Assessment of Neutrophil-Lymphocyte Ratios in Patients with Dry Age-related Macular Degeneration

Purpose: To assess the signicance of neutrophil-lymphocyte ratio (NLR) as an inammatory indicator in patients with dry age-related macular degeneration (AMD). Study design: A retrospective case-control study. Methods: Clinical diagnosis along with complete blood count (CBC) results were extracted from hospital and laboratory information systems for patients with dry-AMD and age/gender-matched controls attending the ophthalmology clinic at King Abdulaziz medical city, Jeddah, Saudi Arabia, between 2018-2020. NLR was calculated by dividing the neutrophil by the lymphocyte count. Results: This study captured 90 patients diagnosed with dry-AMD and 270 control subjects without AMD. The mean of ages 70 and 71 years old for cases and controls, respectively. In univariate analysis, there were no signicant differences in CBC results between cases and control. In NLR, dry-AMD patients have a slightly higher mean than the control group; however, this increase was not statistically signicant (P-value 0.8). In the NLR model, age and gender were statistically signicant factors affecting the NLR values in dry-AMD (P-value 0.03, 0.01 respectively). Conclusion: as a systemic inammatory biomarker, NLR alone could not predict dry-AMD. However, the slight increase in the NLR values may be helpful if augmented with other laboratory measurements to aid in early disease prediction.

The role of NLR as a simple, reliable, and low-cost in ammatory biomarker in ocular conditions is yet unclear. Several reports have demonstrated a correlation between NLR and wet AMD (Reviewed in Niazi et al., 2019). However, the link between NLR and dry AMD is based on a limited number of studies (Ilhan et al., 2015;Kurtul & Ozer, 2016), limiting our insight into the starting phase of the disease. This study aims to assess the levels of NLR in patients with dry AMD.

Study population
Institutional review board and ethics committee approval was obtained from King Abdullah international medical research center (Study number SP21J/083/03). While maintaining participants' con dentiality and anonymity, electronic health records (January 2018 -December 2020) were collected for new patients with dry AMD, ≥50 years, with evidence of macular drusen in at least one eye, with or without signs of geographic atrophy, and complete blood count (CBC) laboratory results. NLR was calculated by dividing the neutrophil (x10 9 /L) by the lymphocyte (x10 9 /L) count. Similar data were collected for age-and sexmatched cataract group without AMD as a control. Electronic health records were compiled from King Abdulaziz medical city (Jeddah, Saudi Arabia), which provides primary, secondary, and tertiary care. The study was performed following the Declaration of Helsinki principles, and informed consent was obtained from all participants.

Sample size calculation
The survivor sampling technique was used with a control group consisting of individuals from the source population who do not have the outcome of interest (with no AMD). For sample size calculation, a matched case-control study was used (the case-control ratio is 1:3).

Statistical analysis
In univariate analysis, associations between demographics and blood results characteristics were assessed across cases and control groups using the chi-square for categorical data and t-test for numeric variables. Multivariate analyses adjusted for age and gender were conducted to examine the differences between cases and control groups. Models were assessed using analysis of covariance according to the nature of our outcome. We checked assumptions of the linear relationship between the dependent variable and the covariate and homogeneity of regression slopes, and all models met the assumptions. Pvalues were two-sided; all con dence intervals were at 95 %. All analyses were conducted using the SAS statistical software version 9.4 (SAS Institute Inc. Cary, NC).

Results
From January 2018 to December 2020, this study captured 90 cases of patients diagnosed with dry AMD and 270 control subjects without AMD. The mean of ages 70 and 71 years old for cases and controls, respectively. CBC results are summarized in table 1.
In univariate analysis (Table 1), there were no signi cant differences in CBC results between cases and control groups. In Platelet results, control groups tend to have a slightly higher mean (268.73) than cases group mean 268.68 with a P-value of 0.0001. In terms of mean corpuscular hemoglobin, the cases group showed a lower mean of 27.9 than the control group, 28.27. In NLR, dry AMD patients have a slightly higher mean than the control group (2.46 and 1.98, respectively). However, this increase was not statistically signi cant (P-value 0.8). The same pattern was observed in mean corpuscular volume, where the case group showed a lower mean than the control group (86.54 and 86.64, respectively). Regression models adjusted for age and gender were summarized in tables 2-3, and gure 1. Age and gender were statistically signi cant factors affecting NLR values in cases compared to the control group (P-value 0.03, 0.01 respectively). No other CBC parameters showed any signi cant difference between cases and the control group.

Discussion
The NLR has been increasingly investigated as a systemic in ammation marker. Several studies have analyzed NLR in in ammatory conditions as an indicator of the disease, progression, and severity. For instance, positive NLR correlation was reported in diabetic retinopathy, worsening renal function in Furthermore, the damaged mitochondria release components such as mitochondrial DNA (mtDNA), which induces in ammatory responses. A study by Lin et al., (2011) has shown that the increase in age-related mtDNA damage can lead to more macula lesions that positively correlate with AMD progression. Sensing mtDNA by cytosolic innate immune sensors, cyclic-GMP-AMP synthase, and the stimulator of interferon genes, lead to the production of Type I interferons (Type I IFNs) and other pro-in ammatory cytokines (Kumar, 2019). Additionally, mtDNA also triggers the assembly of a multiprotein complex known as the in ammasome (Shimada et  Therefore, in ammasome activation by mtDNA and the progression of AMD needs to be investigated. The role of the NLRP3 in ammasome in AMD pathogenesis has been shown using the amyloid-beta (Aβ) component of drusen as a stimulus which resulted in a robust in ammasome activation evident by elevated levels of IL-1β and IL-18. In addition to in ammasome-mediated-cytokines, stimulation with Aβ resulted in upregulation of the pro-in ammatory cytokines, IL-6 and TNF-α (Liu et al., 2013). Therefore, further studies are essential to investigate the levels of in ammatory markers including type I IFNs, IL-1β, IL-18, IL-6, and TNF-α in patients with dry and wet AMD compared to control participants.

Conclusion
As a routine laboratory test, it is important to note that NLR is not speci c for either type of AMD and can be elevated in several diseases and malignancies. The disease is also often associated with other agedependent comorbidities, which could in uence NLR values. Therefore, further studies are essential to generate robust evidence to investigate causality and NLR values clinical utility in line with other in ammatory biomarkers.    Figure 1 Analysis of covariance for NLR