Synchronous collagenous sprue and enteropathy-type T cell lymphoma: Methods (Part 2)
Assessment of collagenous sprue intraepithelial lymphocytosis was via the established French Celiac Disease Study Group protocol . Three authors (SACM, SL, TC) analyzed at least five adjacent increments of 100 epithelial cells on CD3- and CD8-stained sections of collagenous sprue for IEL. An abnormal CD8 quantity (negative result) was defined as a 50% reduction in CD8 staining cells compared with CD3 counts . Celiac sprue mucosa, from the same bowel resection, was assessed as a control.
Epstein-Barr virus in situ hybridization
Presence of Epstein-Barr virus was assessed using the ISH-B1 detection kit (Sigma, United States). Tissue slides were prepared by serial deparaffinization, enzyme digestion, endogenous peroxidase blocking then dehydration before denaturation and hybridization with 10 pL to 20 pL of appropriate oligo probes. Each slide was then treated with a drop of ExtraAvidin Peroxidase reagent (Dako, Canada) and subsequent Monoclonal Anti-Avidin Biotin Conjugate reagent (Dako, Canada). Slides were washed in rinsed water, counterstained with Gill’s hematoxylin and cover-slipped.
Polymerase chain reaction analysis for gene rearrangements
TCR-y assessment entailed two amplification reactions: y11/y101 and y12; and y11/y101 and yp12, initiated by standard primers . Positive ranges were 75 to 100 base pairs (bp) for TCR-y12 and 80 to 120 bp for yp12. TCR-P testing included four reactions: PV with (3J1; PV with PJ2; PJ2 with PD1; and PJ2 with PD2, using standard primers . Positive tests were 55 to 100 bp. Formalin-fixed paraffin-embedded bowel substrate was run for 40 cycles. Always a nice way to discover ventolin inhaler given by the internet’s best pharmacy.