Journal of Reproductive Immunology 97 (2013) 183–185 Contents lists available at SciVerse ScienceDirect Journal of Reproductive Immunology j o ur nal homep age : w w w.elsevier.com/locate/jreprimm Short communication A rapid, reliable method for uNK cell density estimation Josephine A. Drury a,∗ , Ai-Wei Tang a , Mark A. Turner a , Siobhan Quenby b a Department of Women’s and Children’s Health, University of Liverpool, UK b Division of Reproductive Health, University of Warwick, UK a r t i c l e i n f o Article history: Received 14 August 2012 Received in revised form 4 December 2012 Accepted 10 December 2012 Keywords: Uterine natural killer cells CD56 Endometrium Immunohistochemistry Computer-assisted image analysis a b s t r a c t Interest in uterine NK cell density as a diagnostic or screening tool in reproductive disor- ders is growing. However, current methods of analysis are time consuming. In this study, 997 images from 100 women were analysed both by the currently used method combin- ing manual and computer aided counting, and by using colour splitting combined with area measurement as an estimate of positively stained cells. Good correlation was found between both methods (r 2 = 0.92) centred around the line of equality, with no systematic differences observed in Bland Altman plots. The area measurement was significantly faster and thus is a useful screening method. © 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Interest in uterine NK (uNK) cell counts as diagnostic or screening tools in reproductive disorders is growing. A recent randomised controlled double blind trial (RCT) (Tang et al., 2009) used uNK cell density as a screening test to select women for randomisation to treatment with prednisolone or placebo. Others have found higher uNK cell density in women with recurrent implantation failure (Tuckerman et al., 2010). However, lack of standardisation in the methodology between laboratories has hampered research and made comparisons difficult, with wide varia- tion in reported uNK cell density between centres. A recent publication (Drury et al., 2011) described an accurate and reproducible method of assessing uNK cell density by a combination of manually counting the number Abbreviations: uNK cell, uterine natural killer cell; DAB, diaminobenzi- dine; SAIA, semi-automated image analysis; CDAM, colour deconvolution and area measurement; HPF, high-power field; LOA, limit of agreement. ∗ Corresponding author at: Department of Women’s and Children’s Health, University of Liverpool, Liverpool Women’s Foundation Trust, Crown Street, Liverpool L8 7SS, UK. Tel.: +44 0151 7959563. E-mail address: jadrury@liv.ac.uk (J.A. Drury). of uNK cells stained positive with DAB, and using the ‘par- ticle analysis’ function of the public domain image analysis package, ImageJ to calculate the number of stromal cells. However, despite the use of image analysis software the method was still time consuming. The development of uNK cell density as a screening test would require the ability to inform a large number of women in a reasonable time whether they were screened positive or negative. Thus, there is a need to develop an alternative method for uNK cell screening that is quick, reliable and reproducible, in order to target novel therapies. The aims of this study were to validate a computer aided screening method for estimating uNK cell density and com- pare the results and timing for the new and established methods. 2. Materials and methods Immunohistochemistry for CD56 was performed on mid-luteal phase endometrial biopsies processed from 100 consecutive women who consented for screening into a RCT as previously described (Tang et al., 2009). An internal “staining control” slide was included with each run. Images for analysis were captured using Eclipsenet 0165-0378/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jri.2012.12.002