Aim: High-resolution HLA typing is critical to the further improvement of solid organ transplant outcomes by increasing the accuracy of virtual crossmatch and facilitating the application of eplet matching in the organ allocation scheme. Commonly available NGS-based typing platforms are not suitable for Organ Procurement Organization (OPO) labs due to time constraints of the donor workup process and the labor intensiveness of the assays. We report here our experience in the evaluation, implementation and optimization of a rapid NGS-based HLA typing platform in a high-volume OPO-based lab.
Methods: High-resolution typing was performed using the GenDx NGS-Turbo® kit and the Oxford Nanopore Technologies (ONT) MinION Mk1B sequencer and flow cell. ONT MinKNOW software was used for data acquisition and GenDx NGSengine®-Turbo software was used for data analysis.
Results: The GenDx NGS-Turbo typing kit can produce 4-field typing at all 11 HLA loci with an average turnaround time of <4 hours from sample accessioning to result reporting which includes a 95-min whole gene amplification, a 30-min library preparation, a 20-60 min read time and 5-10 mins of analysis. The total hands-on time is only 45-60 mins. We have not encountered any ambiguities that involved CIWD alleles and have not had to confirm any donor typing using other methodologies. Some less common alleles sequenced thus far include C*05:07N, DRB4*02:01N, DPB1*352:01 and DPB1*416:01. Based on our experience, the flow cell can be reused up to 8 times with proper washing and storage which helps reduce the overall typing cost to $450/donor. Due to a broader region of HLA genes being sequenced, periodic discovery of novel alleles is anticipated. OPO labs utilizing this testing platform would need to develop a standardized approach for converting novel alleles to serological equivalents especially for after-hour reporting of deceased donor typing.
Conclusion: Deceased donor HLA typing is frequently performed after hours with technologists constantly struggling with multitasking and time constraints. The ONT platform appears to be an excellent fit for the donor HLA typing workflow in OPO labs. Together with the GenDx NGS-Turbo typing kit, the typing system requires minimal hands-on time and can produce rapid and accurate high-resolution HLA typing. Eplet-based matching will hopefully become a reality soon for deceased donor organ allocation.
