Perinatal factors affecting platelet parameters in late preterm and term neonates.

Platelets parameters including platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV) and platelet distribution width (PDW) are associated with various physiological and pathological functions in various disease. However, few studies have addressed whether perinatal factors may be associated with platelet parameters at birth in a large cohort of late preterm and term neonates. The aim of this study to investigate perinatal factors affecting platelet parameters in late preterm and term neonates. We retrospectively investigated platelet parameters including PLT, PCT, MPV, and PDW on the first day of life in 142 late preterm and 258 term neonates admitted to our NICU from 2006 through 2020. PLT, MPV, PCT, PDW on Day 0 did not significantly differ between the two groups. In term neonates, multivariate analysis revealed that PCT correlated with being small for gestational age (SGA) (β = -0.168, P = 0.006), pregnancy induced hypertension (PIH) (β = -0.135, P = 0.026) and male sex (β = -0.185, P = 0.002). PLT was associated with SGA (β = -0.186, P = 0.002), PIH (β = -0.137, P = 0.024) and male sex (β = -0.166, P = 0.006). In late preterm neonates, multivariate analysis revealed that PLT were associated with PIH, whereas no factors associated with PDW and MPV were found. In all patients studied, chorioamnionitis (CAM) was significantly associated with MPV (CAM = 10.3 fL vs. no CAM = 9.7 fL, P<0.001). Multivariate analysis showed that SGA, male sex and PIH were associated with PCT and PLT. This study demonstrates that different maternal and neonatal complications affect platelet parameters in late preterm and term neonates.

Introduction

Platelets parameters including platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV) and platelet distribution width (PDW) are associated with various physiological and pathological functions in various disease [1-3]. Platelet production is a complex process arising from the proliferation and differentiation of megakaryocytes under the stimulating influence of thrombopoietin. Platelet function in premature neonates is immature. Previous research indicated that low birth weight infants were at almost 2.5-fold increased risk for thrombocytopenia [4]. On the other hand, MPV, PDW, PCT are associated with neonatal disease such as sepsis, fungal infection and intraventricular hemorrhage [5-8]. Among extremely low birth weight neonates born to mothers with preeclampsia, the MPV/platelet count (PLT) ratio at birth significantly correlated with mortality [9]. Also, changes of PDW during the neonatal period were associated with thrombocytopenia and with neonatal sepsis in very low birth weight infants [10]. We previously reported that higher MPV correlates with mortality among those born at < 32weeks’ gestation [11].

On the other hand, Alicja et al reported decreased PCT and PLT in late preterm neonates compared with term neonates [12]. However, few studies have addressed whether perinatal factors may be associated with platelet parameters (PCT, PLT, MPV and PDW) at birth in a large cohort of late preterm and term neonates. Recently, some studies have been associated maternal complication such as premature rupture of membranes (PROM), chorioamnionitis (CAM), and pregnancy induced hypertension (PIH) were associated with platelet parameters [13-15]. Therefore, we hypothesized that platelet parameters such as MPV, PCT and PDW could be affected by perinatal factors. The objective of this study was to investigate the factors affecting platelet parameters at birth in late preterm and term neonates.

Materials and methods

Study design and population

This retrospective, single center, cohort study examined records from 2006 through 2020 at the neonatal intensive care unit (NICU) of Fukushima Medical University Hospital. The study protocol was approved by our institutional review board, The Ethics Committee of Fukushima Medical University, Fukushima, Japan. After deliberation, the board decided that no written consent was needed. Of 1789 neonates admitted to our NICU between January 2006 and Mar 2020, there were 400 born at term or late preterm (weeks 35–36). Late preterm neonates and full term neonates numbered 142 and 258, respectively (Fig 1). Exclusion criteria were congenital anomalies, neonatal alloimmune thrombocytopenia, and neonates not tested within 12 hours of birth.

Fig 1

Flowchart showing enrollment of study subjects.

Platelet parameter measurements

Blood samples were collected through peripheral venipuncture to measure PLT, PCT, MPV and PDW of each newborn. Complete blood counts were measured using a Sysmex CS-5100 coagulation analyzer (Sysmex, Kobe, Japan) on admission.

Prenatal and postnatal risk factors

Platelet parameters were compared with demographic variables including gender, birth weight (BW), and gestational age (GA). Furthermore, we considered pregnancy-induced hypertension (PIH), chorioamnionitis (CAM, defined clinically, with histopathological confirmation in pre-term placentas) [16]. premature rupture of membranes (PROM), and placental abruption (PA) as possible prenatal risk factors, and small for gestational age (SGA), respiratory distress syndrome (RDS), and Apgar scores as possibly associated with platelet parameters. Infants whose birth weight and height were below the 10th percentile of the normal curve at each GA were classified as SGA.

Statistical analysis

Platelet parameters from medical records were rendered into median and interquartile range (IQR) as non-normally distributed continuous variables. Differences in categorical variables and continuous variables were assessed for significance using Chi-square and Mann–Whitney U test. Likewise, correlations between BW, GA, and Apgar scores versus platelet parameters were also investigated using Spearman’s rank correlation (r) with P < 0.05 considered to be statistically significant. Moreover, the variables at P < 0.05 in univariate analysis were entered into a multiple regression analysis to identify independent prognostic factors.

Data analysis was performed with SPSS for Mac, release 25.0 (SPSS, Chicago, IL) and Prism 8 (GraphPad Software, San Diego, CA).

Results

Table 1 shows platelet parameters in late preterm and term neonates. There are no significant differences in platelet parameters between late preterm and term neonates. The median of sampling time was 82 min as shown in Fig 2. Fig 3A and 3B show no significant correlation between BW, GA and PLT. Likewise, Fig 3C and 3D show no significant correlation between BW, GA and PCT in late preterm and term neonates.

Table 1

Platelet parameters in late preterm and term neonates.

	Late preterm (n = 142) Median [IQR]	Term (n = 258) Median [IQR]	p-value
GA (weeks)	36.0 [35.4–36.4]	38.2 [37.5–39.3]	<0.001
BW (grams)	2180 [1802–2602]	2758 [2463–3106]	<0.001
SGA n (%)	57 (39.8%)	52 (20.2%)	<0.001
PCT (%)	0.25 [0.21–0.31]	0.26 [0.21–0.30]	0.661
PDW	10.9 [10.0–12.7]	10.9 [10.2–12.2]	0.810
MPV (fL)	9.9 [9.2–10.3]	9.7 [9.3–10.3]	0.792
PLT (×103/μL)	26.6 [22.2–32.3]	26.4 [21.5–31.4]	0.807

GA, gestational age; BW, birth weight; PCT, plateletcrit; PDW, platelet distribution width; MPV, mean platelet volume; PLT, platelet count; IQR, median interquartile range.

