Issue link: https://ahint.epubxp.com/i/574474
19 ASHI Quarterly Third Quarter 2015 The flow cytometric crossmatch (FCXM) assay is used by the majority of histocompatibility laboratories in North America as part of pre-transplant risk assessment to detect donor specific anti-HLA antibodies 1 The FCXM assay is significantly more sensitive than cytotoxic crossmatch 2 allowing better detection of low level HLA antibodies, thereby improving assessment of pre- transplant immunological risk 3-5 The standard three-color FCXM protocol currently used by the majority of labs was described over 25 years ago 6,7 The protocol can be divided into two parts The first part consists of donor lymphocyte enrichment using ficoll based centrifugation, 6 followed by treatment of donor cells with pronase and DNase to reduce Fc receptor expression and remove dead and dying cells 8 The second part consists of the crossmatch assay and antibody detection by flow cytometry 6 The entire protocol is time intensive, typically requiring four to five hours to complete, and can lead to significant transplant delays and compromise organ quality 9 The goal of this study was to develop an expeditious FCXM procedure without compromising quality or sensitivity of the assay In the first part of the study we investigated the impact of various assay parameters on FCXM results Subsequently, by modifying several of these parameters, we developed and validated a rapid optimized FCXM protocol, which takes 35-40 minutes to perform (a 60% decrease compared to standard protocol) while maintaining excellent assay sensitivity Finally, we adopted the EasySep Direct lymphocyte enrichment technique from StemCell Technologies Inc 10 to improve lymphocyte purity (>90%) and isolation time to less than 60 minutes Materials and Methods Flow Cytometric Crossmatch (FCXM) Assay Reagents All washes in FCXM assays were performed using the flow wash buffer (FWB) composed of phosphate buffered saline (PBS; Life Technologies Inc , Burlington ON, Canada; www lifetechnologies com ) with 2% (v/v) fetal calf serum (Life Technologies Inc ) Anti-CD3-PerCP (cat# 347344) and anti-CD19-PE (cat# 340364) monoclonal antibodies were purchased from BD Biosciences (Mississauga, ON, Canada; www bdbiosciences com ) Goat anti- human IgG-FITC polyclonal antibody (IgG-FITC; cat# 109-096- 098) was purchased from Jackson ImmunoResearch Laboratories Inc (West Grove, PA; www jacksonimmuno com ) Patient Sera All flow cytometric crossmatch (FCXM) optimization experiments were performed using pooled positive control sera (PC; a pool of 20 highly sensitized patient sera) and pooled negative control sera (NC; a pool of 10 non-sensitized patient sera) For the Halifax FCXM and Halifaster FCXM protocol validation studies, patient sera were selected based on virtual crossmatches predicted to result in negative or positive FCXM reactions Virtual crossmatches were considered positive when one or more donor specific HLA antibody(ies), >5000 MFI, were identified by the LABScreen single antigen bead (SAB) assay (One Lambda, Canoga Park, CA; www onelambda com ), and negative when no donor specific HLA antibodies were present (<500 MFI) Donor Cells For FCXM optimization experiments, cells were obtained from volunteer donors For the Halifax FCXM and Halifaster FCXM protocol validation experiments, donor cells were obtained from volunteer, live, and deceased donors as indicated in the results section Peripheral Blood Mononuclear Cell Enrichment Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation Briefly, 12 ml of acid dextrose citrate (ACD) anti-coagulated donor blood were diluted in 12 ml of PBS The blood mixture was then overlayed on top of 18 ml of Lympholyte-H density medium (Cedarlane, Burlington, Ontario, Canada; www cedarlanelabs com ) in a 50 ml Falcon tube and centrifuged at 400 x g for 30 minutes with no brake Mononuclear cell layer was collected, transferred into a fresh 15 ml Falcon tube and washed three times in 10 ml PBS for 10 minutes at 400 x g Lymphocyte Isolation Lymphocytes were purified from donor blood using EasySep TM Direct Human Total Lymphocyte Isolation Kit (STEMCELL Technologies Inc , Vancouver, BC, Canada; http://www stemcell com/ ) according to manufacturer instructions Following isolation, lymphocytes were washed once in 10 ml PBS at 400 x g for five minutes and washed again in 1 ml PBS at 400 x g for one minute S C I E N T I F I C C O M M U N I C A T I O N S Development and Validation of a Rapid Optimized Flow Cytometry Crossmatch (FCXM) Assay, the Halifax and Halifaster FCXM Protocols Robert S Liwski, MD, PhD, FRCPC Robert A Bray, PhD, D(ABHI) Howard M Gebel, PhD, D(ABHI)