Introduction

The neurological sensorimotor disorder Restless Legs Syndrome (RLS) causes a persistent and irresistible urge to move one’s legs that occurs or worsens in the evening and night, thereby often impacting quality of sleep in sufferers [1]. While the disorder is common, with a prevalence between 5 to 19% in European populations [2–5], options for treatment are currently limited and address symptoms rather than the underlying cause of RLS [6, 7]. The pathophysiology of RLS is poorly understood but increased understanding of it may inspire new and more effective treatment strategies. Current accepted pathways involved in the development of RLS include genetic predisposition, iron dysregulation in the central nervous system, and dopaminergic dysfunction [8, 9]. Inflammation has also been proposed as being involved in the pathophysiology of RLS as many conditions and diseases that are highly associated with RLS also have links to inflammation [10]. However, to date, only a limited number of studies have investigated links between inflammation and RLS. The neutrophil-to-lymphocyte ratio (NLR) can be used as an inflammation marker but results from small studies have been inconclusive in determining whether NLR is associated with RLS. Varım, Acar [11] found in a study of 75 RLS cases and 56 controls that NLR was higher in RLS cases than in controls, and suggested that NLR may be used as a predictor of RLS. On the contrary, Tak and Sengul [12] found no difference in NLR between RLS cases and controls in a similarly small-sized study (62 RLS cases, 40 controls). Due to these discrepant findings in small studies, we aimed to assess whether an association between NLR and RLS exists in a large cohort of 13,055 otherwise healthy individuals, including 661 RLS cases. As RLS is known to impact quality of sleep, and sleep disturbances have been shown to increase systemic inflammation markers [13], we also investigated whether NLRs are increased in donors reporting having difficulty sleeping. We also evaluated the associations with RLS for nine other haematological measurements, including white blood cell (WBC) count, platelets and platelet-to-lymphocyte ratio (PLR).

Methods

Results

Neutrophil and lymphocyte data were available in 13,055 participants, of which 661 individuals were determined as current RLS cases (5.1%). RLS cases included a higher proportion of females (62.5% vs 47.5%; P<0.001) and were older than controls (median age 40.9 years in RLS cases vs 38.0 years in controls; P<0.001). No differences in BMI, smoking or alcohol consumption were found between RLS cases and controls (Table 1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Demographic descriptive statistics for RLS cases and controls in the Danish Blood Donor Study with neutrophil, lymphocyte and NLR data available (N = 13,055).

https://doi.org/10.1371/journal.pone.0259681.t001

NLR was higher in RLS cases (median NLR: 1.80) compared to controls (median NLR: 1.72) (P = 0.033). Furthermore, RLS cases had higher absolute number of neutrophils (median neutrophil count: 3.51×10⁹/L in RLS vs 3.40×10⁹/L in controls; P = 0.007). No differences between the two groups were observed for absolute lymphocyte count or in the proportion of individuals with NLR above 3.5 (Table 1). To ensure that the increase in the NLR in RLS cases is not be due to secondary insomnia, we examined whether donors with self-reported difficulties sleeping had higher NLRs (Table 2 and S2 Fig). Of the 13,055 participants with NLR and RLS data available, only 39 (0.3%) did not answer the question on sleeping difficulty. We found that there was no significant difference (P = 0.772) in the NLR between donors having difficulty sleeping (NLR: 1.73 (IQR: 1.36–2.21)) and donors who do not (NLR: 1.73 (1.35–2.19)). In regard to RLS, we observed a significantly higher proportion of RLS cases reporting having difficulty sleeping (10.6%) compared to controls (5.0%) (P<0.001) (S1 Table and S3 and S4 Figs).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Median NLR in blood donors reporting difficulty sleeping in the DBDS NLR-RLS dataset, excluding 39 who did not answer the question (N = 13,016).

https://doi.org/10.1371/journal.pone.0259681.t002

We performed logistic regression models using neutrophils, lymphocytes and NLR as the predictor variable and RLS status as the outcome (Fig 1). We found that an increased NLR was associated with RLS in the unadjusted model (OR for RLS = 1.10 per NLR unit increase [95%CI:1.01–1.20]; P = 0.032); however, the association was not significant after adjusting for sex and age (OR = 1.08 [0.99–1.19]; P = 0.094) or sex, age, alcohol consumption, smoking status and BMI (OR = 1.08 [0.98–1.18]; P = 0.107). Likewise, the association between absolute neutrophil count and RLS was not significant after adjusting for sex and age (OR = 1.04 per 1×10⁹/L increase [0.97:1.10]; P = 0.270).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Investigating the NLR in RLS.

