Severity of myxomatous mitral valve disease in dogs may be predicted using neutrophil-to-lymphocyte and monocyte-to-lymphocyte ratio

Dayoung Ku Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Yeon Chae Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Chaerin Kim Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Yoonhoi Koo Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Dohee Lee Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Taesik Yun Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Dongwoo Chang Department of Veterinary Imaging, Veterinary Teaching Hospital, College of Veterinary Medicine, Chungbuk National University Cheongju, Chungbuk, South Korea

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Byeong-Teck Kang Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Mhan-Pyo Yang Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Hakhyun Kim Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea

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Abstract

OBJECTIVE

To investigate the neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR) in dogs with myxomatous mitral valve disease (MMVD).

ANIMALS

106 dogs with MMVD and 22 healthy dogs were included in the study.

PROCEDURES

CBC data were obtained retrospectively, and NLR, MLR, and PLR were compared between dogs with MMVD and healthy dogs. The ratios were also analyzed according to MMVD severity.

RESULTS

NLR and MLR were significantly higher in dogs with MMVD C and D (NLR of 4.99 [3.69–7.27]; MLR of 0.56 [0.36–0.74]) than in healthy dogs (NLR: 3.05 [1.82–3.37], P < .001; MLR: 0.21 [0.14–0.32], P < .001), MMVD stage B1 (NLR: 3.15 [2.15–3.86], P < .001; MLR: 0.26 [0.20–0.36], P < .001), and MMVD stage B2 dogs (NLR: 3.22 [2.45–3.85], P < .001; MLR: 0.30 [0.19–0.37], P < .001). The area under the receiver operating characteristic curves of the NLR and MLR to distinguish dogs with MMVD C and D from those with MMVD B were 0.84 and 0.89, respectively. The optimal cutoff value for NLR was 4.296 (sensitivity, 68%; specificity, 83.95%), and the MLR value was 0.322 (sensitivity, 96%; specificity, 66.67%). NLR and MLR were significantly decreased after treatment in dogs with congestive heart failure (CHF).

CLINICAL RELEVANCE

NLR and MLR can be used as adjunctive indicators of CHF in dogs.

Abstract

OBJECTIVE

To investigate the neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR) in dogs with myxomatous mitral valve disease (MMVD).

ANIMALS

106 dogs with MMVD and 22 healthy dogs were included in the study.

PROCEDURES

CBC data were obtained retrospectively, and NLR, MLR, and PLR were compared between dogs with MMVD and healthy dogs. The ratios were also analyzed according to MMVD severity.

RESULTS

NLR and MLR were significantly higher in dogs with MMVD C and D (NLR of 4.99 [3.69–7.27]; MLR of 0.56 [0.36–0.74]) than in healthy dogs (NLR: 3.05 [1.82–3.37], P < .001; MLR: 0.21 [0.14–0.32], P < .001), MMVD stage B1 (NLR: 3.15 [2.15–3.86], P < .001; MLR: 0.26 [0.20–0.36], P < .001), and MMVD stage B2 dogs (NLR: 3.22 [2.45–3.85], P < .001; MLR: 0.30 [0.19–0.37], P < .001). The area under the receiver operating characteristic curves of the NLR and MLR to distinguish dogs with MMVD C and D from those with MMVD B were 0.84 and 0.89, respectively. The optimal cutoff value for NLR was 4.296 (sensitivity, 68%; specificity, 83.95%), and the MLR value was 0.322 (sensitivity, 96%; specificity, 66.67%). NLR and MLR were significantly decreased after treatment in dogs with congestive heart failure (CHF).

CLINICAL RELEVANCE

NLR and MLR can be used as adjunctive indicators of CHF in dogs.

Cardiovascular disease is associated with inflammation, and congestive heart failure (CHF) is particularly considered an inflammatory status.1 In dogs, pro-inflammatory cytokines (eg, tumor necrosis factor-alpha [TNF-α], interleukin-1, and interleukin-6) are elevated in CHF.2 These factors contribute to the development of inflammatory processes in CHF by altering the cardiomyocyte phenotype and function, inhibiting cardiomyocyte contractile function, inducing macrophage activation, and causing microvascular inflammation and dysfunction.2,3

