Superfrost plus adhesion slides

RBCs were fixed in suspension for 15 min at RT in a solution containing 4% paraformaldehyde and 0.05% glutaraldehyde (v:v). Fixed RBCs were then washed and dropped off on PLL-coated coverslips for 8 min. Coverslips were finally mounted with Dako (Invitrogen) on SuperFrost Plus adhesion slides (VWR) and observed by light microscopy. RBCs were classified into discocytes, echinocytes and spherocytes. Their respective abundance was assessed through manual counting and expressed as % of the total RBC population.

All anonymised FFPE blocks used within the study were sectioned by author RS over the course of 4 batches which were carried out on 4 consecutive days with key factors controlled for, such as, water bath set to 37°C, same equipment used each day, and same batch numbers for reagents used. This was to reduce batch-to-batch variability.
All slides were sectioned at 3 µm and picked up on either TOMO^® IHC adhesive slides (Solmedia Ltd., Shrewsbury, United Kingdom) for IHC labelling with positive control material or on SuperFrost®Plus Adhesion Slides (VWR^®, Leicestershire, United Kingdom) for H&E sections which will have been consistent to the original H&E slides for reporting.
H&E staining was performed using the automated Leica Autostainer XL [Leica Microsystems (UK) Ltd. Milton Keynes, United Kingdom], using a commercially available Harris’ haematoxylin and a 1% aqueous Eosin [Leica Microsystems (UK) Ltd. Milton Keynes, United Kingdom]. H&E staining was carried out using the same routine protocol that is used within the laboratory.
Cut slides for IHC were left to dry in a 37°C oven for 10 min before being transferred to a 60°C oven to bake for 1 h. Slides were then labelled according to the anonymised format given and loaded onto the IHC labelling platform on the same day that each batch was produced.

RBCs were fixed in suspension for 15 min at RT in a solution containing 4% paraformaldehyde and 0.05% glutaraldehyde (v:v). Fixed RBCs were then washed and dropped off on PLL-coated coverslips for 8 min. Coverslips were finally mounted with Dako (Invitrogen) on SuperFrost Plus adhesion slides (VWR) and observed by light microscopy. RBCs were classified into discocytes, echinocytes and spherocytes. Their respective abundance was assessed through manual counting and expressed as % of the total RBC population.