Neutrophil-to-Lymphocyte Ratio as a Potential Biomarker to Managing Type 2 Diabetes Mellitus and Predicting Disease Progression

Introduction Diabetes is a chronic disease that causes dysregulation of blood glucose. Type 2 diabetes mellitus (T2DM) could result in long-term inflammatory conditions that affect different organs of the body. Despite the availability of diagnostic markers like glycated hemoglobin (HbA1c) for T2DM, it is essential to find an appropriate marker that could predict long-term complications. This study evaluates the potential role of neutrophil-to-lymphocyte ratio (NLR) in predicting disease progression and treatment responses. Methods This case-control study was carried out among 160 T2DM patients and 132 non-diabetic persons. Blood samples were collected from each participant and were processed for hemoglobin, HbA1c, iron, ferritin, and complete blood picture (NLR). Results The study showed that there was a significant variation in the serum levels of ferritin (264.8±611.6 ng/ml versus 168.3±364.7 ng/ml, p=0.392), iron (4.095±8.851 mcg/dl versus 55.20±37.62 mcg/dl, p=0.0111), and HbA1c (8.169±1.635% versus 5.668±0.5260% p<0.0001) among T2DM patients compared to non-diabetic persons. The NLR values (4.189±4.154 versus 4.095±8.851, p=0.009) among patients with T2DM significantly varied with that of non-diabetic persons. A significant negative correlation was noticed between the serum levels of iron and NLR (r=-0.17, p=0.014) and a positive correlation was noticed between HbA1c and NLR (r=0.19, p=0.014). The serum levels of iron revealed a significant positive correlation with the serum levels of ferritin (r=0.24, p=0.002) and hemoglobin percentage (r=0.41, p=0.008). HbA1c revealed a significant positive correlation with NLR (r=0.19, p=0.014). Additionally, a significant negative correlation was observed between iron with NLR (r=-0.17, p=0.029) and hemoglobin percentage with NLR (r=-0.30, p=0.005). However, no such correlation was demonstrated among non-diabetic persons. With an accuracy of 89.85% and high sensitivity and specificity, NLR showed diagnostic accuracy like HbA1c. Conclusions NLR demonstrated equivalent efficacy to HbA1c in predicting glycemic control. Since diabetes affects different organs of the body, evaluating NLR probably predicts inflammation. Therefore, NLR could be useful in the management of T2DM and in predicting long-term complications.


Introduction
Diabetes is a chronic illness characterized by elevated blood glucose/sugar levels.Uncontrolled diabetes over a long period could affect different organs of the body like the heart, eyes, and kidneys.Diabetes also disturbs the metabolism of a person and disrupts the nervous system.According to the World Health Organization (WHO), more than 420 million people could be living with diabetes.Recently, there has been an increasing prevalence of diabetes throughout the world, especially among people living in low-and middle-economy countries like India [1].
There are two types of diabetes; diabetes mellitus (DM) and diabetes insipidus.DM is the most predominant type of diabetes which is further classified into type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and gestational diabetes among other types.T2DM is the most common type of diabetes which is Diabetes was diagnosed based on the current American Diabetes Association (ADA) diagnostic criteria for diabetes, which suggests HbA1c levels of 6.5% or more as diabetes.A venous blood sample was collected from all study participants.In this study, 5 mL of blood (3 mL drawn into a plain vacutainer-red cap and 2 mL drawn into an ethylenediamine tetraacetic acid (EDTA) vacutainer-lavender cap) was collected from each participant for a complete blood picture and estimation of HbA1C, serum ferritin, serum iron.HbA1c was measured by high-performance liquid chromatography method using a D-10 analyzer (Bio-Rad Laboratories, Inc., Hercules, California, United States).Hemoglobin estimation was by spectrophotometry and neutrophil and lymphocyte count was carried out by the DxH 800 autoanalyzer (Beckman Coulter, Inc., Brea, California, United States), NLR was calculated and serum ferritin estimation was performed by chemiluminescence method using the DxI 600 analyzer (Beckman Coulter, Inc.), serum Iron was estimated by 2,4,6-tripyridyl-Striazine (TPTZ) using the DxC 700 AU analyzer (Beckman Coulter, Inc.).

Results
The mean age of male T2DM patients was 65.80 ± 10.74 years, while that of female T2DM patients was 59.55 ± 13.28 years and that of male non-diabetic individuals was 53.03 ± 15.82 years.The mean values of HbA1C in the non-diabetic male population (5.692 ± 0.5186 %) and diabetic males (8.178 ± 1.499 %) were found to be statistically significant (p < 0.05).Moreover, there was a significant variation (p < 0.05) in the Iron levels between T2DM patients (47.12 ± 33.23 mcg/dl) and non-diabetic controls (62.67 ± 35.82 mcg/dL).The detailed depiction of HbA1c, hemoglobin level (Hb%), ferritin, iron, and NLR among T2DM patients and non-diabetic participants is shown in Table 1.A significant negative correlation was noticed between the serum levels of iron and NLR (r=-0.17,p=0.014) and a positive correlation was noticed between HbA1c and NLR (r=0.19,p=0.014).The serum levels of iron revealed a significant positive correlation with the serum levels of ferritin (r=0.24,p=0.002) and Hb% (r=0.41,p=0.008).HbA1c revealed a significant positive correlation with NLR (r=0.19,p=0.014).Additionally, a significant negative correlation was observed between iron and NLR (r=-0.17,p=0.029) and Hb% and NLR (r=-0.30,p=0.005).The parameters were tested and their correlation with NLR are given below in Table 3.The ROC curve analysis to evaluate the diagnostic efficacy of NLR in comparison with HbA1c among T2DM patients showed an accuracy of 89.85% and superior sensitivity (85.46%) and specificity (94.23%), as shown in Figure 1, which is marginally better compared to ROC in non-diabetic participants (Figure 2).The ROC curve analysis to evaluate the diagnostic efficacy of NLR in comparison with HbA1c among the non-diabetic participants showed an accuracy of 87.82% and superior sensitivity (82.55%) and specificity (93.1%).