Fig 2

Peripheral blood sampling time distribution.

Fig 3

Correlation between platelet count, plateletcrit, GA, and BW.

R is Spearman’s correlation coefficient.

Tables 2 and 3 outline the results of univariate and multivariate analyses for the correlation between perinatal factors and platelet parameters in preterm and term neonates, respectively.

Table 2

Factors affecting platelet parameters in term neonates.

(n)	PCT (%)		PDW (%)	MPV (fL)	PLT (×103/μL)
	univariate analysis	multivariate analysis	univariate analysis	univariate analysis	univariate analysis	multivariate analysis
	Median [IQR]	p-value (β)	Median [IQR]	Median [IQR]	Median [IQR]	p-value (β)
Male (131)	0.24 [0.20–0.29]		10.8 [10.1–12.0]	9.7 [9.3–10.2]	25.9 [20.6–29.3]
Female (127)	0.27 [0.22–0.32]		11.1 [10.3–12.5]	9.8 [9.4–10.4]	28.8 [22.6–32.8]
	P = 0.005	P = 0.002 (-0.185)	P = 0.053	P = 0.139	P = 0.009	P = 0.006 (-0.166)
RDS (18)	0.26 [0.22–0.29]		10.9 [10.0–13.2]	9.4 [8.9–10.4]	26.3 [22.9–34.9]
non-RDS (240)	0.24 [0.21–0.30]		10.9 [10.0–12.3]	9.8 [9.3–10.3]	26.6 [21.4–31.3]
	P = 0.720		P = 0.842	P = 0.193	P = 0.580
SGA (52)	0.23 [0.16–0.29]		11.0 [10.4–12.5]	10.0 [9.4–10.4]	24.8 [17.4–30.5]
non-SGA (206)	0.26 [0.22–0.30]		10.9 [10.9–12.1]	9.7 [9.3–10.3]	26.7 [22.3–31.8]
	P = 0.024	P = 0.006 (-0.168)	P = 0.299	P = 0.193	P = 0.023	P = 0.002 (-0.186)
PROM (8)	0.22 [0.21–0.25]		10.3 [9.8–10.9]	9.4 [9.3–9.8]	23.8 [20.5–28.1]
non-PROM (250)	0.26 [0.21–0.30]		10.9 [10.2–12.3]	9.7 [9.3–10.3]	26.5 [21.5–31.5]
	P = 0.186		P = 0.194	P = 0.286	P = 0.230
CAM (9)	0.21 [0.19–0.29]		11.3 [11.0–12.1]	10.4 [10.1–10.9]	22.5 [20.1–28.2]
non-CAM (249)	0.26 [0.21–0.30]		10.9 [10.2–12.3]	9.7 [9.3–10.2]	26.5 [21.7–31.5]
	P = 0.553		P = 0.144	P = 0.005	P = 0.199
PA (5)	0.21 [0.19–0.24]		11.1 [9.8–14.9]	10.2 [8.1–10.3]	23.1 [20.5–27.8]
non-PA (243)	0.26 [0.21–0.30]		10.9 [10.2–12.3]	9.7 [9.3–10.3]	26.5 [21.7–31.4]
	P = 0.086		P = 0.725	P = 0.913	P = 0.319
PIH (4)	0.19 [0.12–0.30]		11.1 [10.9–12.2]	9.9 [9.3–10.3]	17.4 [12.3–19.8]
non-PIH (244)	0.50 [0.12–0.19]		10.9 [10.2–12.3]	9.7 [9.5–10.2]	26.5 [21.9–31.4]
	P = 0.009	P = 0.026 (-0.135)	P = 0.447	P = 0.683	P = 0.007	P = 0.024 (-0.137)
GA	r = -0.121		r = -0.003	r = 0.048	r = -0.072
	P = 0.052		P = 0.958	P = 0.446	P = 0.149
BW	r = 0.070		r = -0.053	r = 0.027	r = 0.020
	P = 0.260		P = 0.400	P = 0.666	P = 0.753
AP 1	r = 0.026		r = -0.039	r = -0.052	r = 0.022
	P = 0.677		P = 0.530	P = 0.409	P = 0.730
AP5	r = 0.072		r = -0.074	r = -0.048	r = 0.087
	P = 0.251		P = 0.235	P = 0.445	P = 0.165

PCT, platelet count; PDW, platelet distribution width; MPV, mean platelet volume; PLT, platelet count; GA, gestational age; BW, birth weight; RDS, respiratory distress syndrome; SGA, small for gestational age; AP1, Apgar score at 1 min.; AP5, Apgar score at 5 min.; PROM, premature rupture of membranes; CAM, chorioamnionitis; PA, placental abruption; PIH, pregnancy-induced hypertension; IQR, median interquartile range. Significant correlation between GA, BW, Apgar score and platelet parameters were analyzed using Spearman’s rank correlation (r). β means standardized regression coefficient.

Table 3

Factors affecting platelet parameters in late preterm neonates.