Results from the logistic regression models using neutrophils, lymphocytes and NLR as the predictor variable and RLS status as the outcome in the DBDS cohort. Model 0 = Crude association. Model 1 = adjusting for sex and age. Model 2 = adjusting for sex, age, alcohol consumption, smoking and BMI. Data are presented as odd ratios (OR) with 95% confidence intervals and an asterisk (*) denotes a P value < 0.05.

https://doi.org/10.1371/journal.pone.0259681.g001

We additionally analysed seven other haematological measurements in 28,871 participants (S2 Table). Compared to controls, RLS cases had higher mean corpuscular volumes (P = 0.002) and lower mean corpuscular haemoglobin concentrations (P<0.001). However, as with NLR, the associations were no longer significant after adjusting for sex and age (S5 Fig).

Discussion

We aimed to assess the association between NLR and RLS in a large cohort of healthy Danish blood donors. Although a significant difference in NLR was observed between RLS cases and controls, the association was not significant in the logistic regression model when adjusting for sex and age or when adjusting for sex, age, alcohol, smoking and BMI.

NLR has previously been associated with RLS in a small study with 75 RLS cases and 56 controls [11]. However, a later study with 62 RLS cases and 40 controls could not replicate the association [12]. Our study is strengthened by our superior sample size of 13,055 individuals including 661 RLS cases, which provides strong evidence against any association between NLR and RLS after only correcting for sex and age. Importantly, none of the former studies adjusted for age and sex [11, 12]. Using a uniformly healthy study population is also a clear strength of our study, as blood donors are thoroughly screened at every visit to the blood bank. RLS can be categorised as either primary/idiopathic or secondary, in which secondary RLS has an identifiable cause such as kidney disease or pregnancy [18]. We therefore assume that RLS cases in the DBDS are predominantly idiopathic as eligible donors must be healthy which in turn reduces the presence of comorbidities associated with secondary RLS. Distinguishing between primary/idiopathic and secondary RLS may therefore explain why associations have previously been observed between inflammatory disorders and RLS [10]. However, it must be noted that CH-RLSq was used to determine RLS status in contrast to the International Restless Legs Syndrome Study Group (IRLSSG) questionnaire used in the previous studies. Whether our alternative method for RLS assessment impacted our results is unknown, though the CH-RLSq is also based on the IRLSSG criteria, and may also have additional strengths as it includes additional items and has been validated in several population settings (diagnostic sensitivity 87.2% and specificity 94.0%) [15].

Our study also found no difference in the NLR between donors having difficulty sleeping and those who do not. Although sleep disturbances are associated with increased inflammation, a large meta-analysis on sleep disturbance, sleep duration, and inflammation in adults concluded that short sleep duration is not associated with increases in systemic inflammation markers [13], which is in line with our findings.

Despite our findings that NLR was not associated with RLS after correcting for sex and age, a link between primary/idiopathic RLS and inflammation may still exist and may therefore be discoverable using other inflammatory markers than NLR. Associations between RLS and circulating levels of soluble inflammatory markers have been found previously in small studies, including C-reactive protein (CRP) [19, 20], interleukin-6 (IL-6) [21], interleukin-1beta [21] and tumour-necrosis factor alpha [21]. However, some studies have not been able to replicate the associations with CRP [21, 22] and IL-6 [22], emphasizing the need for further studies with larger samples sizes investigating inflammatory markers in RLS.

In conclusion, we found no association between RLS and the inflammatory marker NLR among Danish blood donors after adjusting for sex, age, alcohol consumption, smoking status and BMI, indicating that NLR has no role as a marker of RLS. Further studies exploring the potential link between RLS and inflammation investigating different inflammatory markers are warranted, to determine whether inflammation is involved in the pathophysiology of RLS.

Supporting information

Acknowledgments

We thank the Danish blood donors for their valuable participation in the Danish Blood Donor Study as well as the staff at the blood centres making this study possible.