Among these inflammatory markers, WBCs and their subtypes are easy and cheap to measure and have been extensively studied in human patients with heart disease. In human patients with acute myocardial infarction, neutrophilia is linked to the development of acute heart failure,4 and lymphopenia is associated with poor prognosis in patients with heart failure.5 The neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR) are newly proposed inflammatory biomarkers for patients with heart disease.6,7 Myxomatous mitral valve disease (MMVD) is the most common acquired heart disease in dogs and one of the most common causes of CHF. According to the American College of Veterinary Internal Medicine (ACVIM) consensus, thoracic radiography and echocardiography should be performed for the diagnosis and staging of MMVD.8 However, such diagnostic tools cannot be immediately used in dyspneic dogs with suspected CHF. Therefore, a simple and prompt examination is necessary for rapid emergency treatment, such as lung ultrasound, which is a non-invasive diagnostic tool for detecting CHF in dogs with MMVD. However, additional supporting tools for detecting CHF could be helpful because the sensitivity of the lung ultrasound examination is approximately 90%.9 One such tool is the plasma NT-proBNP, which can distinguish dogs with cardiac dyspnea from those with non-cardiogenic dyspnea.10 Unfortunately, this biomarker could be not available in an emergency.

A human study showed that patients with CHF had a significantly higher NLR than control.11 Neutrophils and monocytes were significantly increased, and lymphocytes were significantly decreased in dogs with CHF.3 However, the CHF group in the above study included MMVD and dilated cardiomyopathy. Moreover, there was a difference in age between the control and CHF groups, which may have affected the WBC count.3 Therefore, in this study, excluding the effects of age, the NLR, MLR, and PLR of dogs with MMVD were examined to confirm whether they can be used as indicators of congestive heart failure occurrence and treatment response.

Materials and Methods

Animals

This study was a retrospective review of the medical records of 128 client-owned dogs over 5 years from May 2017 to May 2022 at the Chungbuk National University Veterinary Teaching Hospital, which was approved by the local ethics committee [CBNUA-2000-22-02]. Data on dogs with MMVD or signs of acute CHF were obtained. Dogs with obvious tracheal or bronchial collapse, other systemic inflammatory diseases, life-limiting diseases (eg, neoplasia), or treated with corticosteroids within the previous 3 months were excluded. Additionally, dogs without CBC tests or echocardiographic examinations were excluded from the study. Data extracted from the medical records included signalment, body weight, heart rate, systolic blood pressure, respiratory rate, murmur grade, vertebral heart score (VHS), left atrial to aortic root ratio (LA:Ao), left ventricular end-diastolic diameter normalized for body weight (LVIDdN), NLR, MLR, and PLR (Table 1).

Table 1

Demographic, physical examination, radiographic, echocardiographic, and hematologic data for 128 dogs.

Healthy Stage B1 MMVD Stage B2 MMVD Stage C, D MMVD P value
No. of dogs 22 42 39 25
Sex (m/f) 15/7 23/19 23/16 13/12 .683
Age (years) 10 (8.50–12.00) 10 (8.52–12.81) 11 (9.00–13.17) 11.17 (9.92–14.04) .116
Weight (kg) 4.08 (2.82–5.31) 4.26 (3.25–5.42) 4.8 (3.42–6.12) 3.98 (2.88–5.72) .576
Heart rate (per minute) 135 (120–151.5) 144 (126–164.5) 156 (143–180) 168 (147–185)a,b < .001
Respiratory rate (per minute) 36 (30–42) 36 (30-60) 30 (24–60) 60 (45–80)a,b,c < .001
Systolic blood pressure (mmHg) 142.5 (128.8–152.8) 140 (128.5–152.5) 132 (125.5–150) 120 (110–147.5) .041
Murmur grade 0 (0–0) 3 (3–4)a 4 (4–4)a,b 4 (4–5)a,b < .001
VHS 10.27 ± 0.72 10.41 ± 0.88 11.24 ± 0.92a,b 11.93 ± 1.22a,b,c < .001
LA:Ao ratio 1.56 (1.44–1.73) 1.96 (1.78–2.5)b 2.3 (2.00–2.56)b < .001
LVIDdN 1.43 ± 0.18 1.82 ± 0.25b 1.74 ± 0.31b < .001
NLR 3.05 (1.82–3.37) 3.15 (2.15–3.86) 3.22 (2.45–3.85) 4.99 (3.69–7.29)a,b,c < .001
MLR 0.21 (0.14–0.32) 0.26 (0.20–0.36) 0.30 (0.19–0.37) 0.56 (0.36–0.74)a,b,c < .001
PLR 200.1 (135.6–242.4) 208.0 (154.4–284.6) 200.8 (133.7–270.3) 246.5 (165.5–333.4) .341

Ao = aorta. LA = left atrium. LVIDdN = left ventricular end-diastolic diameter normalized for body weight. MLR = monocyte-to-lymphocyte ratio. MMVD = myxomatous mitral valve disease. NLR = neutrophil-to-lymphocyte ratio. PLR = platelet-to-lymphocyte ratio. VHS = vertebral heart score. — = not applicable. The mean ± standard deviation was used to describe normally distributed data, and the median (interquartile range) was used to represent non-normally distributed data.

a

P < .05 vs healthy.

b

P < .05 vs stage B1 MMVD.

c

P < .05 vs stage B2 MMVD.

