S901 COMBINED GENE PANEL SEQUENCING AND EKTACYTOMETRY ‐ THE NEXT GENERATION OF RED CELL DIAGNOSTICS

Background: The Red Cell Gene Panel next generation sequencing (NGS) service has been offered at the Viapath Red Cell Centre at Kings College Hospital since 2016. To date we have reported 1500 diagnostic cases in the following categories: unexplained anaemia (membranopathy, enzymopathy, haemoglobinopathy and congenital dyserythropoietic anaemia), inherited bone marrow failure, congenital erythrocytosis and other (iron regulation and porphyria). The diagnostic yield of the unexplained anaemias was 46% in the first 1000 cases (patients in which a clear genetic diagnosis was found). However, in a further 34% of cases, a genetic variant of uncertain clinical significance (VUS) was detected and therefore the diagnosis was unclear. Within haemolytic anaemia, blood film, EMA dye binding and enzyme assays have had a positive impact on the diagnostic yield. The membranopathy (spherocytosis, elliptocytosis and stomatocytosis) genes have yielded the largest amount of clear diagnoses. However, due to the large nature of the membrane genes ( PIEZO1 , ankyrin, alpha and beta spectrin, etc.) and their propensity for variation, they have also produced the largest amount of uncertain results. A further functional assay that could help to discriminate between the disease causing genetic variants and benign variation was sought to clarify cases with VUSs. Osmotic gradient ektacytometry presents us with such a methodology for looking at the functional properties of the red cell membrane. Aims: Here we present data from the evaluation and validation study of the LoRRca Maxsis (RR Mechatronics) at aiding clinical interpretation of genomic variants. By using ektacytometry we aim to clarify the clinical significance of VUSs in cases where a diagnosis would previously have been uncertain. Methods: Patients that were referred for EMA, membranopathy genetic analysis and those with a diagnosis of unexplained haemolytic anaemia were used in the validation study. All samples had genetic analysis and ektacytometry. These data were combined and the clinical utility of having genetic and ektacytometry results was assessed. Results: A total of 96 patients to date have been analysed using genetic data and ektacytometry, 80 patients had the NGS panel performed and a further 16 were analysed solely for the familial variant detected in the proband. Whilst many cases that had a clear genetic diagnosis have shown concordant ektacytometry results, here we present 5 cases that we believe show a clear diagnostic benefit of this combined approach. The new phenotypic information has informed the classification of the genetic variants detected. This includes a suspected case of di‐genic inheritance of variants in the SPTA1 and GLUT1 ( SLC2A1 ) genes and cases where multiple variants have been found and the pathogenic variant has been elucidated. Summary/Conclusion: This study has shown a good clinical utility for a combined NGS and ektacytometry approach and we are moving towards ISO15189 diagnostic accreditation for this testing to be offered alongside genetic analysis and as a standalone test. This testing has shown the potential to improve our discrimination value in variant interpretation for the red cell gene panel. We have also used this approach to aid designation of candidate variants from whole genome sequencing through the 100,000 genomes project. Investigation into the utility of this analysis for discriminating between variants in the PIEZO1 gene that cause dominant dehydrated stomatocytosis and recessive hereditary lymphoedema is also ongoing.