Technical note Duration and method of tissue storage alters placental morphology e Implications for clinical and research practice A. Garrod a , G. Batra b , I. Ptacek a , A.E.P. Heazell a, c, * a Maternal and Fetal Health Research Centre, Institute of Human Development, University of Manchester, Manchester, UK b Department of Paediatric Histopathology, Royal Manchester Children’s Hospital, Manchester Academic Health Science Centre, Manchester, M13 9WL Manchester, UK c St Mary’s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, M13 9WL Manchester, UK article info Article history: Accepted 25 July 2013 Keywords: Placental morphometry Tissue storage Placental histopathology Stillbirth abstract We assessed whether placental morphology is affected by placental storage before fixation. Fresh tissue from uncomplicated pregnancies (n ¼ 10) was fixed immediately and further samples were stored dry, in PBS or culture medium for 24, 48 or 72 h at 4  C. Placental morphology quantified using image analysis software found no difference in syncytial nuclear aggregates, cytokeratin 7, CD45 or Ki67 immuno- staining irrespective of duration or mode of storage. The number of blood vessels per villus (CD31) was reduced in all conditions after 72 h (p < 0.05). Distal villous hypoplasia increased after 72 h (p < 0.05). Ideally, storage time should be minimised to 48 h prior to morphological or qualitative analysis. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction There is international consensus that histological examination of the placenta should be performed to determine the cause of stillbirth [1]. Fixation prior to macroscopic examination is not recommended as this makes assessment more difficult [2]. Instead, dry storage in plastic buckets at 4  C is recommended, during which the placenta is reported to be preserved for meaningful examina- tion for many days with minimal autolysis. However, in a review of placental examination Naeye et al. states that “troublesome arte- facts” can appear after 48 h of refrigeration [3]. As modern classi- fication systems for stillbirth place increased emphasis on placental pathology [4] it is important to resolve whether the method or duration of placental storage alters placental morphology. We hypothesised that placental morphology would be affected by the duration of storage. 2. Methods Placental tissue was obtained following written informed consent from women with uncomplicated pregnancies delivering after 37 weeks gestation (North West Research Ethics Committee 08/H1010/55); demographic characteristics of partici- pants are shown in Supplementary Table 1. Unless otherwise stated, reagents were obtained from SigmaeAldrich Chemical Company, Dorset, UK. Ten 1 cm 3 samples of placental parenchyma were taken from the middle of the placenta then washed in phosphate buffered saline (PBS). One sample of fresh tissue was fixed in 4% neutral buffered formalin for 24 h. The remaining samples were placed dry, in PBS or sup- plemented CMRL-1066 culture medium [5] at 4  C for 24, 48 or 72 h. After this period, samples were fixed using the same method for fresh tissue. Tissue samples were then dehydrated and wax-embedded prior to analysis. Placental proliferation, leukocyte infiltration, trophoblast area and vascularity were assessed by immunoperoxidase staining in 5 mm tissue sections using anti- bodies specific for Ki67 (Dako, Ely, Cambridgeshire, UK; 0.16 mg/ml), CD45 (Dako; 3.5 mg/ml), cytokeratin 7 (Dako; 1.1 mg/ml) and CD31 (Dako; 5.15 mg/ml) respectively. Negative controls were performed using non-immune mouse IgG (Dako) at matching concentrations to the primary antibody. Immunoperoxidase staining was performed as previously described [6]. Haematoxylin and eosin staining was per- formed to allow quantification of syncytial nuclear aggregates (SNAs) and qualitative assessment. Dewaxed and rehydrated sections were stained with Harris’s haema- toxylin for 10 min before differentiation in acid-alcohol. Slides were stained with eosin for 2 min, rinsed in cold tap water, and dehydrated and mounted as described above. Images were captured using an Olympus BX41 light microscope (Southend-on- Sea, UK) and QIcam Fast 1394 (QImaging, BC, Canada) and Image Pro Plus 6.0 (Media Cybernetics Inc, MD, USA). For every section, 5 random images of terminal villi were taken at 100 magnification. Morphometry was performed using image analysis software for proliferative index, number of CD45 positive cells, trophoblast area, density of SNAs and villous vascularity as previously described [6]. In addition, sections stained with haematoxylin and eosin were assessed by a perinatal histo- pathologist (GB) blinded to storage conditions. Each sample was qualitatively assessed for the presence of infarction, excessive syncytial knots, distal villous hy- poplasia, villous immaturity, villitis and other incidental abnormalities. Statistical analysis was performed using GraphPad InStat 3.0 (GraphPad Software, La Jolla, CA, * Corresponding author. Maternal and Fetal Health Research Centre, 5th Floor (Research), University of Manchester, St Mary’s Hospital, Oxford Road, Manchester M13 9WL, UK. Tel.: þ44 161 701 0889; fax: þ44 161 276 6134. E-mail addresses: alex_heazell@talk21.com, alexander.heazell@ manchester.ac.uk (A.E.P. Heazell). Contents lists available at ScienceDirect Placenta journal homepage: www.elsevier.com/locate/placenta 0143-4004/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.07.067 Placenta 34 (2013) 1116e1119