The healthy control consisted of clinically healthy dogs based on all diagnostic tests, including blood analysis, urinalysis, radiography, and abdominal ultrasonography, within the normal range in dogs presented for a health examination (data not shown). MMVD was diagnosed based on the presence of a typical heart murmur of regurgitation due to mitral valve degeneration confirmed by echocardiographic examination.8 The severity of MMVD was classified according to the 2019 ACVIM consensus criteria for MMVD: MMVD B1 was defined when a heart murmur was identified; however, structural remodeling of the heart was not confirmed by radiography and echocardiography. MMVD B2 was defined when structural remodeling of the heart was confirmed.8 Cardiac remodeling was considered present when VHS, which represents the total heart size, was > 10.5 after radiography, and LA:Ao > 1.6, evidence of left atrial dilatation; and LVIDdN > 1.7, evidence of left ventricular dilatation, on echocardiography.8 Heart failure with clinical signs such as tachypnea and dyspnea was diagnosed as MMVD C when there was evidence of cardiac enlargement (VHS > 10.5, lateral view), pulmonary edema on thoracic radiographs, and resolution of the clinical signs after cardiac medications, including diuretics.8 Furthermore, MMVD D was diagnosed when refractory CHF occurred despite diuretic treatment.8

NLR, MLR, and PLR measurement

Blood samples were obtained from the jugular or cephalic veins of all dogs, and samples were collected in EDTA tubes. CBC was performed within 30 minutes at 20 °C using the IDEXX ProCyte Dx (IDEXX Laboratories, Inc). The CBC included measurements of neutrophils (X 103/μL), lymphocytes (X 103/μL), monocytes (X 103/μL), and platelets (X 106/μL).12 The measured values for the study dogs are presented elsewhere (Supplementary Table S1). The NLR, MLR, and PLR were then calculated by dividing the neutrophil, monocyte, and platelet counts by the lymphocyte count, respectively.

Thoracic radiography

Thoracic radiography was performed on all dogs, and right-lateral and dorsoventral inspiratory images were obtained. The right lateral inspiratory view was used to measure VHS. Right lateral and dorsoventral inspiratory images were used to determine the presence or absence of CHF, respectively. As previously described, VHS measurements were derived using a commercial digital radiography system (RadiAnt DICOM Viewer Version 2020.2, Medixant Corporation).13 All measurements were determined by 2 investigators, and the averages are presented.

Echocardiography

From May 2017 to March 2021, echocardiographic evaluations were performed using the Aloka Prosound Alpha 7 device (Wallingford). However, from April 2021 to May 2022, another echocardiographic device, Philips EPIQ7 (Bothell), was used. LA:Ao, LVIDdN, ejection fraction (EF), fractional shortening (FS), E-wave velocity, and A-wave velocity were measured.

Right parasternal short-axis echocardiography was used to measure LA:Ao14 and to assess the left ventricular end-diastolic diameter (LVIDd) and systolic diameter (LVIDs) using right parasternal short-axis echocardiography and M-mode echocardiography.15,16 Using LVIDd and body weight, the left ventricular internal dimension at end-diastole normalized to body weight was determined (LVIDdN = LVIDd (cm)/body weight (kg)0.294).17 The following formula was used to determine FS: (FS = [LVIDd − LVIDs]/LVIDd X 100).18 End-systolic volume (ESV) and end-diastolic volume (EDV) were calculated using the Teicholz method: ESV = (7 X LVIDs3)/(2.4 + LVIDs) and EDV = (7 X LVIDd3)/(2.4 + LVIDd).19 EF was determined using the following formula: (EF = [EDV − ESV]/EDV X 100).18 The early diastolic flow wave (E wave) was assigned to the first diastolic flow, while the atrial contraction flow wave (A wave) was assigned to the second diastolic flow.20 Peak E- and A-wave velocities were measured in meters per second (m/s). All the parameters were averaged after 2 measurements.

