Capillary blood reference intervals for platelet parameters in healthy full-term neonates in China

The study commenced after obtaining Institutional Ethical Committee approval (TJ-IRB20190308). The subjects were prospectively enrolled into this study, and all of them were healthy full-term (259 to 293 days (37 to 41 weeks) of gestation) neonates born at two hospitals from November 2018 to April 2019. Major inclusion/exclusion criteria are listed as follows. As listed in Chang et al. [6] for mothers, maternal age ranged from 20 to 40 years old and they had a normal health check-up without unfavourable past history during pregnancy, such as smoking, infectious diseases (hepatitis B/C, HIV, syphilis infections), idiopathic thrombocytopenic purpura, malignancies and chronic diseases (diabetes mellitus, autoimmune disease, immunodeficiencies and thrombotic disorders); mothers did not receive aspirin during pregnancy; a vaginal delivery or caesarean section was event-free. Professional obstetricians were responsible for the data of this part. As listed in Chang et al. [6] and Wasiluk [15] for neonates, they fitted all clinical state criteria for full-term neonates; they were born with normal birth weight (2500-4000 g) appropriate for gestational age and normal Apgar score (8–10 points at 1–5 min of life); physical examinations of the subjects were normal without any signs of infection or congenital anomalies at the time of sampling. And we excluded all neonates with an unfavourable perinatal history of premature rupture of membrane more than 24 h, abnormal placenta, placenta abruption, chorioamnionitis or meconium stain, or with a medical history of hospitalizing in the neonatal intensive care unit. All subjects are Han ethnicity.

After written informed consent was obtained from a parent or guardian for all subjects, we collected about 70 μL capillary whole blood of the subjects during the first 4 days of life (12 to 84 h old), by heel prick with the automated incision device into BD Microtainer tubes (spec, 0.5 mL; Becton Dickinson and Company, USA) containing K2-ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Samples were obtained by experienced technicians at the same time of blood sampling for the screening of congenital hypothyroidism and phenylketonuria or the routine serum bilirubin in morning hours (1–2 h later after last food intake). The first-second drop of blood was discarded and each collection time was less than 1 min. Samples were stored at ambient temperature for a maximum of 4 h before the assay was performed.

All capillary blood samples were analysed by SYSMEX XN-9000 haematology automatic analyser (Sysmex Corporation, Kobe, Japan) in the pre-dilution mode (dilution ratio, complete blood: PK dilution = 1:6) to measure PLT (× 10⁹/L) and related parameters, including mean platelet volume (MPV, fL), plateletcrit level (PCT, %), platelet size distribution width (PDW, fL) and platelet large cell ratio (P-LCR, %). As listed in Kaito et al. [16] for the detection principle of platelet parameters, MPV was calculated by a formula (PCT / PLT × 10⁴), and PDW and P-LCR were analysed from a histogram of platelet size distribution. Quality controls were run during every shift.

Data management and analyses were performed using SPSS 25.0 software (SPSS Inc., Chicago, USA). The data distribution was evaluated using Q-Q plots, histograms and Shapiro-Wilk test. We built Box plot (with Tukey variation) to identify the outliers using GraphPad Prism 8.2.1 (GraphPad software, La Jolla, CA, USA). An outlier was defined as a value < Q₁–1.5 × IQR (Q₁: 25th percentiles, IQR: interquartile range), or > Q₃ + 1.5 × IQR (Q₃: 75th percentiles). Quantitative data were expressed as either mean (± standard deviation, SD) for normally distributed data or median (IQR) for data not normally distributed. RIs for platelet parameters were defined by an interval of 2.5th – 97.5th percentiles. We performed Student’s t-test or Mann-Whitney U test as appropriate in order to evaluate influence of sex on platelet parameters. Pearson’s or Spearman’s correlation coefficients was performed as appropriate to evaluate correlations between platelet parameters and correlation factors. Two-tailed P values of less than 0.05 were set as statistical significance.

Totally 594 neonates were enrolled in present study. Demographic data are shown in Table 1. Several values were identified as outliers in platelet parameters and we excluded cases test-by-test in data analyses (see Fig. 1). Except for PLT, other platelet indices were not normally distributed. RIs for platelet parameters represent the central 95 % out of all data (Table 2). As a result, capillary blood RI of PLT ranged from 152 to 464 (× 10⁹/L).

Female neonates had higher capillary blood PLT values (310 (±77) × 10⁹/L) than male neonates (299 (±77) × 10⁹/L) but this difference was not statistically significant (p-value = 0.082). Expectedly, other platelet parameters were not significantly affected by sex (all p-values > 0.05) (Table 2). In order to evaluate correlations between platelet parameters and gestational age (259 to 293 days of gestation) and postnatal age (12 to 81 h old), we performed Pearson’s correlation coefficient in PLT and Spearman’s correlation coefficient in platelet indices (Table 3). Significant negative correlations between gestational age and platelet parameters were observed (p-value < 0.05). However, in view of the low coefficients of correlation in PLT and PDW across gestational age, a biologic relationship between them is likely absent. Moreover, correlations between postnatal age and platelet parameters (except for PCT) were significant but very mild as all of absolute values of correlation coefficients were below 0.2.