(n)	PCT (%)		PDW (%)	MPV (fL)	PLT (×103/μL)
	univariate analysis Median [IQR]	multivariate analysis p-value (β)	univariate analysis Median [IQR]	univariate analysis Median [IQR]	univariate analysis Median [IQR]	multivariate analysis p-value (β)
Male (76)	0.25 [0.19–0.30]		10.9 [9.9–13.5]	9.8 [9.0–10.2]	26.9 [22.0–31.7]
Female (66)	0.25 [0.21–0.31]		10.9 [0.3–12.6]	9.9 [9.5–10.4]	25.8 [22.6–32.7]
	P = 0.568		P = 0.373	P = 0.167	P = 0.995
RDS (13)	0.22 [0.16–0.26]		11.2 [10.0–13.3]	9.6 [9.0–10.1]	23.9 [18.6–28.9]
non-RDS (129)	0.25 [0.21–0.31]		10.9 [10.0–12.5]	9.9 [9.2–10.3]	27.0 [22.1–32.6]
	P = 0.050		P = 0.883	P = 0.374	P = 0.138
SGA (57)	0.24 [0.17–0.28]		10.9 [10.1–13.5]	9.9 [9.2–10.4]	24.4 [16.9–29.8]
non-SGA (85)	0.26 [0.22–0.31]		11.0 [10.0–12.5]	9.8 [9.1–10.2]	28.0 [23.8–32.8]
	P = 0.020	P = 0.357 (β = -0.116)	P = 0.533	P = 0.454	P = 0.010	P = 0.165 (β = -0.172)
PROM (33)	0.25 [0.20–0.32]		11.3 [10.5–12.0]	10.1 [9.8–10.4]	25.9 [20.1–32.5]
non-PROM (109)	0.25 [0.20–0.30]		10.9 [10.0–13.1]	9.8 [9.2–10.4]	26.7 [22.9–32.2]
	P = 0.847		P = 0.496	P = 0.044	P = 0.673
CAM (7)	0.26 [0.21–0.31]		11.2 [10.0–12.0]	10.3 [9.8–10.6]	28.0 [24.1–32.4]
non-CAM (135)	0.25 [0.20–0.31]		10.9 [10.0–13.1]	9.9 [9.0–10.2]	26.6 [22.1–32.4]
	P = 0.752		P = 0.936	P = 0.087	P = 0.588
PA (9)	0.25 [0.12–0.36]		10.1 [10.0–13.5]	9.8 [9.5–10.6]	26.6 [11.3–37.2]
non-PA (133)	0.25 [0.21–0.30]		10.9 [10.2–12.5]	9.9 [9.1–10.3]	26.7 [22.7–32.0]
	P = 0.937		P = 0.566	P = 0.586	P = 0.834
PIH (24)	0.23 [0.21–0.32]		10.9 [9.9–12.2]	9.9 [9.1–10.3]	23.7 [15.6–29.1]
non-PIH (118)	0.25 [0.16–0.27]		10.9 [10.0–13.1]	9.8 [9.4–10.4]	27.1 [23.1–32.9]
	P = 0.036	P = 0.051 (β = -0.170)	P = 0.685	P = 0.564	P = 0.007	P = 0.023 (β = -0.194)
GA	r = 0.011		r = 0.030	r = 0.053	r = 0.026
	P = 0.895		P = 0.722	P = 0.533	P = 0.757
BW	r = 0.180		r = -0.040	r = -0.046	r = 0.173
	P = 0.032	P = 0.929 (β = -0.011)	P = 0.637	P = 0.590	P = 0.039	P = 0.960 (β = -0.006)
AP 1	r = 0.028		r = 0.115	r = 0.046	r = 0.049
	P = 0.744		P = 0.172	P = 0.583	P = 0.565
AP5	r = -0.002		r = 0.117	r = 0.122	r = -0.006
	P = 0.978		P = 0.164	P = 0.149	P = 0.941

PCT, platelet count; PDW, platelet distribution width; MPV, mean platelet volume; PLT, platelet count; GA, gestational age; BW, birth weight; RDS, respiratory distress syndrome; SGA, small for gestational age; AP1, Apgar score at 1 min.; AP5, Apgar score at 5 min.; PROM, premature rupture of membranes; CAM, chorioamnionitis; PA, placental abruption; PIH, pregnancy-induced hypertension; IQR, median interquartile range. Significant correlation between GA, BW, Apgar score and platelet parameters were analyzed using Spearman’s rank correlation (r). β means standardized regression coefficient.

As shown in Table 2, in term neonates, MPV was associated with CAM (CAM = 10.4 fL vs. no CAM = 9.7 fL, P = 0.005). Furthermore, PCT was associated with male sex (male = 0.24% vs. female = 0.27%, P = 0.005), SGA (SGA = 0.23% vs. no SGA = 0.26%, P = 0.024) and PIH (PIH = 0.19% vs. no PIH = 0.50%, P = 0.009), respectively. PLT was also associated with male sex (male = 25.9×103/μL vs. female = 28.8×103/μL, P = 0.009), SGA (SGA = 24.8×103/μL vs. no SGA = 26.7×103/μL, P = 0.023) and PIH (PIH = 17.4×103/μL vs. no PIH = 26.5×103/μL, P = 0.007), respectively. Multivariate analysis revealed that PCT correlated with male sex (β = -0.185, P = 0.002), SGA (β = -0.168, P = 0.006) and PIH (β = -0.135, P = 0.026). PLT was associated with male sex (β = -0.166, P = 0.006), SGA (β = -0.186, P = 0.002) and PIH (β = -0.137, P = 0.024).

As shown in Table 3, univariate analysis revealed that MPV was correlated with PROM in late preterm neonates (PROM = 10.1 fL vs. no PROM = 9.8 fL, P = 0.044). Multivariate analysis associated PLT with PIH (β = -0.194, P = 0.023), whereas no associations with PCT were found in late preterm neonates.

When all patients were studied, multivariate analysis showed that SGA, male sex and PIH were associated with PCT and PLT (S1 Table). Furthermore, MPV was significantly associated with CAM (CAM = 10.3 fL vs. no CAM = 9.7 fL, P = 0.001) and male sex (male = 9.7 fL vs. female = 9.9 fL, P = 0.011).

Next, we compared the differences of platelet parameters, sex and PIH between SGA and not SGA (S2 Table). S2 Table showed that SGA was significantly associated with PIH in preterm neonates. Furthermore, PCT and PLT were significantly decreased in SGA neonates compared with not SGA neonates. All data pertaining to our analyses are contained within the S1 Text.

Discussion

This large cohort showed no differences in platelet parameters at birth between late preterm and term neonates. Previous reports indicated that PLT and PCT were lower in late preterm neonates compared with term neonates [12, 17]. Furthermore, they suggested that GA was significantly correlated with PLT, however, in our study, GA did not correlate with PLT [12]. Instead, there was positive correlation between BW and PLT in late preterm neonates by univariate analysis. These differences may be due to differences in sample size and time of sampling. Other research included 129 neonates: 58 late preterm and 71 full term. Moreover, they found that PLT measured using cord blood was associated with GA and BW in late preterm and term neonates. In our study, we used venous blood at admission to NICU. The mean time of sampling in this study was just under 2 hours after birth.

