The role of lymphocyte-monocyte ratio on ankylosing spondylitis diagnosis and sacroiliitis staging  


 Background: Ankylosing spondylitis (AS)is a chronic inflammatory disorder involving the sacroiliac joints, that could lead to disability due to the failure of timely treatment. Therefore, early diagnosis is essential to for AS treatment. The lymphocyte-to-monocyte ratio (LMR) is an indicator of disease progression. However, its role in AS remains unclear. The aim of this study was to investigate the role of LMR in AS diagnosis, disease activity classification and sacroiliitis staging. Methods: Seventy-eight AS patients and 78 sex and age matched healthy controls (HCs) were enrolled in this study. The diagnosis of AS was performed according to the New York criteria or the Assessment of SpondyloArthritis international Society (ASAS) classification criteria, whereas the staging of sacroiliitis in AS patients was determined by X-ray examination. Comparisons of LMR levels between groups were performed using t test. Pearson or Spearman correlation analysis were used to assess correlations between LMR and other indicators. Receiver operating characteristic (ROC) curves were used to determine the role of LMR in the diagnosis of AS.Results: Higher neutrophil-to-lymphocyte ratio(NLR), red blood cell distribution width(RDW), platelet-to-lymphocyte ratio(PLR), mean platelet volume(MPV), erythrocyte sedimentation rate (ESR), and C-reactive protein(CRP) levels and lower red blood cell (RBC), hemoglobin (Hb), Hematocrit (Hct), LMR, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and albumin/globulin (A/G) levels were noted in AS patients compared to HCs. Positive correlations were observed between LMR and RBC, Hb, Hct and A/G, whereas negative correlations were found between LMR and NLR, PLR, AST, and TBIL (P< 0.05). ROC curves showed that the area under the curve(AUC) for LMR in the diagnosis of ankylosing spondylitis was 0.803 (95% CI =0.734-0.872) with a sensitivity and specificity of 62.8% and 87.2%, respectively, and the AUC (95% CI) for the combination of ESR, CRP and LMR was 0.975 (0.948-1.000) with a sensitivity and specificity of 94.9% and 97.4%, respectively. LMR levels were lower (P<0.05) and significant differences in LMR values were observed among different stages (P<0.05). Conclusions: Our study suggested that LMR could be an important inflammatory marker that can be used to diagnosis AS and identify disease activity and X-ray stage of sacroiliitis.


Background
Ankylosing spondylitis (AS), an immune-mediated chronic in ammatory rheumatic disease with unknown etiology, mainly affects the axial bone and articular structures, but also extra-articular manifestations, such as enthesitis, nger arthritis and dactylitis with extraarticular manifestations, such as uveitis. [1].The prevalence of AS is approximately 0.2-0.3%, and this condition primarily occurs in males aged 20-30 years. Without effective treatment, severe disabilities could develop in approximately one-third of patients [2].To date, the pathophysiology of AS is not completely understood. Risk factors associated with heredity factors, immunity and in ammation are considered the most important factors in the pathogenesis of AS. The Assessment in Spondyloarthritis International Society (ASAS) provides recommendations for the management of AS, including classi cation criteria, magnetic resonance imaging (MRI), X-rays and laboratory indicators for sacroiliac joints and the spine.
In current clinical practice, HLA-B27 is helpful in diagnosis of AS given its high prevalence (90%-95%) in AS and its direct role in the onset of AS [3]. In addition to HLA-B27, imaging modalities such as X-ray, computed tomography (CT), ultrasonography and MRI are typically employed in the diagnosis of AS [4]. Xray imaging is used to diagnose typical sacroiliitis, but early sacroiliitis is easily missed by this methodology. CT can satisfactorily reveal the sacroiliac joint space and articular surface bone, and reveal slight articular surface bone erosion and subchondral cystic change that X-rays cannot show.
Ultrasonography can show peripheral arthritis and enthesitis, but it cannot evaluate axial manifestations. MRI can directly display articular cartilage, and is superior to CT in the early detection of sacroiliac joint cartilage changes, evaluation of sacroiliitis conditions and curative effect judgments. [5] Different imaging modalities have different characteristics and unique advantages and limitations, including radioactive properties, relative costs, long turnaround time and limited use in speci c patients (such as pregnant women). Therefore, speci c and sensitive biochemical markers for auxiliary diagnosis, treatment guidance and prognosis monitoring of AS are urgently needed.
Bath ankylosing spondylitis disease activity index (BASDAI), bath ankylosing spondylitis functional index (BASFI) and ankylosing spondylitis disease activity score (ASDAS) are common disease activity scoring systems for ankylosing spondylitis [6]. Among them, BASDAI and BASFI are based on the patient's own feelings and do not include the doctor's evaluation of the patient's condition and laboratory indicators.
ASDAS re ects the subjective feelings of patients and doctors, and includes in ammatory indicators, so this metric is more suitable for clinical practice. However, due to other complications, the increase in in ammatory markers can affect the evaluation of disease activity. Commonly used in ammatory markers, including erythrocyte sedimentation rate(ESR) and C-reactive protein(CRP) [7], have been veri ed to be related to AS disease severity. ESR × duration of disease and CRP × duration of disease exhibit a good correlation with poor physical activity of AS patients [8]. In recent years, some new in ammatory markers, such as the neutrophil to lymphocyte ratio (NLR) and red blood cell distribution width (RDW), have also been found to be associated with AS disease activity. In AS patients, NLR exhibits a good correlation with ESR and CRP, and increased NLR was found in patients with high disease activity [9]. In addition, different NLR levels were found in patients subject to different treatments, such as anti-TNFalpha therapy, and nonsteroidal anti-in ammatory drugs [10]. Moreover, a signi cant difference in RDW was noted between patients with BASDAI index > 4 and < 4. RDW was positively correlated with BASDAI index as well as ESR and CRP levels [9]. Based on these ndings, routine blood test indexes could represent potential resource for novel and effective marker exploration for AS.
Like RDW and NLR, the Lymphocyte to monocyte ratio (LMR), is also a common blood routine indicator. LMR has been a subject of great interest in a wide range of elds such as in ammation, immunology and carcinoma for a long period of time. Recent data from several studies suggested that LMR was associated with diagnostic, pretreatment and prognostic statue of diseases. A genome-wide association study has con rmed that mutations in ITGA4 and HLA-DRB1 genes could affect LMR levels, and these genes have been widely recognized as susceptibility genes for autoimmune diseases, such as rheumatoid arthritis (RA) [11], suggesting their potential value in AS diagnosis and prognostic evaluation.
To date, few studies have investigated the association between LMR and AS. Therefore, the aim of this study was to explore the diagnostic value of LMR in AS and its role in re ecting disease activity and X-ray staging of sacroiliitis.