Discussion
It is essential to manage T2DM patients to minimize long-term health complications.Despite neutrophil and lymphocyte counts being individually measured through the complete blood picture, the estimation of NLR has been suggested as a potential biomarker that can assess systemic inflammation and stress [7].This is evident by the fact that neutrophils constitute the innate immune responses, and lymphocytes represent the adaptive/acquired immune responses.Among healthy individuals, the NLR ratio (1-2) is maintained.However, during infections and inflammatory or non-infectious conditions like cancers and other host disturbances, the NLR changes (>3 or <0.7).
In recent times, especially during the coronavirus disease 2019 (COVID-19) pandemic, NLR has been used as a biomarker to manage COVID-19 patients.This was due to the evidence linking inflammatory status to abnormal NLR [8].A very high NLR among COVID-19 patients suggested severe complications, hospitalizations, and mortality.
The role of variations in the NLR has been explored in various non-infectious diseases like psoriasis, multiple sclerosis, neuromyelitis optica spectrum disorders, and breast cancer [9][10][11][12].Similarly, the NLR ratio has been evaluated for its usefulness in managing infectious conditions like COVID-19, pneumonia, pertussis, diabetic foot ulcers/wounds, and urinary tract infections, especially among diabetic persons [13,14].
Additionally, T2DM is a known risk factor for infections and non-infectious conditions like invasive fungal diseases and chronic kidney and cardiovascular diseases [15][16][17].
T2DM is generally diagnosed and managed by biomarkers like the analysis of fasting blood glucose and HbA1C activity.However, there is emerging evidence of the utility of novel biomarkers like C-peptide and others like serum 25-hydroxyvitamin D (25(OH)D) in the diagnosis and management of T2DM [18,19].
T2DM predisposes people to complications like kidney diseases, cardiovascular complications, and other infectious and non-infectious outcomes.The utility of traditional biomarkers like estimation of blood glucose and HbA1c to manage T2DM appears to be limited.This is evident from the reports that suggest the potential application of NLR to manage T2DM and predict long-term complications like stroke and kidney and cardiovascular diseases, among others [20,21].
Only a few studies are available that have examined the role of NLR in managing T2DM patients.A previous study from Pakistan showed that an increased NLR correlated with abnormal glycaemic control, as evidenced by elevated HbA1c activity [22].A recent systematic review and meta-analysis (SRMA) study by Adane et al. confirmed the role of NLR in predicting glycemic control among T2DM patients [23].A study from China that included T2DM patients has positively evaluated the efficacy of NLR in predicting the prognosis of diabetic foot [24].

Study limitations
This hospital-based study included patients attending a tertiary care teaching hospital.The study included T2DM patients and healthy non-diabetic controls.The study results did not include variations in the NLR based on the co-morbidities present among the diabetic population.Therefore, NLR variations among T2DM patients were not attributed to a particular complication.Additionally, this study's results could have been influenced by the medication taken by the diabetic persons.Further studies in this regard are warranted to improve the understanding of the role played by NLR in the management of T2DM patients, especially with co-morbidities, including both infectious and non-infectious conditions.

Conclusions
Despite the availability of traditional markers like HbA1c and blood glucose to diagnose and manage T2DM patients, they may be inappropriate to assess the overall health and predict long-term complications of T2DM.According to the results of the current study, there was a significant correlation between HbA1c and NLR among T2DM patients, unlike in non-diabetic persons.Moreover, the NLR significantly varied between T2DM patients and non-diabetic persons.The results of the current study support the role played by the NLR to manage T2DM patients.Alternatively, a more comprehensive evaluation of T2DM patients is possible with the estimation of NLR.Unlike the traditional biomarkers, NLR could be used to predict potential complications among T2DM patients.

TABLE 3 : Parameters tested and their co-relation with NLR among the diabetic subjects (N=160)
r=Pearsons correlation, *Statistically significant (p<0.05)NLR:neutrophil-to-lymphocyte ratio; HbA1c: glycated hemoglobin; Hb%: hemoglobin level However, no such correlation was demonstrated among non-diabetic persons.The serum activities of iron positively correlated with ferritin (r=0.323,p=0.0002)and Hb% (r=0.291,p=0.001).Additionally, there was a significant negative correlation of Hb% with NLR (r=-0.230,p=0.008)and a significant positive correlation of Hb% with serum iron (r=0.291,p=0.0007)among non-diabetic participants.The details involving the inter-correlation between the parameters tested among non-diabetic persons are detailed inTable 4.