Methodological considerations in estrogen assays of breast fluid and breast tissue Robert T. Chatterton a,⇑ , Miguel Muzzio b , Richard Heinz c , Peter H. Gann d , Seema A. Khan a a Northwestern Feinberg School of Medicine, United States b Illinois Institute of Technology Research Institute, United States c University of Colorado Anschutz Medical Campus, United States d University of Illinois College of Medicine, United States article info Article history: Received 1 May 2014 Accepted 16 June 2014 Available online 24 August 2014 Keywords: Estradiol Breast Tissue Serum Immunoassay Tandem mass spectroscopy abstract Estradiol (E2) in nipple aspirate fluid (NAF), ductal lavage fluid (DLF), and random fine needle aspirates (rFNA) are compared. Quantification was by immunoassay or tandem MS. The percent of women yielding NAF varied between 24% and 48% and for DLF was 86.3%. Variation between ducts within a breast was not less than variation between breasts within women but variation between breasts and within women over time was significantly less than variation between women. Serum E2 was highly significantly different among phases of the menstrual cycle but NAF E2 was not different. The correlation between serum and breast fluid E2 concentrations in premenopausal women had coefficients of determination of less than 15%. The correlation between serum and NAF in studies of postmenopausal women varied greatly and may depend on patient selection. The difference between NAF E2 between pre- and postmenopausal women was only 22%; for rFNA it was non-significantly 44% lower in a similar group of postmenopausal women. Progesterone was 96% and 98% lower in postmenopausal NAF and rFNA samples, respectively. Measurements of E2 in breast fluid or breast tissue appears to provide similar estimates of E2 exposure. E2 levels in breast fluid do not reflect the rapid changes that occur in serum and, thus, serum availability of E2 is only one factor determining its levels in the breast. The similarity of levels between breasts and between ducts suggests that estimates of estrogen exposure does not require multiple samples, however, unavailability of fluid may require rFNA in some cases. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction There are a number of reasons to suggest that serum concentra- tions of estrogens may not be representative of the concentrations of estrogens available to the breast parenchyma. Both sulfation and hydrolysis of estrogens sulfates occurs in the breast [1–3] and changes in metabolism may result in alterations in tissue concen- trations. Uptake of sulfated estrogens may also be affected [4,5]. Earlier studies of nipple aspirate fluid indicated that the concentra- tion of estradiol is higher in the breast than in serum, and the coef- ficients of determination (the percent of the variance in breast fluid or tissue that can be accounted for by variation in serum) were generally less than 0.5 [6–8]. Nevertheless, the concentrations remain sufficiently constant over time within individuals to be useful as indicators of exposure of individuals [9]. 2. Experimental 2.1. Collection of NAF and DLF The methods for collection of NAF and DLF have been described in detail in previous publications [9,10]. Briefly, after warming and massage of the breast, droplets of NAF are collected from the nip- ple in calibrated capillary tubes. The volume is measured, the sam- ple is flushed out with 200 lL of phosphate-buffered saline, and the diluted sample is sealed and stored at À80 °C. Lavage for DLF was performed as described previously [10]. The breast was mas- saged, and the Cytyc aspirator (Cytyc Corp., Boxborough, MA) was used to elicit nipple aspirate fluid. Lavage of fluid-yielding ducts and visualized-non-fluid yielding ducts was performed through a microcatheter (Cytyc), using plasmalyte, an isotonic electrolyte solution (Baxter Healthcare Corp., Deerfield, IL). The http://dx.doi.org/10.1016/j.steroids.2014.08.002 0039-128X/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author at: Department of Ob/Gyn, Northwestern University Feinberg School of Medicine, 710 N Fairbanks Court, Chicago, IL 60611, United States. Tel.: +1 312 503 5272. E-mail address: chat@northwestern.edu (R.T. Chatterton). Steroids 99 (2015) 103–107 Contents lists available at ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids