The effect of leucocytosis on retinopathy of prematurity

Postnatal leukocytosis reflects the general condition of inflammatory. Infection and inflammatory reaction have been proven to affect the occurrence of ROP and other visual dysfunction. Infants with a gestational age of < 28 weeks who were less than three days of age and admitted to the hospital between September 2015 and March 2021 were included in the study. Infants with a white blood cell (WBC) count ≥ 30 × 109/L were assigned to the leucocytosis group (n = 82). Gestational age- and weight-matched infants without leucocytosis were included as a control group (n = 85). The incidence and prognosis of ROP in preterm infants were compared between the groups. Receiver operating characteristic (ROC) curves were used to analyse the correlation between the WBC count and severe ROP. Compared to the infants in the control group, those in the leucocytosis group had lower 1-min Apgar scores (p < 0.001); higher C-reactive protein (p < 0.001) and procalcitonin (p < 0.001); and higher incidences of intracranial haemorrhage (p = 0.007), leukomalacia (p = 0.045), sepsis (p = 0.006), bronchopulmonary dysplasia (p = 0.017). The maternal age was higher in the leucocytosis group (p < 0.001). After adjusting for gestational age at 45 weeks, the incidence of severe ROP (p = 0.001) and the requirement for ranibizumab injections (p = 0.004) were higher in the leucocytosis group. The cut-off WBC count was determined to be 19.1 × 109/L, with a sensitivity of 88.6%, a specificity of 77.3%, and an area under the curve of 0.941 (95% confidence interval: 0.904–0.978) for the detection of severe ROP. Leucocytosis may be associated with severe ROP in premature infants.


Statistical methods
Continuous variables are expressed as mean ± SD or as median and range.Categorical variables are expressed as number and frequency.Continuous variables were compared using the t-test, while categorical variables were compared using the chi-squared or Fisher's exact tests, as appropriate.For confounding factor analyses, logical regression analyses were used.The correlation between the WBC count and severe ROP was analysed using the receiver operator characteristic curve to predict the cut-off value of an abnormal WBC count.SPSS (version 21.0, IMB Corp., New York, USA) was used to conduct the analyses.Statistical significance was set at P < 0.05.

Ethics approval and consent to participate
All methods in this study were carried out in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.The Life Science Ethics Committee of Children's Hospital affiliated to Zhengzhou University approved the study.Written informed consent was obtained from all participants' parents.

Demographic data
During the study period, 430 premature infants born at a gestational age < 28 weeks were admitted to the neonatal intensive care unit, including 105 with a WBC count ≥ 30 × 10 9 /L.Nine patients with incomplete data, six for whom treatment was discontinued, five who died, and three with congenital malformations or genetic metabolic diseases were excluded from the study.The final analysis included 82 premature infants with leucocytosis (50 males and 32 females) born at a mean gestational age of 26.9 ± 1.8 weeks (range: 24-28 weeks).The mean birth weight was 1110 ± 212 g (range: 540-1500 g).The control group included 85 patients (49 males and 46 females), with a mean gestational age of 27.1 ± 1.7 weeks (range: 24-28 weeks).The mean birth weight was 1107 ± 221 g (range: 550-1490 g).The baseline clinical characteristics of the two groups were not significantly different (Table 1).

Risk factor analysis
Leucocytosis was identified as a risk factor for severe ROP (statistical information; Table 3).

ROP
At a corrected gestational age of 45 weeks, the incidence of severe ROP was significantly higher in the leucocytosis group than in the control group (p = 0.001) (Table 4 and Fig. 1).The use of laser therapy was not significantly

Correlation between leucocytosis and severe ROP
The cut-off value of an abnormal WBC count was 19.1 × 10 9 / L, with a sensitivity of 88.6%, a specificity of 77.3%, and an area under the curve of 0.941 (95% confidence interval 0.904-0.978)(Fig. 2).