This study also demonstrates that various maternal and neonatal factors affect platelet parameters in late preterm and term neonates. Especially, late preterm and term SGA neonates had lower PLT and PCT compared with not SGA neonates as previously described [18, 19]. Lower PLT in SGA indicates the immaturity of thrombopoiesis [20, 21]. Roberts suggested that reduced platelet production was characteristic of preterm neonates and they had fewer circulating megakaryocytes [22]. Another study reported that plasma thrombopoietin concentrations were low in thrombocytopenic newborns, indicative of inadequate up-regulation of TPO production [23]. Platelets are produced in the fetal liver, with production transferred to bone marrow in the third trimester [24]. The liver is one of the first organs to be affected by growth restriction. Furthermore, in this study, PIH was associated with lower PLT and PCT. Moreover, PIH is also a significant risk factor for SGA in this study as previously reported. Maternal PIH can result in neonatal thrombocytopenia [13, 25]. However, the pathogenesis of thrombocytopenia among infants born to mothers with PIH remain unknown. One potential mechanism is that maternal hypertension and resultant fetal hypoxia affects megakaryocyte proliferation [13, 26].

The present study suggested that CAM was associated with MPV in term neonates. However, PROM is not associated with MPV as in a previous study [27]. Recently, a retrospective study has reported significant correlation between MPV and intrauterine infection in neonates with thrombocytopenia and leukopenia at birth [23, 28]. Since CAM was indicative of intrauterine infection, our results are consonant with previous studies. Furthermore, PDW in SGA neonates did not differ from those of not SGA neonates. This result agrees with a previous study [29]. Recently, elevated PDW was associated with poor prognosis of various conditions such as carcinoma and cardiovascular disease [1, 30, 31]. Although, Patrick et al reported that high MPV and PDW showed high specificity in neonates with late sepsis [32], there are no late sepsis cases in this study.

Another important finding of the present study concerns PLT differences by sex. Although there are few reports about the relationships between PLT and sex in neonates, a previous study reported that school-age females had higher PLT than their male counterparts [33]. However, the mechanisms responsible for PLT differences by sex are unknown.

Our study has several limitations. First, we used samples of venous blood on admission. The timing of samples might affect platelet parameters. Second, we could not investigate the relationship between platelet parameters and sepsis because few of our preterm infants had sepsis at birth and we did not investigate changes of platelet parameters during the postnatal period. Some studies associated sepsis with elevated MPV in neonates during the postnatal period [32, 34]. Third, Apgar score was not associated with lower PLT in late preterm and term neonates in this study. Some studies previously showed that low Apgar score at 5 minutes was related to thrombocytopenia [35, 36]. Mario et al showed that SGA, low Apgar score at 5 minutes, cesarean delivery and lower gestational age were risk factors for thrombocytopenia in preterm deliveries between 27 and 35 weeks of gestation [35]. In murine models, it is known that hypoxia affects megakaryocyte progenitors and induces thrombocytopenia [37, 38]. Castle et al reported that severe hypoxia significantly shortened the survival of platelets in a murine model [39]. In this study, there were too few subjects with severe birth asphyxia to make any inferences. Since very low birth weight neonatal platelets were markedly less reactive than adult platelets to ADP/epinephrine, further study to investigate the relationship between severe birth asphyxia requiring epinephrine and PLT reactivity is needed [40]. Finally, the subjects of term neonates admitted to our NICU in this study have various diseases such as transient tachypnea of newborn, pneumothorax and RDS or hypoglycemia or birth asphyxia. Although RDS is not associated with platelet indices in this study, these term neonates strictly differ from healthy term neonates.

In summary, this study demonstrates that various maternal and neonatal factors affect platelet parameters in late preterm and term neonates. In particular, SGA male sex, and PIH were associated with lower PLT and PCT in late preterm and term neonates. Furthermore, CAM was significantly associated with MPV. Thus, this study demonstrates that different maternal and neonatal complications affect platelet parameters in late preterm and term neonates.

Factors affecting platelet parameters in late preterm and term neonates.

(DOCX)

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Platelet parameters in SGA and not neonates.

(DOCX)

Click here for additional data file.

This study’s data set.

(XLSX)

Click here for additional data file.

13 Apr 2020

PONE-D-19-33766

Perinatal factors affecting platelet parameters in late preterm and term neonates.

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Reviewer 1:

Authors have chosen interesting subject for research.

However, there are many unanswered questions in the mansucript-

1. The study is retrospective in nature; what were the indications for doing all the studied platelet parameters in these late preterm and term neonates at the first place? Is that a routine practice in their unit to screen all babies for platelet issues? Is it justified ethically?

2.All late preterms and term neonates admitted during the study period (491 total) were included in the study and there is no exclusion.How is the data managed in the unit?

3. The data shown in table 1 describes platelet parameters between late PT and term neonates. The given data is highly inadequate. There is no mention whether it is mean or median and no standard deviation or IQR is shown.Further except for GA, BW and PCT , other values are exactly the same which further raise queries on data collection and analysis.

4.Again in table 2 and 3, there is no mention of measures of variation.In multivariate analysis , odds ratio should have been shown instead of just p value.

5.Authors mention use of cord blood or venous blood at admission for study.What site of cord blood was used whether maternal side or neonatal side?Further the mean sampling time was under 2 hours , the studied parameters might reflect maternal characteristics. It would have been clinically relevant if postnatal changes also studied simultaneously.

6.Authors rightly acknowledged their limitations about sepsis and its effect on platelet parameters, which is a major factor which affect platelets

7.Typographical error in the introduction first line -"....in various disease diseases"; remove disease.

Reviewer 2:

General Comments:

This manuscript attempts to provide a cross sectional observation of the platelet’s morphology parameters associated with late preterm and term births. While compelling, the manuscript is lacking in both writing and grammatical issues as well as is lacking in some important analysis. Overall if these issues are addressed this could be an important contribution to the base of knowledge in neonatal intensive care.

The most obvious omission is the lack of connection between the existing base of knowledge on this topic om platelet function and how this would attribute to a clinical outcome. What is the hypothesis? How would a change in platelet number or size influence clinical care?

Better definition of clinical variables needs to be presented. The AGA and SGA descriptors need to be updated. It is important to note in the demographic table 1, what is the proportion of SGA’s in the term and late preterm population.

