Each participant interpreted the HER2 IHC score according to the ASCO-CAP guidelines [7]. Figure 1 Workflow of the EQA program. A. EQA HER2 immunostaining: specimens were selected and sent by the Coordinating Center (CC) to the 16 PCs. B. EQA HER2 interpretation: specimens were selected and sent by the CC to the 16 PCs grouped into 3 sets. The Tariquidar order study was reviewed and approved by the Ethics Committee of the Regina

Elena National Cancer Institute and a signed informed consent was obtained from all patients. Statistics In the EQA HER2 immunostaining step, the performance of each laboratory was evaluated by comparing the reviewer’s interpretation of the slides stained by each laboratory according to the reference values. In addition, in order to evaluate the contribution of each scoring category to the overall agreement (i.e. the agreement between the score given by the reviewers on the slides stained by each laboratory in accordance with the reference values) the kappa category-specific (kcs) statistic [19], and its 95% confidence interval obtained by means of the Jackknife method [20], were calculated as previously Liproxstatin-1 described [21, 22]. To this end, the slides stained by all the

participants were jointly considered. Each kcs value was interpreted in a qualitative manner based on the Landis and Koch classification criteria PF-573228 in vivo [23]. In the EQA HER2 interpretation step, the level of agreement of each laboratory according to the reference values was evaluated by computing the weighted kappa statistic (kw) and its 95% Jackknife confidence interval as previously described. In line with our previous experience with

EQA programs, the agreement was considered fully satisfactory only when the lower limit of the 95% Jackknife confidence interval was equal to or greater than 0.80. For each participant the kcs statistic and its 95% Jackknife confidence interval were also computed. Statistical analyses were performed with the SAS software (Version 9.2.; SAS Institute Inc., Cary, NC). Results Questionnaire The results of the questionnaire are reported in Table 1. Frequency distribution of the responses indicates moderate methodological heterogeneity between the 16 laboratories. All the PCs used Thiamet G paraffin embedded tissue and the DAB chromogen in their routine. Most PCs adopted buffered formalin during fixation. Twenty-four hours was the modal fixation time and also the modal time elapsing between cutting to IHC. For more than two thirds of participants, the slides were stored at room temperature. Only 5 PCs used the manual immunostaining procedure. The polyclonal antibody A0485 purchased by Dako was the most commonly used reagent. The majority of PCs used a heat retrieval in an automated immunostainer. Only one participant used an image analyzer for evaluating the sample in addition to the optical microscope in their routine.