Ventana ish iview blue detection kit

Hematoxylin-and-eosin-stained sections of 3–4 µm thickness were prepared after retrieving the formalin-fixed paraffin-embedded (FFPE) tissue blocks from the histopathology laboratory. EBV status was assessed by EBER detection through in situ hybridization (ISH) using the Ventana^® ISH iView Blue Detection Kit (Roche^® Diagnostics GmbH, Germany) and automated BenchMarck^® Ultra staining module according to manufacturer’s instructions (Ventana^® Medical Systems Inc., United States). EBER status was classified as positive or negative, as defined by the presence or absence of deep dark blue signal in nuclei of tumor cells seen under light microscopy.

In order select EBV-positive AIL samples, the EBV was detected by in situ hybridization (ISH). The formalin-fixed paraffin-embedded (FFPE) tissue sections used for this detection were initially deparaffinized, then rehydrated in a graded solution of xylene and alcohol and deproteinized with proteinase K (ThermoFisher Scientific, Illkirch-Graffenstaden, France). They were subsequently incubated with the Ventana EBER 1 DNP Probe^® (Roche Diagnostics, Meylan, France; catalog number 760-1209) used for EBER hybridization, followed by staining with the Ventana ISH iVIEW blue detection kit^® (Roche Diagnostics; catalog number 760-097). Images of the EBER staining obtained for control 1 (absence of EBER detection) and for a patient with AITL are visible in the Figure S1.

EBER in situ hybridization was performed using a commercially available kit. Briefly, formalin-fixed, paraffin-embedded sections were hybridized with the Inform EBER probe (#05278660001, Roche Diagnostics) and the VENTANA ISH iView Blue Detection Kit was used for visualization (#05278511001, Roche Diagnostics). The staining procedures were performed in a Ventana BenchMark ULTRA IHC/ISH System (Roche Diagnostics) according to guidelines from the supplier.

An independent review of BM dysplasia was performed by two hematopathologists for detection of MDS. MDS based on criteria established by the WHO⁵² (≥ 10% dysplasia of ≥ 1 lineage). MDS was confirmed when both hematopathologists reached agreement. Anti-CD3 (DAKO, USA), CD20 (DAKO), CD4 (Leica biosystem, USA), CD8 (Roche, Switzerland), granzyme B (Roche), and CD56 antibodies (Roche), EBER in-situ hybridization (Ventana ISH iView Blue Detection Kit, Roche) were used with automatic staining (Ventana Benchmark Ultra, Roche) for exclusion of other mature T cell neoplasm and assessment of T cell expansion in BM. Most of T-LGL patients (36/41, 87.8%) expressed CD8, 2 patients (4.9%) expressed CD4, 1 patient (2.4%) were CD4/8 coexpressed and the remaining 1 patient (2.4%) showed undetermined phenotype due to poor stainability. Analysis of rearrangements of TCR gene beta and gamma chain was performed using the BIOMED-2 assay (Invivoscribe, USA) according to the manufacturer’s instructions.