Specific comments follow below:

Page: 12

1. The opening sentence of the Introduction is very vague and not compelling. Please be more specific and deliberate in your writing.

2. What is your hypothesis? It’s not clear what you are adding to the existing body of knowledge.

3. What factors? What comparisons? What are the variables you are interested in studying? At this point, this sounds like an observational study, so please state that explicitly.

4. Why was analysis on preterm babies not performed? That data is discussed in the introduction, but you do not provide a rationale for why it is not included in this study. Why did you limit to term and late preterm?

Page 13:

1. When were these samples collected? Beginning of Day of Life 1? At the end? Any time in the first 24 hours? This will confound your results significantly.

2. One of the factors you have failed to examine is how many babies required resuscitation at birth with epinephrine? Epinephrine has been reported to cause platelet aggregation as well as recruitment into the circulation. See Kjeldsen SE, Weder AB, Egan B, Neubig R, Zweifler AJ, Julius S. Effect of circulating epinephrine on platelet function and hematocrit. Hypertension. 1995;25(5):1096–1105.

3. Furthermore, reactivity to epinephrine via ADP and thromboxane differs between neonates and adults. Rajasekhar D, Barnard MR, Bednarek FJ, Michelson AD. Platelet hyporeactivity in very low birth weight neonates. Thromb Haemost. 1997;77(5):1002–1007.

4. Therefore, it is important to note what the exposure to resuscitation and epinephrine is in these subjects.

Page 14:

1. It would have been nice if platelet morphology was examined via microscopy on peripheral smears

Page 15:

1. Here it would be very important to separate the term and late preterm subjects by AGA and SGA status. So, have four columns instead of two.

Page 17:

1. What is your definition of SGA?

Page 18:

1. You have omitted definitions for several of these variables. Is CAM clinical or culture proven? What is the definition of clinical chorio at your institution?

Page 24:

1. Timing of sampling needs to be clearly stated in the methods.

2. You also need to differentiate which values were collected from cord and which were from venous samples. Were these values compared to one another? If not, it would be vital to know if there is a difference in parameters from these two methods of sampling.

Page 25:

1. Determining exposure to epinephrine and resuscitation may help understand this better. See comment above.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General Comments:

This manuscript attempts to provide a cross sectional observation of the platelet’s morphology parameters associated with late preterm and term births. While compelling, the manuscript is lacking in both writing and grammatical issues as well as is lacking in some important analysis. Overall if these issues are addressed this could be an important contribution to the base of knowledge in neonatal intensive care.

The most obvious omission is the lack of connection between the existing base of knowledge on this topic om platelet function and how this would attribute to a clinical outcome. What is the hypothesis? How would a change in platelet number or size influence clinical care?

Better definition of clinical variables needs to be presented. The AGA and SGA descriptors need to be updated. It is important to note in the demographic table 1, what is the proportion of SGA’s in the term and late preterm population.

Specific comments follow below:

Page: 12

1. The opening sentence of the Introduction is very vague and not compelling. Please be more specific and deliberate in your writing.

2. What is your hypothesis? It’s not clear what you are adding to the existing body of knowledge.

3. What factors? What comparisons? What are the variables you are interested in studying? At this point, this sounds like an observational study, so please state that explicitly.

4. Why was analysis on preterm babies not performed? That data is discussed in the introduction, but you do not provide a rationale for why it is not included in this study. Why did you limit to term and late preterm?

Page 13:

1. When were these samples collected? Beginning of Day of Life 1? At the end? Any time in the first 24 hours? This will confound your results significantly.

2. One of the factors you have failed to examine is how many babies required resuscitation at birth with epinephrine? Epinephrine has been reported to cause platelet aggregation as well as recruitment into the circulation. See Kjeldsen SE, Weder AB, Egan B, Neubig R, Zweifler AJ, Julius S. Effect of circulating epinephrine on platelet function and hematocrit. Hypertension. 1995;25(5):1096–1105.

3. Furthermore, reactivity to epinephrine via ADP and thromboxane differs between neonates and adults. Rajasekhar D, Barnard MR, Bednarek FJ, Michelson AD. Platelet hyporeactivity in very low birth weight neonates. Thromb Haemost. 1997;77(5):1002–1007.

4. Therefore, it is important to note what the exposure to resuscitation and epinephrine is in these subjects.

Page 14:

1. It would have been nice if platelet morphology was examined via microscopy on peripheral smears

Page 15:

1. Here it would be very important to separate the term and late preterm subjects by AGA and SGA status. So, have four columns instead of two.

Page 17:

1. What is your definition of SGA?

Page 18:

1. You have omitted definitions for several of these variables. Is CAM clinical or culture proven? What is the definition of clinical chorio at your institution?

Page 24:

1. Timing of sampling needs to be clearly stated in the methods.

2. You also need to differentiate which values were collected from cord and which were from venous samples. Were these values compared to one another? If not, it would be vital to know if there is a difference in parameters from these two methods of sampling.

Page 25:

1. Determining exposure to epinephrine and resuscitation may help understand this better. See comment above.

Reviewer #2: Authors have chosen interesting subject for research.

However, there are many unanswered questions in the mansucript-

1. The study is retrospective in nature; what were the indications for doing all the studied platelet parameters in these late preterm and term neonates at the first place? Is that a routine practice in their unit to screen all babies for platelet issues? Is it justified ethically?

2.All late preterms and term neonates admitted during the study period (491 total) were included in the study and there is no exclusion.How is the data managed in the unit?

3. The data shown in table 1 describes platelet parameters between late PT and term neonates. The given data is highly inadequate. There is no mention whether it is mean or median and no standard deviation or IQR is shown.Further except for GA, BW and PCT , other values are exactly the same which further raise queries on data collection and analysis.

4.Again in table 2 and 3, there is no mention of measures of variation.In multivariate analysis , odds ratio should have been shown instead of just p value.

5.Authors mention use of cord blood or venous blood at admission for study.What site of cord blood was used whether maternal side or neonatal side?Further the mean sampling time was under 2 hours , the studied parameters might reflect maternal characteristics. It would have been clinically relevant if postnatal changes also studied simultaneously.

