify both exogenous and endogenous lipid deposits. In surgical pathology, the Oil Red O stain has been used to identify lipid within parathyroid and liver tissue, tumors (e.g., renal cell carcinoma), fat emboli, cardiomyopathy, atherosclerotic lesions, and polyethylene debris within tissue procured after arthroplasty. It has also been used in forensic investigations to identify the presence of fingerprints on wet surfaces. In cytopathology, Oil red O staining is not commonly used in practice. One of the reasons for this is that the staining results are often unreliable. Nonetheless, the major indication for Oil red O staining at present is to detect and enumerate lipid-laden alveolar macrophages (LLAM) in respiratory specimens from the lower airways of young children. Requests from clinicians to look for LLAM in their pediatric patients persist. That is why cytologists should at least be aware of the practice of staining these respiratory specimens with Oil red O. Childhood pulmonary aspiration of food or gastric contents can occur secondary to a structural and/or medical condition, such as swallowing dysfunction or gastroesophageal reflux. The diagnosis of aspiration in the pediatric patient can be challenging, as it is generally accepted that no ‘‘gold-standard’’ test exists. As alveolar macrophages phagocytose aspirated material, an increased lipid content in these macrophages from a bronchoalveolar lavage (BAL) or tracheal specimen may serve as a useful noninvasive indicator of pulmonary aspiration or lipoid pneumonia. Oil red O staining of respiratory aspirates with the subsequent finding of abundant LLAM with intracellular lipid droplets (Fig. C-1) is accordingly used as a positive indicator of suspected aspiration. However, this finding is not specific for aspiration. For example, Oil red O-positive macrophages in BAL specimens can also be observed in trauma patients. A semiquantitative LLAM index (LLMI) has been described in which macrophages are graded by evaluating the amount of intracellular lipid present in each macrophage with a score of 0–4 (Table I). To determine the grade, it is recommended that at least 100 macrophages be evaluated in each specimen. Indices may thus range from 0–400. If the LLMI is above 85, aspiration should be suspected, in the appropriate clinical setting. The exact cutoff score is best determined through study in one’s own laboratory, correlating with clinical data. The significance of lower values is unclear. Moreover, the interobserver coefficient of variation for the LLMI is poor (>50%). The LLMI has not proven to be clinically useful when applied to sputa. Others have assigned grades of absent (grade 1⁄4 0), positive (grade 1: 1–25 lipid-laden macrophages), moderate positive (grade 2: 26–50 lipid-laden macrophages), and high positive (grade 3: > 50 lipid-laden macrophages). Granules in neutrophils may also stain with Oil red O, and as such must be excluded from the count. Therefore, we suggest that in specific clinical situations, such as pediatric cases with suspected aspiration, an air-dried preparation of a respiratory specimen be prepared to preserve and stain for lipid in macrophages, if requested by the clinician. Oil red O staining in cytopathology has rarely been used in other instances, such as assisting in the diagnosis of lipid-rich tumors. Staining an air-dried cytologic smear or touch preparation of nipple discharge fluid to examine for lipid material can be used to confirm galactorrhea (a milky discharge unassociated with childbirth or nursing). A nipple discharge due to galactorrhea, such as breast milk, is rich in lipids. Hence, galactorrhea can be Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, Springfield, MA *Correspondence to: Liron Pantanowitz, M.D., Department of Pathology, Baystate Medical Center, 759 Chestnut Street, Springfield, MA 01199. E-mail: liron.pantanowitz@baystatehealth.org Received 12 November 2009; Accepted 17 February 2010 DOI 10.1002/dc.21390 Published online 14 May 2010 in Wiley Online Library (wileyonlinelibrary.com).