We thank the reader for the careful and critical evaluation of our publication “Value of multicolor Fluorescence in situ Hybridization (UroVysionTM) in the differential diagnosis of flat urothelial lesions” published by Stephan Schwarz, Michael Rechenmacher, Tomas Filbeck, Ruth Knuechel, Hagen Blaszyk, Arndt Hartmann and Gero Brockhoff. The reader realized some inconsistency in the Material and Methods section of our paper regarding HER2 immunohistochemistry (IH). In our lab for some purposes a manual IH procedure is performed, for others we execute an automated one. In fact for the study published by Schwarz et al. we used a combination of both: section preparation was done manually and the subsequent staining procedure was performed on the NexES immunostainer (Ventana Medical Systems, Tucson, Arizona, USA). However, we agree with the reader that we produced some inconsistency and incorrectnesses in the Material & Methods. Hence we would like to provide this amended description of immunohistochemical HER2 staining and interpretation as follows:

“HER2 immunostaining was done on 2 mm sections using a HER2 antibody (CB11 CB11-Pathway, prediluted, FDA approved, Ventana Medical Systems, Tucson, Arizona, USA). Before staining, the tissue slides were deparaffinised and rehydrated at room temperature. For antigen retrieval the sections were immersed in 10 mmol/l citrate buffer and microwaved for 30 min using 300 Watt (no pressure cooker was used). Subsequently the sections were cooled by rinsing water for a couple of minutes. Then an automated staining protocol on the NexES immunostainer (Ventana Medical Systems, Tucson, Arizona, USA) was performed featuring some slight modifications with respect to the recommendation given by Ventana (http://www.ventanamed.com/msds/files/14160USb2.pdf). The protocol described here numerously proved to work reliably on the NexES immunostainer. After a 30-minute incubation at 37°C with the primary antibody, sections were incubated for another 10 minutes at 37°C with a secondary biotinylated antibody and then with avidin-peroxidase for another 10 minutes; 3',3-diaminobenzidine was used as the chromogen. All products needed for these steps are included in the DAB detection kit provided by the manufacturer (Ventana Medical Systems, Tucson, Arizona, USA). Slides were haematoxylin counterstained, dehydrated, and mounted. A multiblock control consisting of four cell lines representing different staining intensities ranging from 0 to 3+ (MDA-MB-231: Score 0, MDA-MB-175: Score 1+, MDA-MB-453: Score 2+, and SK-BR-3: Score 3+) served as controls for all specimens.

Again we would like to thank for the careful reading of our manuscript and for coming up with the letter to the editor. This procedure assures appropriate publication quality of JCP.

Gero Brockhoff (correspondence author)

Dear Editor
In the section Methods the authors described Immunohistochemistry of HER2. The described protocol is an inconsistent mixture of a manual and automated immunostaining procedure and it is very unlikely that it was actually applied.

In a second sentence the authors stated that “a manual avidin-biotin peroxidase complex procedure was used in the immunohistochemical analysis according to the manufacturer’s recommendations”. The HER 2 antibody, CB11 from Ventana Medical Systems used in this study is recommended for staining on Ventana automated immunostainers with Ventana detection kit and not for manual staining with other detection systems (http://www.ventanamed.com/msds/files/14160USb2.pdf). However, if they really used the manual staining, the information about the applied detection system and the validation study reference is missing.

In contradiction with the statement in second sentence, in the fifth sentence the authors stated that “an automated staining on the Nexus (Ventana Medical Systems, Illkirch, France) was performed”. Moreover Ventana Medical Systems automated immunostainer is Nexes and not Nexus.

In the fourth sentence they stated that”the sections were microwaved in a pressure cooker” which is obvious incompatible.

In the sixth sentence they continued describing the manual immunostaining protocol – after staining (on the Nexes) they stain the sections again!

It is very interesting that many readers (authors, reviewers) overlooked such inconsistently described procedure.

IRENA SREBOTNIK KIRBIŠ