6.Authors rightly acknowledged their limitations about sepsis and its effect on platelet parameters, which is a major factor which affect platelets

7.Typographical error in the introduction first line -"....in various disease diseases"; remove disease.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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Submitted filename: Summary of Comments on PONE-D-19-33766_reviewer_Bhatti comments.pdf

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Submitted filename: Summary of Comments on PONE-D-19-33766_reviewer_Bhatti comments.docx

Click here for additional data file.

28 Jul 2020

June 9, 2020

Anna Palatnik, MD

Academic Editor, PLOS ONE

RE:　Revised Manuscript PONE-D-19-33766

Title: "Perinatal factors affecting platelet parameters in late preterm and term neonates."

Dear Dr. Palatnik,

Thank you for your letter of April 13, 2020 regarding manuscript PONE-D-19-33766. We appreciate the opportunity to respond to reviewers’ comments, and thank everyone for insightful guidance that has helped us significantly improve the paper.

Below, please find our responses to peer review, incorporated by reference into this cover letter, including required text pertaining to financial disclosure.

It will be a privilege to earn your further consideration toward the publication of this manuscript.

Sincerely,

Hayato Go, MD, PhD

Department of Pediatrics, Fukushima Medical University School of Medicine, Fukushima, 960-1295, Japan

#Editor’s comments

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response

Thank you for your suggestion. We revised the manuscript to meet PLOS ONE’s style requirements.

2. In your ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records used in your retrospective study. Specifically, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

Please also clarify whether blood collection from the umbilical cord blood or peripheral venipuncture was a part of routine and standard care or whether it was collected specifically for this study.

Response

Thank you for your suggestion.

Following this guidance, we added information about patient records in Supplemental Table 1.

In our NICU, complete blood count (CBC) is routinely performed on admission. However, most of the samples were from peripheral venipuncture and only two samples were from umbilical cord blood. We removed the cases using umbilical cord blood.

3. Thank you for stating the following financial disclosure: "NO - Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

a. Please provide an amended Funding Statement that declares *and fully names all* the funding or sources of support received during this specific study (whether external or internal to your organization) as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response

Thank you, we attest to the following as true:

“This research proceeded without the benefit of grant money or any other external financial support. Accordingly, funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response

Thank you for your suggestion.

Following this guidance, we added information about patient records in Supporting Table 1.

5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response

Thank you for your suggestion.

Following this guidance, we added information about patient records in Supporting Table 1. We added the mean time of sampling after birth in the results (new Fig 2) and deleted the words “data not shown” from our discussion.

We also added the following sentence in results section.

“The median sampling time was 82 min as shown in Fig 2.”

Reviewer 1:

Authors have chosen interesting subject for research.

However, there are many unanswered questions in the mansucript-

1. The study is retrospective in nature; what were the indications for doing all the studied platelet parameters in these late preterm and term neonates at the first place? Is that a routine practice in their unit to screen all babies for platelet issues? Is it justified ethically?

Response

Thank you for these questions.

In our NICU, complete blood count (CBC) is routinely performed on all babies upon admission. Most late preterm and term neonates admitted to our NICU have various disease such as transient tachypnea of the newborn, pneumothorax, and respiratory distress syndrome, hypoglycemia, or birth asphyxia, etc. So, these term neonates differ from healthy term neonates. Ethically, we are trying to exercise an abundance of caution by collecting data that might guide and improve patient care.

Therefore, we also added the following sentence in discussion.

“Finally, term neonates admitted to our NICU in this study have various diseases such as transient tachypnea of the newborn, pneumothorax, RDS, hypoglycemia, or birth asphyxia. Although RDS is not associated with platelet indices in this study, these term neonates differ from healthy term neonates.”

2.All late preterm and term neonates admitted during the study period (491 total) were included in the study and there is no exclusion. How is the data managed in the unit?

Response

Thank you. We added a flow chart indicative of our data management. We found that the original data set included some cases such as congenital heart disease and chromosomal abnormalities. After excluding these, the total number of subjects decreased from 491 to 400. We reanalyzed the data using these 400 total neonates.

The flow chart appears in new Fig 1.

3. The data shown in table 1 describes platelet parameters between late PT and term neonates. The given data is highly inadequate. There is no mention whether it is mean or median and no standard deviation or IQR is shown. Further except for GA, BW and PCT, other values are exactly the same which further raise queries on data collection and analysis.

Response

We agree with reviewer’s comment.

We reanalyzed the data in Table 1 and added IQR.

4.Again in table 2 and 3, there is no mention of measures of variation. In multivariate analysis, odds ratio should have been shown instead of just p value.

Response

We agree with reviewer’s comment.

We reanalyzed the data in Table 2 and 3 and added IQR in each Table. Furthermore, in terms of multivariate analysis, we used multiple regression analysis. Since platelet parameters were continuous variables, we mentioned both of p value and standardized coefficients. We are open to further advice about the necessity of 95% Confidence interval for unstandardized coefficients.

5.Authors mention use of cord blood or venous blood at admission for study. What site of cord blood was used whether maternal side or neonatal side? Further the mean sampling time was under 2 hours , the studied parameters might reflect maternal characteristics. It would have been clinically relevant if postnatal changes also studied simultaneously.

Response

We agree with reviewer’s comment.

When collecting cord blood, we typically use the neonatal side. However, in this study, there were only two samples using cord blood. So, we deleted these samples from our analysis.

On the other hand, we reanalyzed the sampling time. The median of sampling time was 82.0 min (IQR 50-120). Data was shown in new Fig 2. The data discussed in this study have been deposited in Supplemental Table 1.

6.Authors rightly acknowledged their limitations about sepsis and its effect on platelet parameters, which is a major factor which affect platelets

Response

We agree with reviewer’s comment.

In this study, we could not find out which factors of SGA, PIH, and sepsis affect platelet parameters. While we are at a loss to explain this, we suspect that SGA, PIH, and sepsis are associated with platelet parameters via various mechanisms.

7.Typographical error in the introduction first line -"....in various disease diseases"; remove disease.

Response

Thank you, we removed the sentence.

Reviewer 2:

General Comments:

This manuscript attempts to provide a cross sectional observation of the platelet’s morphology parameters associated with late preterm and term births. While compelling, the manuscript is lacking in both writing and grammatical issues as well as is lacking in some important analysis. Overall if these issues are addressed this could be an important contribution to the base of knowledge in neonatal intensive care.

