GENType: all-in-one preimplantation genetic testing by pedigree haplotyping and copy number profiling suitable for third-party reproduction.

STUDY QUESTION: Is it possible to develop a comprehensive pipeline for all-in-one preimplantation genetic testing (PGT), also suitable for parents-only haplotyping and, for the first time, third-party reproduction? SUMMARY ANSWER: Optimized reduced representation sequencing (RRS) by GENType, along with a novel analysis platform (Hopla), enables cheap, accurate and comprehensive PGT of blastocysts, even without the inclusion of additional family members or both biological parents for genome-wide embryo haplotyping. WHAT IS KNOWN ALREADY: Several haplotyping strategies have proven to be effective for comprehensive PGT. However, these methods often rely on microarray technology, whole-genome sequencing (WGS) or a combination of strategies, hindering sample throughput and cost-efficiency. Moreover, existing tools (including other RRS-based strategies) require both prospective biological parents for embryo haplotyping, impeding application in a third-party reproduction setting. STUDY DESIGN, SIZE, DURATION: This study included a total of 257 samples. Preliminary technical validation was performed on 81 samples handpicked from commercially available cell lines. Subsequently, a clinical validation was performed on a total of 72 trophectoderm biopsies from 24 blastocysts, tested for a monogenic disorder (PGT-M) (n = 15) and/or (sub)chromosomal aneuploidy (PGT-SR/PGT-A) (n = 9). Once validated, our pipeline was implemented in a diagnostic setting on 104 blastocysts for comprehensive PGT. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were whole-genome amplified (WGA) and processed by GENType. Quality metrics, genome-wide haplotypes, b-allele frequencies (BAFs) and copy number profiles were generated by Hopla. PGT-M results were deduced from relative haplotypes, while PGT-SR/PGT-A results were inferred from read-count analysis and BAF profiles. Parents-only haplotyping was assessed by excluding additional family members from analysis and using an independently diagnosed embryo as phasing reference. Suitability for third-party reproduction through single-parent haplotyping was evaluated by excluding one biological parent from analysis. Results were validated against reference PGT methods. MAIN RESULTS AND THE ROLE OF CHANCE: Genome-wide haplotypes of single cells were highly accurate (mean > 99%) compared to bulk DNA. Unbalanced chromosomal abnormalities (>5 Mb) were detected by GENType. For both PGT-M as well as PGT-SR/PGT-A, our technology demonstrated 100% concordance with reference PGT methods for diverse WGA methods. Equally, for parents-only haplotyping and single-parent haplotyping (of autosomal dominant disorders and X-linked disorders), PGT-M results were fully concordant. Furthermore, the origin of trisomies in PGT-M embryos was correctly deciphered by Hopla. LIMITATIONS, REASONS FOR CAUTION: Intrinsic to linkage-analysis strategies, de novo single-nucleotide variants remain elusive. Moreover, parents-only haplotyping is not a stand-alone approach and requires prior diagnosis of at least one reference embryo by an independent technology (i.e. direct mutation analysis) for haplotype phasing. Using a haplotyping approach, the presence of a homologous recombination site across the chromosome is biologically required to distinguish meiotic II errors from mitotic errors during trisomy origin investigation. WIDER IMPLICATIONS OF THE FINDINGS: We offer a generic, fully automatable and accurate pipeline for PGT-M, PGT-A and PGT-SR as well as trisomy origin investigation without the need for personalized assays, microarray technology or WGS. The unique ability to perform single-parent assisted haplotyping of embryos paves the way for cost-effective PGT in a third-party reproduction setting. STUDY FUNDING/COMPETING INTEREST(S): L.D.W. is supported by the Research Foundation Flanders (FWO; 1S74619N). L.R. and B.M. are funded by Ghent University and M.B., S.S., K.T., F.V.M. and A.D. are supported by Ghent University Hospital. Research in the N.C. lab was funded by Ghent University, VIB and Kom op Tegen Kanker. A.D.K and N.C. are co-inventors of patent WO2017162754A1. The other authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Establishing local reference intervals for full blood count and white blood cell differential counts in Cape Town, South Africa.

BACKGROUND: Accurate laboratory reference intervals (RIs) are essential to differentiate between health and disease. There are variations in haematological indices within populations relating to gender, age, ethnicity and environment. Iron deficiency is common, has a wide range of clinical morbidities and affects red cell indices. Locally derived RIs for full blood count (FBC) parameters are needed for the Western Cape region of South Africa, after the exclusion of iron deficiency. In addition, information regarding the prevalence of iron deficiency in first-time blood donors would inform blood transfusion services regarding policies to screen for and treat iron deficiency. OBJECTIVES: To establish locally derived RIs for FBC and white blood cell (WBC) differential count parameters in healthy adults in the Cape Town area, by including first-time blood donors and excluding those with iron deficiency and thalassaemic indices. These new locally established RIs could update those in use by the local National Health Laboratory Service. A secondary objective was to establish the prevalence of iron deficiency in first-time blood donors. This would inform blood donation policies regarding screening and appropriate iron supplementation in high-risk groups prior to blood donation. METHODS: This was a prospective, descriptive study with direct convenience sampling. Participants were prospective voluntary blood donors aged between 18 and 60 years, presenting for first-time blood donation. Ethnicity was self-identified. Participants who tested positive for HIV or hepatitis B and/or C viruses were excluded. Prospective participants with iron deficiency, defined by serum ferritin levels below the RI, and those with red cell indices suggestive of an underlying thalassaemia trait were excluded. FBC samples were analysed using a Sysmex XN-1000 cell counter. Statistical non-parametric methods were used to calculate the RIs, according to international guidelines. RESULTS: Of the 774 participants screened, 82 (11%) had iron deficiency and were excluded. Six hundred and sixty-two patients were included for analysis, 409 (62%) female and 253 (38%) male. The majority of the participants, 348 (53%), were between 20 and 29 years of age, with a mean age of 29 years for females and 28 years for males. Participants comprised a mix of the various ethnic groups residing in Western Cape Province. The mean haemoglobin concentration for females was lower than that for males (p<0.0001). There were significant gender differences for total WBC count, absolute neutrophil count and platelet count, with females having higher counts than males. CONCLUSIONS: Locally established, population-specific RIs are essential for the accurate interpretation of haematological indices. This study established locally derived gender-specific RIs for the Cape Town region, after exclusion of iron deficiency. These new RIs have implications for the accurate diagnoses of cytopenias, cytoses and other blood count abnormalities. Iron deficiency is common in first-time blood donors, and screening for iron deficiency using point-of-care testing should be considered.