Patients with AS
A total of 78 patients with AS [51males and 27 females; mean age 41.0 (29-52) years] were enrolled in this cross-sectional study. These patients received treatment at the Department of Endocrinology and Rheumatology, Taizhou Hospital (Zhejiang, China). All patients ful lled the AS criteria prescribed by the New York criteria of 1984 [12] or the ASAS classi cation criteria of 2009 [13]. All patients were exclusively treated by nonsteroidal anti-in ammatory drugs, NSAIDs are the rst choice for AS patients, and other drugs, such as sulfasalazine, methotrexate, and Tripterygium wilfordii, and biological agents may affect the hematopoietic function of bone marrow and have a greater impact on peripheral blood cell counts. Patients with autoimmune diseases such as Sjogren's syndrome (SS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and psoriasis, malignant diseases, end-stage kidney diseases, liver diseases, acute myocardial infarction, hypertension, diabetes, cerebrovascular diseases were excluded.

Sacroiliitis X-ray staging of the AS Patients
The stage of sacroiliitis was assessed using X-ray and staged from I to IV as follows: stage I with suspicious sacroiliitis; stage II with vague margin of sacroiliac joint, slightly sclerotic and minimally invasive lesions, and unchanged joint space; stage III with moderate or progressive sacroiliitis, accompanied by one or more following changes, including sclerosis of proximal articular area, narrowing/widening of joint space, bone destruction or partial ankylosis; and stage IV with complete joint fusion or ankylosis with or without sclerosis.

Healthy Controls
Healthy controls (HCs) included 55 males and 23 females with a mean age of 40 (30-53) years. These subjects were selected from individuals who underwent a physical examination at the Physical Examination Center of Taizhou Hospital (Zhejiang, China) and the sex and age of these subjects were matched with AS patients. All subjects were healthy without any disease and were not taking drugs that affect bone metabolism, such as hormone replacement therapy.

Biological detection and Imaging system
Fasting blood samples were obtained from all included subjects, whereas X-ray imaging was acquired simultaneously from AS patients. Blood routine tests were assessed using the Mindray BC6800-plus (China) automatic blood analyzer. ESR was detected using the ALifax Tes1(Italy) automatic blood analyzer. CRP was detected by Immage 800 (Beckman Coulter, USA). ALT, AST, TBIL and Alb/Globin (A/G) were detected using the AU5800 (Beckman Coulter, USA) automatic biochemical analyzer. X-ray images were obtained using the Digital X-ray imaging system (DR) (Philips, Holland).