The most obvious omission is the lack of connection between the existing base of knowledge on this topic om platelet function and how this would attribute to a clinical outcome. What is the hypothesis? How would a change in platelet number or size influence clinical care?

Response

Thank you, yes, we are eager to advance knowledge to improve neonatal care. This includes sharing data for which explanations might ultimately emerge from other groups looking at our data in the context of their own research. We have added text and references to put this research in a broader context of existing knowledge. In addition to this and other revisions suggested by the editor and reviewers, our co-author whose first language is English has indeed noticed and corrected some linguistic irregularities that were in the original submission.

Specific comments follow below:

Page: 12

1. The opening sentence of the Introduction is very vague and not compelling. Please be more specific and deliberate in your writing.

Response

Thank you for the excellent suggestion. We added text to the beginning and end of the first paragraph of our introduction.

2. What is your hypothesis? It’s not clear what you are adding to the existing body of knowledge.

Response

Thank you. Now, in the last sentence of the second paragraph or our introduction, we hypothesize that “that platelet parameters such as MPV, PCT, and PDW could be affected by perinatal factors. The objective of this study was to investigate the factors affecting platelet parameters at birth in late preterm and term neonates.”

3. What factors? What comparisons? What are the variables you are interested in studying? At this point, this sounds like an observational study, so please state that explicitly.

Response

Thank you, this, too has been incorporated into our revised first paragraph.

4. Why was analysis on preterm babies not performed? That data is discussed in the introduction, but you do not provide a rationale for why it is not included in this study. Why did you limit to term and late preterm?

Response

Thank you for these questions. We previously reported the association between very low birth weight and platelet parameters using the data collected from 2006 to 2017. So, we added the sentence of our previous study in the introduction, below.

“We previously reported that higher MPV correlates with mortality among those born at <32 weeks’ gestation (11).”

Page 13:

1. When were these samples collected? Beginning of Day of Life 1? At the end? Any time in the first 24 hours? This will confound your results significantly.

Response

Thank you. We collected these samples at admission, with a time distribution described in the text and in the new Figure 2.

2. One of the factors you have failed to examine is how many babies required resuscitation at birth with epinephrine? Epinephrine has been reported to cause platelet aggregation as well as recruitment into the circulation. See Kjeldsen SE, Weder AB, Egan B, Neubig R, Zweifler AJ, Julius S. Effect of circulating epinephrine on platelet function and hematocrit. Hypertension. 1995;25(5):1096–1105.

Response

Thank you for this reference. Among our cases, two babies required resuscitation at birth with epinephrine. Although we could not investigate the correlation between epinephrine and platelet count, in these two cases we measured 22.9 ×103/μL and 24.5 ×103/μL.

Furthermore, we found that some cases included congenital anomalies such as congenital heart disease and chromosomal abnormalities. After excluding these neonates (new Figure 1), the total number of patients decreased from 491 to 400. After reanalyzing the data using just these 400, Apgar score was not associated with platelet parameters. This is possibly because severe birth asphyxia cases were decreased.

We added to the discussion as follows:

“Third, Apgar score was not associated with lower PLT in late preterm and term neonates in this study. Some studies previously showed that low Apgar score at 5 minutes was related to thrombocytopenia (35,36). Mario et al showed that SGA, low Apgar score at 5 minutes, cesarean delivery and lower gestational age were risk factors for thrombocytopenia in preterm deliveries between 27 and 35 weeks of gestation (35). In murine models, it is known that hypoxia affects megakaryocyte progenitors and induces thrombocytopenia (37,38). Castle et al reported that severe hypoxia significantly shortened the survival of platelets in a murine model (39). In this study, there were too few subjects with severe birth asphyxia to make any inferences. Since very low birth weight neonatal platelets were markedly less reactive than adult platelets to ADP/epinephrine, further study to investigate the relationship between severe birth asphyxia requiring epinephrine and PLT reactivity is needed (40).”

3. Furthermore, reactivity to epinephrine via ADP and thromboxane differs between neonates and adults. Rajasekhar D, Barnard MR, Bednarek FJ, Michelson AD. Platelet hyporeactivity in very low birth weight neonates. Thromb Haemost. 1997;77(5):1002–1007. Therefore, it is important to note what the exposure to resuscitation and epinephrine is in these subjects.

Thank you for the citations and guidance. Pertinent to this is the new final sentence in the text above, repeated here: “Since very low birth weight neonatal platelets were markedly less reactive than adult platelets to ADP/epinephrine, further study to investigate the relationship between severe birth asphyxia requiring epinephrine and PLT reactivity is needed (40).”

Page 14:

1. It would have been nice if platelet morphology was examined via microscopy on peripheral smears.

Response

We agree, but did not assess the platelet morphology, and so we cannot add any associations between platelet morphology and other parameters. We hope to do this in the future, and hope also to see such data from other investigators.

Page 15:

1. Here it would be very important to separate the term and late preterm subjects by AGA and SGA status. So, have four columns instead of two.

Response

We agree, and reanalyzed the data after separating the term and preterm subjects by SGA and AGA (re-branded as “not SGA”), now in revised tables.

Page 17: What is your definition of SGA? Better definition of clinical variables needs to be presented. The AGA and SGA descriptors need to be updated. It is important to note in the demographic table 1, what is the proportion of SGA’s in the term and late preterm population.

Response

Thank you for your suggestion. We added the definition of SGA in Methods as below.

“Infants whose birth weight and height were below the 10th percentile of the normal curve at each GA were classified as SGA.”

Furthermore, we changed the word “AGA” to “not SGA”.

Page 18:

1. You have omitted definitions for several of these variables. Is CAM clinical or culture proven? What is the definition of clinical chorio at your institution?

Response

Thank you. We followed standard clinical diagnostic criteria for CAM (Tita AT, Andrew WW. Diagnosis and management of clinical chorioamnionitis. Clinics in Perinatology. 2010;37(2):339-354), with histopathological confirmation in the case of preterm births, for which placentas are routinely sent to pathology. Cultures were not performed to diagnose CAM. Text has been added to the “Prenatal and postnatal risk factors” subsection of our Methods.

Page 24:

1. Timing of sampling needs to be clearly stated in the methods.

Response

Thank you for your suggestion. We added the mean sampling time in Fig 2.

