Tissue microarray and digital image analysis: a methodological study with special reference to the microenvironment in Hodgkin lymphoma Ingrid Glimelius, 1, * Fredrik Qvarnstro ¨m, 1, * Martin Simonsson, 1 Anders Ekwall, 1 Karin E Smedby, 2 Daniel Molin 1 & Rose-Marie Amini 3 1 Department of Radiology, Oncology and Radiation Science, Uppsala University, Rudbeck Laboratory, Uppsala, 2 Department of Medicine Solna, Clinical Epidemiology Unit, Karolinska Institutet, Stockholm, and 3 Department of Immunology, Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Uppsala, Sweden Date of submission 19 September 2011 Accepted for publication 18 December 2011 Glimelius I, Qvarnstro ¨m F, Simonsson M, Ekwall A, Smedby K E, Molin D & Amini R-M (2012) Histopathology Tissue microarray and digital image analysis: a methodological study with special reference to the microenvironment in Hodgkin lymphoma Aim: Cancer research has moved from solely investi- gating the tumour cells to also including analysis of the tumour microenvironment; however, the methods utilized have not been evaluated for this change. The aim of this study was to compare tissue microarrays (TMA) to whole tissue sections (WS) with regard to cells in the tumour microenvironment. Manual evalu- ation and digital image analyses (DIA) were utilized and also compared. Methods and results: TMA slides from 117 Hodgkin lymphoma patients were immunostained for forkhead box protein 3 (FoxP3) [identifying regulatory T cells (T reg )], and 39 corresponding WS were also analysed. Manual evaluation and DIA were utilized for all patients on both the TMA and the WS. A correlation coefficient of 0.83 was obtained for the proportion of T reg in TMA versus WS using manual evaluation and a correlation coefficient of 0.77 with DIA. T reg counts using manual evaluation correlated in turn with DIA, with a coefficient of 0.79 for the 117 TMA sections and 0.65 for the 39 WS. Conclusion: Because a high correlation was observed between TMA and WS, TMA can be utilized when evaluating cells in the tumour microenvironment. DIA appears to provide a reliable measurement method, provided that manual control of the tumour slides is conducted. Keywords: digital imaging, Hodgkin lymphoma, microenvironment, regulatory T cells, tissue microarray Abbreviations: DIA, digital image analyses; FoxP3, forkhead box protein 3; HL, Hodgkin lymphoma; HRS, Hodgkin and Reed-Sternberg; TMA, tissue microarrays; T reg, regulatory T cells; WS, whole tissue sections Introduction Studies of inflammatory cells in cancer have gained increasing interest, particularly with regard to tumour invasiveness and metastasis. The composition of the microenvironment is of prognostic importance in several tumour types, 1 and new drugs targeting the microenvironment are currently under development. 2 Thus, new methods are needed to study this important compartment within tumour tissue and will help to improve our understanding of tumorigenesis. In Hodgkin lymphoma (HL), the malignant Hodgkin and Reed-Sternberg (HRS) cells are in a minority in the tumour tissue. 3 For this reason, HL provides a well-suited model system for the study of tumour-surrounding inflammatory cells (the microenvironment). The inflam- matory infiltrate consists of different B- and T lympho- cytes, eosinophils, mast cells, macrophages, plasma cells, Address for correspondence: I Glimelius MD, PhD, Department of Radiology, Oncology and Radiation Science, Uppsala University Hospital, 751 85 Uppsala, Sweden. e-mail: ingrid.glimelius@onkologi.uu.se *These authors contributed equally to this study. Ó 2012 Blackwell Publishing Limited. Histopathology 2012 DOI: 10.1111/j.1365-2559.2012.04185.x