Statistical analyses
All statistical analyses were performed using SPSS version 19.0 (SPSS Inc., Chicago, IL), and all graphs were drawn using GraphPad Prism 8. Continuous variables that conform to a normal distribution were expressed as the mean ± standard deviation, and t tests were used for comparison between groups. The median (P25-P75) was used for non-normal distributions, and Mann Whitney U test and Kruskal Wallis tests were used for comparisons between groups. Categorical variables were expressed as numbers (percentage), and comparisons between groups were performed using chi-square or Fisher's exact tests.
Receiver operating characteristic (ROC) curve analysis with calculation of area under curve (AUC) and 95% con dential interval (CI) was used to determine the role of LMR in the diagnosis of AS. Moreover, the optimal cut-off value was calculated using Youden's index based on speci city and sensitivity. The correlations between LMR and other indicators were assessed using Pearson correlation or Spearman correlation analysis for normal and non-normally distributed datas, respectively. P ≤0.05 was considered to indicate statistical signi cance.

Results
Baseline characteristics of the included subjects Seventy-eight AS patients [51 males, 27 females; mean age (range): 41 (29-52) years] and 78 healthy controls [55 males and 23 females with a mean age (range) of 40 (30-53) years] were included in this study. Blood-routine test indexes, ESR and the serum levels of hs-CRP in both groups were assayed, and compared between AS and HCs. Higher NLR, RDW, PLR, MPV, ESR, and CRP and lower RBC, Hb, Hct, LMR, ALT, AST, TBIL and A/G levels were noted in the AS group compared to healthy controls (P<0.05), and the differences were signi cant. (Table 1)   (Fig.2). Comparison of characteristics between the low X-ray stage and high X-ray stage group A total of 43 and 35 patients were included in the low X-ray stage group (stage I-) and high X-ray stage group (stage -), respectively. The comparison results revealed higher WBC [7.7 (6.0-9.5) vs. 4

LMR in different X-ray stages of AS Patients
Further staging of AS patients based on X-ray imaging resulted in the allocation of 16, 27, 30 and 5 patients into stages I, II, III, and IV, respectively. Statistical differences in LMR values were observed among patients at different stages (P<0.05). Speci cally, the higher the stage, the lower the LMR ( Figure  3).

