Automated immunostainer

The Wright-Giemsa stained aspirate smears and haematoxylin and eosin (H&E) stained core bone marrow biopsies were reviewed. Immunohistochemistry studies were performed on formalin fixed, paraffin-embedded tissue with monoclonal antibodies using an automated immunostainer (Dako Omnis) using the manufacturer's instructions. Quality controls for each immunohistochemistry stain were reviewed. Flow cytometry was performed as part of routine case workup.
Standard PCR-based and cytogenetic analysis was performed at the Associated Regional and University Pathologists, Inc., ARUP Laboratories (Salt Lake City, Utah). Detection of the FLT3 TKD and IKD mutation was performed on isolated DNA using targeted fluorescent PCR primers for sequence amplification. The TKD products were cut using the EcoRV restriction enzyme and the resultant amplified ITD and TKD sequences were analyzed for base pair length on an ABI 3500xl genetic analyzer. A fragment of NPM1 exon 12 was also analyzed by targeted PCR amplification and interpreted using capillary electrophoresis. For cytogenetic studies, chromosomes were prepared from a nondiluted bone marrow aspirate collected in a heparinized syringe. The specimens were transported within 48 hours to ARUP laboratories. Each sample was cultured and suspended in metaphase. Giemsa-banded karyotyping was performed and interpreted with ISCN 2013.

For α‐SMA and Sox9 quantification, paraffin‐embedded mouse liver sections were stained on an automated immunostainer (Dako), using a mouse on mouse ImmPRESS HRP kit (Vector Laboratories) according to the manufacturer's instructions, using anti‐mouse α‐SMA (1:200; ASM‐1; Vector Laboratories) or anti‐rabbit Sox9 (1:500; Millipore) with a Vector ImmPRESS horseradish peroxidase rabbit secondary antibody. Ten non‐overlapping fields (×20 objective) from each section were captured using a light microscope (Carl Zeiss) with identical illumination and exposure. Digital image analysis was performed using ImageJ software for α‐SMA quantification. Sox9⁺ cells were counted manually. For collagen quantification, paraffin‐embedded mouse liver sections or snap‐frozen human liver sections were stained with Sirius red, using a standard protocol. Collagen values were expressed as the percentage of the total area of a section occupied by Sirius red staining.

Immunohistochemistry was carried out on formalin-fixed paraffin-embedded (FFPE) sections using an automated immunostainer (Dako Omnis). A primary antibody directed against GFAP (polyclonal, prediluited, high pH, DAKO) was applied. GFAP stained tissue slices were acquired using the Nanozoomer (Hamamatsu, RRID : SCR_022537) instrument. Slides were scanned at 40X and images were saved into.ndpi format and viewed using the NDPIv2 software (Hamamatsu). 3 random images at 20X magnification were exported from 11 PDHGG patient tissues (n=5 for H3.1 K27M; n=6 for H3.3 K27M) as.TIFF file and analyzed using the ImageJ software (RRID : SCR_003070, http://imagej.nih.gov/ij/) as described in Negm et al. (31 (link)). The mean intensity feature evaluated for each image was normalized over the number of manually counted nuclei for each image.

Immunohistochemistry on cell block and FFPE capsular specimens was performed using an automated immunostainer (Dako, Glostrup, Denmark) with the following primary antibodies: CD30 (clone Ber-H2), CD3 (clone F7.2.38), CD4 (clone 4B12), CD8 (clone C8/144B), CD68 (clone PG1), CD15 (clone Car-b), Granzyme B (clone GrB-7), IRF-4 (clone MUM1) (Dako), CD25 (clone 4C9, Novocastra, Newcastle Upon Tyne, UK), PAX5 (clone SP34, Thermo Scientific, Waltham, USA). Paraffin sections were pretreated using EnVision^™ FLEX Target Retrieval Solution (Dako) and incubated with an optimal dilution of the primary antibody. The reaction was visualized with the EnVision Detection Kit (Dako) using 3–3’-diaminobenzidine chromogenic substrate. Sections were counterstained with EnVision FLEX Hematoxylin (Link) (Dako).
For every marker, positive cells were counted out of 10 non-overlapping randomly selected high-power microscopic fields (HPFs, 40×10), and the mean number of positively stained cells per HPF was recorded. The percentage of positive cells was calculated as the ratio between the mean number of stained cells per HPF and the mean number of total cells per HPF.