Discussion
This study aimed to raise awareness among neonatologists about potentially considering leucocytosis a means for early identification of severe ROP.We selected WBC in this study as a predictor owing to its wide use in clinical settings; this will further promote effortless application of this screening process among neonatologists.Many factors affect WBC count including infection, inflammation, stress, and medications 2 .The incidence of confirmed infection among infants with leucocytosis is 22% 1 , which is similar to that in adults 13 .Healthy new-borns and premature infants have a peak WBC count after birth 14 which may be due to an increase in the number of cells in the marginal granulocyte pool mediated by catecholamines or the production and release of immature granulocytes in the bone marrow induced by postpartum stress.
There are a number of factors that can lead to elevated leukocytes, infection being one of the most common.Clinical chorioamnionitis leads to elevated white blood cells and fetal inflammatory response syndrome (FIRS), and it has been shown that babies born to mothers with histologic and clinical chorioamnionitis have a higher incidence of ROP 15,16 .Prenatal administration of steroids reduces the risk of ROP development and progression to severe ROP, but at the same time higher leukocyte counts were observed in newborns from postnatal day 7 to day 14, probably achieved by reducing the excessive inflammatory response, which can still be realized by the production of inflammatory factors, inducing endothelial growth factor, when excess leukocytes are present in leukocytosis 17,18 .In addition, transient myelopoietic abnormalities due to Down's syndrome are also a factor in leukocytosis 19 .Inflammatory mediators stimulate monocytes and macrophages to produce granulocyte colonystimulating factor, increasing the WBC count 20 ..While maternal infections lead to reduced immunity in the newborn and increase the possibility of infection and inflammatory storms 22 , sepsis in the infant leads to decreased IGF-1 (insulin-like growth factor-1) 23 .A previous study suggested that IGF-1 deficiency is related to insufficient vascular growth and subsequent proliferative ROP 24 .
During the first stage of ROP, inflammation inhibits the development of retinal nerves and vessels, leading to increased vascular occlusion resulting in increased retinal hypoxia.These factors promote abnormal neovascularization during the second stage of ROP and increase the risk of severe ROP 4 .Inflammation may increase the risk of ROP by sensitizing the developing retina and neovascularization to oxygen-induced growth factors.In contrast, the stress state of the retina can promote the activation of inflammatory cells and the production of inflammatory cytokines, ultimately affecting neovascularization 4 .
Increased levels of inflammatory biomarkers, including cytokines and C-reactive protein, during the first few weeks after birth, are associated with the development of ROP at a later stage 25 .A previous study reported that histologic chorioamnionitis and funisitis are unrelated to the occurrence and development of ROP, though maternal chorioamnionitis and leucocytosis may increase the risk of ROP 26 .In this study, the incidence of severe ROP in infants with leucocytosis was significantly higher than that in the control group, and the cut-off WBC count had high sensitivity, specificity, and area under the curve.
Ashki et al. 27 reported that the infiltration of macrophages, monocytes, and leukocytes led to the release of nitric oxide (NO) and superoxide anions from tissues, resulting in the conversion of NO to peroxynitrite, leading to an increase in angiogenic growth factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and hypoxia-inducible factor.This mechanism may also be involved in the development of ROP 28 .
The lymphocyte-to-monocyte ratio (LMR) and monocyte counts in preterm infants at 4 weeks postnatally are independent risk factors for the development of ROP.Increased WBC and neutrophil counts increase the risk of ROP and the LMR can be used to predict the occurrence of ROP within 24 h after birth 29,30 .The NLR was not a risk factor for ROP development but was a risk factor for ROP treatment 31 .In this study, a WBC ≥ 30 × 10 9 /L was closely related to the increased risk of severe ROP in ultra-preterm infants and may be a predictor of severe ROP in this population.

Conclusions
The WBC count is a simple, economical, and widely used clinical parameter.Neonatologists can use the WBC count to predict the development of severe ROP, allowing for timely screening evaluations and interventions, which will ultimately reduce the risk of developing severe ROP.A prospective study with a larger patient population is required to further investigate the correlation between leucocytosis and severe ROP.

Figure 1 .
Figure 1.A girl, gestational age 30 weeks, birth weight 1200 g, severe ROP at 42 weeks of corrected gestational age, stage 4 AOP (partial retinal detachment) in the right eye and stage 5 ROP (total retinal detachment) in the left eye.

Figure 2 .
Figure 2. Working characteristic curve of white blood cell count and subject with severe retinopathy of prematurity.

Table 1 .
Comparison of baseline data of the two groups.a is represented by example (%), the statistical value is χ2 value, and b is represented by the statistical value is the t value.

Table 2 .
Comparison of clinical data of the two groups.RDS is respiratory distress syndrome, BPD is bronchopulmonary dysplasia, IVH is intracranial hemorrhage, NEC is necrotizing enterocolitis, CRP is C-reactive protein, PCT is procalcitonin; a is represented by example (%), the statistical value is χ2 value, and b is represented by the statistical value is the t value.Control group N = 85 Leukocytosis group N = 82 Statistical values P value

Table 4 .
Comparison of severe ROP between leukocytosis group and control group at 45 weeks of gestational age correction.