Discussion
In previous studies, recently developed in ammatory and immunological indicators such as NLR and the platelet-to-lymphocyte ratio (PLR) have been veri ed as diagnostic makers of disease activity and severity in various disorders. Peng et al. indicated that the combined use of NLR, PLR and CEA could represent a good diagnostic biomarkers for colorectal cancer, and positive correlations were found between the TNM stage and NLR or PLR [15]. In addition, Zhao et al. also observed that NLR was correlated with knee recurrence after arthroscopic surgery combined with local radiotherapy [16]. A recent study of rheumatoid arthritis (RA) patients with and without rheumatoid arthritis-associated interstitial lung disease (RA-ILD) by Chen et al. revealed that PLR and NLR exhibited a statistically signi cant positive correlation with DAS28 and PLR could be used to diagnose RA and RA-ILD and distinguish RA-ILD patients from RA patients and healthy subjects [17].
In recent years, another indicator LMR has attracted considerable attention in the diagnosis and prognosis of many diseases such as cancers or various immunological diseases. Rajwa et al. found that urothelial bladder cancer patients treated with radical cystectomy with lower LMR values exhibited a greater risk for developing postoperative in-hospital complications [18]. Du et al. showed that the LMR was an in ammatory marker that is effective in disease activity evaluation in patients with RA and RA differentiation from other arthritis condition [19].
The present study revealed a decreased LMR in AS patients compared to healthy controls, especially in AS patients with high X-ray stages. Furthermore, the correlations between LMR and other AS related indicators revealed that LMR was positively correlated with RBC, Hb, Hct and A/G and negatively correlated with NLR, PLR, AST and TBIL.
Anemia is a common phenomenon in the process of chronic in ammation and is also found in AS patients, and the mechanisms were attributed to the inhibitory effects of cytokines secretion. Tumor necrosis factor alpha(TNF-α) could block the effects of Erythropoietin(EPO) on CD34(+) hematopoietic stem/progenitor cells [20]. Increased hemoglobin level were observed in AS patients with signi cant improvement of physical function and fatigue [21]. Thus, hemoglobin levels could re ect AS activity and severity. Speci cally, reduced Hb levels indicate reduced disease severity.
As a major component in serum protein, serum albumin was used to reveal long-standing malnutrition and was also associated with systemic in ammation [22]. Globulin is the carrier of sex hormones, and globulin levels combined with levels of pro-in ammatory proteins (including complement components, immunoglobulin, CRP, interleukin, TNF) are re ective of the in ammatory state [23]. A/G is based on serum albumin and globulin levels and re ects immunonutritional status and systemic in ammatory reactions with more accuracy compared with either indicator alone.. A higher A/G value indicates a good malnutrition status and low hormone levels. Besides, Lin et al also showed that a low A/G level was signi cantly correlated with high total bilirubin levels but low hemoglobin levels [24]. These results were consistent with the results obtained in this study.
Lymphocytes play an important role in immunology. Although different subsets of T cells are associated with poor tumor prognosis [25,26], high absolute lymphocyte counts are associated with good prognosis in gastric cancer patients [27]. An increased number of monocytes was associated with poor prognosis in various types of tumors [28,29].
Monocytes could differentiate into tumor-associated macrophages (TAMs) in the tumor microenvironment [30]. TAMs could promote tumor angiogenesis and tumor growth by secreting TNFα [31]. Therefore, LMR may be associated with a good AS prognosis because a higher of LMR could result in reduced inhibitory effects of TNF-α on EPO secretion as well as higher hemoglobin levels and A/G ratios. In contrast, lower NLR, PLR, total bilirubin levels and direct bilirubin levels were also observed, supporting the results of this study.
Liver toxic effects could result from systemic rheumatic diseases and therapeutic drugs. Hepatic involvement is a severe type of extra-articular manifestations in various rheumatic diseases. Hepatotoxicity is commonly observed with the use of nonsteroidal anti-in ammatory drugs (NSAIDs) and disease-modi ed anti-rheumatic drugs (DMARD) as immunosuppressants [32]. In our study, we only included AS patients who were treated by NSAIDs; Therefore, the negative correlation between LMR and AST was reasonable.
Regarding the correlations we observed between AS and LMR, we further discussed the diagnostic value of LMR in AS prognosis. ROC curve analysis showed that LMR had a high diagnostic value for AS second only to ESR and CRP. The combined diagnostic AUC of LMR, ESR and CRP for ankylosing spondylitis was 0.975 with a sensitivity and speci city of 94.9% and 97.4%, respectively. Based on X-ray staging, AS patients were divided into low X-ray and high X-ray stages. WBC and NLR levels were higher in the high Xray stage group, whereas LMR levels were lower. Based on these observations, it can be inferred that LMR is associated with the X-ray stage of sacroiliitis in AS patients.
The relationship between LMR and X-ray staging in AS is rarely reported in previous publications. In our research, we classi ed AS patients as stage I to IV according to X-ray imaging to discuss the associations between LMR and the severity of sacroiliitis. The value of LMR decreased as X-ray staging increased, indicating the role of LMR in determining AS severity.
The main limitation of our study was its retrospective design and that it only assessed patients from a single center.. Therefore, multicenter prospective study is needed for further veri cation of our results. Secondly, we could not obtain scoring criteria related to AS activity such as BASDAI and ASDAS activity indexes, given the lack of or incomplete clinical datas of AS patients. Third, the sample size was relatively small given the low prevalence and we only included AS patient treated by NSAIDs in the present study. Fourth, most of AS patients included in the study exhibited a lower stage of activity and disease severity, and more sensitive imaging techniques are needed.

Conclusion
In conclusions, we found that LMR is a rapid, cheap and nonradioactive parameter that can be used to diagnosis AS. Furthermore, LMR may represent an important tool for the assessment of disease activity and X-ray stage of sacroiliitis in AS patients.

Declarations
Acknowledgements: This work was supported by grants from Taizhou Municipal Science and Technology Bureau (CN) (1802KY18). We thank the Endocrinology Department of Taizhou Hospital of Zhejiang Province for their support to our research.
Ethics approval and consent to participate: Medical ethics committee of Taizhou Hospital of Zhejiang Province.
Consent for publication: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Competing interests: The authors declare that they have no nancial or other con icts of interest in relation to this research and its publication.
Funding: This study was supported by grants from Taizhou Science and Technology Plan (1802ky18) (Zhejiang, China). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Authors' contributions: JW, GL conceived, designed and coordinated the study, participated in acquisition and interpretation of data, and drafted the manuscript. X J participated in acquisition of data. Y Y participated in blood and urine determination levels and the interpretation of data. JS, BS participated in the revision of manuscript. All authors have read and approved the manuscript in the "Authors' contributions" section.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  LMR values in AS patients with different X-ray stages. Signi cantly differences were found between patients with different stages.