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Introduction The Sysmex XN-series haematology analyser has newly adopted a fluorescent channel to measure immature platelet fraction (IPF). To promote the clinical utility of this promising parameter, establishing a reliable reference interval is mandatory. According to previous studies, IPF values may be affected by the employed analyser and the ethnic background of the individual, but no differences seem to be found between individuals' genders. Therefore, this study aimed to define the reference interval for IPF in a Spanish population following Clinical and Laboratory Standard Institute (CLSI) guidelines. Materials and methods A total of 153 healthy Caucasian adults from Spain met the inclusion criteria. IPF measurement was performed by means of a Sysmex XN-2000 haematology analyser. A non-parametric percentile method was used to calculate the reference intervals in accordance with CLSI guidelines. Results The obtained reference interval for IPF on the Sysmex XN-2000 was 1.6-9.6% (90% confidence intervals (CIs) were 1.5-1.8 and 9.3-11.5, respectively). No significant gender difference in IPF reference intervals was observed (P = 0.101). Conclusions This study provides, for the first time, a reference interval for IPF using a Sysmex XN-2000 in a Spanish population, ranging from 1.6 to 9.6%. These data are needed to evaluate platelet production in several conditions such as thrombocytopenia, inflammatory states and cardiovascular diseases, as well as for future research.