2. You also need to differentiate which values were collected from cord and which were from venous samples. Were these values compared to one another? If not, it would be vital to know if there is a difference in parameters from these two methods of sampling.

Response

Thank you for your suggestion. We added the mean sampling time in Methods. In this study, there were only two samples of cord blood. So, we deleted these two samples from this study.

Page 25:

1. Determining exposure to epinephrine and resuscitation may help understand this better. See comment above.

Response

Thank you for your suggestion. We added text, as noted above.

21 Oct 2020

PONE-D-19-33766R1

Perinatal factors affecting platelet parameters in late preterm and term neonates.

PLOS ONE

Dear Dr. Go,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please justify why the sample size was reduced to 400 from the original 491 in the first submission.

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We look forward to receiving your revised manuscript.

Kind regards,

Kelli K Ryckman

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Thank you for addressing the comments/reviews you received. Most issues have been adequately addressed. My only remaining comment is that the results section is still difficult to follow with the abundance of abbreviations being used. Table 2 is also very confusing. Perhaps by reversing the X and Y axis, it will be easier to read and follow. This also applies to Table 3 and 4.

Reviewer #2: 1.Although , the authors explain that sampling was done in order to exercise caution , it is still unnecessary to do samplings routinely in newborn and the practice is to avoid unless indicated in order to avoid sampling induced blood loss and consequent anemia.

2. The flow chart has probably typographical error with regards to defining premature birth as > /= 35 weeks .Needs clarification on below which gestation they have defined as premature birth.

3.Regarding the statistical analysis , the authors need to compile the tables more compressed and crisp so that it is reading friendly. Emphasis should be given to what does it imply . The number of tables should be restricted to 2 or 3 maximum.Rest statistical tables can be added as supplementary material

4.Odds ratio may be preferred with 95% CI for interpretation of regression analysis

5.If a complete blood count has also been done along with platelets , then including TLC and ANC as a proxy for sepsis and correlating it with platelet parameters could be of value.

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3 Nov 2020

Nov 3, 2020

Kelli K Ryckman

Editor, PLoS One

RE:　Revised Manuscript PONE-D-19-33766

Title: " Perinatal factors affecting platelet parameters in late preterm and term neonates.

"

Dear Kelli K Ryckman

Thank you for your letter of Oct 21, 2020 regarding our manuscript- PONE-D-19-33766. We appreciate the opportunity to respond to the reviewer’s comments.

We wish to express our appreciation to the reviewers and editor for their insightful comments,

which have helped us significantly improve the paper.

We've checked your submission and before we can proceed, we need you to address the following issues:

1) Please justify why the sample size was reduced to 400 from the original 491 in the first submission.

Response

Thank you. As we mentioned in the last revision, we found that the original data set included some cases such as congenital heart disease and chromosomal abnormalities. After excluding these cases, the total number of subjects decreased from 491 to 400.

Reviewer #1: Thank you for addressing the comments/reviews you received. Most issues have been adequately addressed.

My only remaining comment is that the results section is still difficult to follow with the abundance of abbreviations being used. Table 2 is also very confusing. Perhaps by reversing the X and Y axis, it will be easier to read and follow. This also applies to Table 3 and 4.

Response

Thank you for your suggestion.

We revised Table 2,3,4 so that it will be easier to read and follow.

Reviewer #2:

1.Although, the authors explain that sampling was done in order to exercise caution, it is still unnecessary to do samplings routinely in newborn and the practice is to avoid unless indicated in order to avoid sampling induced blood loss and consequent anemia.

Response

Thank you for this comment.

We concur with modern principles of patient blood management. A complete blood count (CBC) is routinely performed on all admissions to NICU, because all of these late preterm and term neonates have various diseases such as jaundice, transient tachypnea of the newborn, pneumothorax, respiratory distress syndrome, hypoglycemia, birth asphyxia, etc. NICU neonates differ from healthy term neonates. Ethically, our abundance of caution includes using blood samples that need to be drawn for immediate NICU care to also collect data that might guide and improve future patient care.

Therefore, we also added the following sentence in discussion.

“Finally, term neonates admitted to our NICU in this study have various diseases such as transient tachypnea of the newborn, pneumothorax, RDS, hypoglycemia, or birth asphyxia. Although RDS is not associated with platelet indices in this study, these term neonates differ from healthy term neonates.”

2. The flow chart has probably typographical error with regards to defining premature birth as > /= 35 weeks .Needs clarification on below which gestation they have defined as premature birth.

Response

Thank you for your suggestion. We revised it.

3.Regarding the statistical analysis , the authors need to compile the tables more compressed and crisp so that it is reading friendly. Emphasis should be given to what does it imply . The number of tables should be restricted to 2 or 3 maximum.Rest statistical tables can be added as supplementary material

Response

Thank you for your suggestion.

We revised the Tables and convert previous Table 4 and Table 5 to supplemental Table 1 and supplemental Table 2.

4.Odds ratio may be preferred with 95% CI for interpretation of regression analysis

Response

Thank you for your suggestion.

Although we could, in principle, add 95% CI in the Table, we have to maintain readability, so in practice, we do not include it.

5.If a complete blood count has also been done along with platelets, then including TLC and ANC as a proxy for sepsis and correlating it with platelet parameters could be of value.

Response

While agreeing in principle, we encountered few sepsis cases at birth. Therefore, we could not assess any correlations between platelet parameters and sepsis.

Thank you again for reconsidering our manuscript for publication. We greatly appreciate the reviewers’ comments and suggestions. We are confident that the paper is now greatly improved and we hope that it is now acceptable for publication.

Sincerely,

Hayato Go, MD, PhD

Pediatrics School of Medicine

Fukushima Medical University

1st Hikarigaoka

Fukushima Fukushima Japan, 960-1295

phone#: 81-24-547-1295

FAX#: 81-24-548-6578

E-mail: go@fmu.ac.jp

Submitted filename: Response1030 20201102kn.docx

Click here for additional data file.

5 Nov 2020

Perinatal factors affecting platelet parameters in late preterm and term neonates.

PONE-D-19-33766R2

Dear Dr. Go,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Kelli K Ryckman

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

9 Nov 2020

PONE-D-19-33766R2

Perinatal factors affecting platelet parameters in late preterm and term neonates.

Dear Dr. Go:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kelli K Ryckman

Academic Editor

PLOS ONE