CHF treatment

Dogs with MMVD C and D were prescribed diuretics, angiotensin-converting enzyme inhibitors, and pimobendan, according to the ACVIM consensus.8 NLR, MLR, and PLR were compared at the time of CHF onset and after treatment. Post-treatment NLR, MLR, and PLR were obtained with blood tests for at least 3 days and up to 1 month (15.38 ± 10.16 days) after treatment initiation.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc). P values < .05 were considered statistically significant. The Shapiro–Wilk test was used to determine whether the data were normally distributed. Normally distributed continuous data are expressed as means ± standard deviation, and non-normally distributed continuous data are expressed as medians (interquartile ranges). The NLR, MLR, PLR, age, body weight, heart rate, respiratory rate, systolic blood pressure, murmur grade, and LA:Ao were compared healthy, MMVD B1, B2, and C and D groups by using 1-way ANOVA and Tukey’s multiple comparisons tests. The VHS and LVIDdN were compared among the healthy, MMVD B1, B2, and C and D groups using the Kruskal–Wallis and Dunn’s multiple comparisons tests. Mann–Whitney U tests were conducted to compare the NLR, MLR, and PLR between the healthy and MMVD groups. Kruskal-Wallis and Dunn’s multiple comparisons tests were used to compare the NLR, MLR, and PLR among the healthy, MMVD B1, B2, and C and D groups. Each correlation between radiographic index (eg, VHS) and NLR, MLR, and PLR was evaluated, and the correlation between echocardiographic indices (eg, LA:Ao, LVIDdN) and NLR, MLR, and PLR was evaluated. The Pearson correlation test was conducted for normally distributed data, and for non-normally distributed data, the Spearman rank correlation test was used. Receiver operating characteristic (ROC) curve analysis was used to evaluate the ability of NLR and MLR to discriminate the presence of MMVD and CHF. The ideal cutoff value with the highest Youden index (sensitivity + specific-1) was chosen based on sensitivity and specificity calculations.21 Based on the area under the curve (AUC) value, diagnostic accuracy was classified as excellent (0.9–1.0), very good (0.8–0.9), good (0.7–0.8), sufficient (0.6–0.7), or bad (0.5–0.6).21 To confirm the utility of NLR, MLR, and PLR as indicators of treatment response, NLR, MLR, and PLR at the time of CHF onset and after treatment were compared using paired t test or Wilcoxon signed-rank test.

Results

Study population

Overall, 128 dogs were included in this study: 22 healthy dogs and 42 dogs with MMVD stage B1, 39 dogs with MMVD stage B2, and 25 dogs with MMVD stages C and D. The healthy dogs consisted of 5 Maltese, 3 miniature Poodles, 2 mixed breeds, 2 Pomeranians, 3 miniature Pinschers, 2 Chihuahuas, and 1 each of Dachshund, Dalmatian, Golden Retriever, Spitz, and Yorkshire Terrier. The stage B1 MMVD dogs included 8 Pomeranians, 22 Maltese, 6 Shih Tzu, 3 miniature Poodles, and 1 each of mixed breed, Spitz and Yorkshire Terrier. The stage B2 MMVD dogs consisted of 8 Pomeranians, 11 Maltese, 4 miniature Poodles, 3 Japanese Chin, 7 Shih Tzu, and 1 Miniature Pinscher, Miniature Schnauzer, Pekingese, mixed breed, Spitz, and Yorkshire Terrier. Stage C and D MMVD dogs consisted of 5 mixed breeds, 15 Maltese, 2 Shih Tzu, 2 Spitz, and 1 Yorkshire Terrier. The demographic characteristics and physical examination, radiographic, echocardiographic, and blood analysis data (NLR, MLR, and PLR) for the dogs are presented (Table 1).

Comparison of the NLR, MLR, and PLR between healthy and MMVD dogs

The NLR of the MMVD group (3.48 [2.51–4.76]) was significantly higher than that of the healthy group (3.05 [1.82–3.37], P = .019). The MLR of the MMVD group (0.32 [0.21–0.43]) was significantly higher than that of the healthy group (0.21 [0.14–0.32], P = .003). The PLR of the MMVD group (213.4 [151.5–284.3]) was not different from that of the healthy group (200.1 [153.6–242.4], P = .309; Figure 1).

Figure 1
Figure 1

Scatterplot comparing the NLR (A), MLR (B), and PLR (C) of the healthy group (n = 22) and MMVD group (n = 106). The horizontal bars show the medians and interquartile ranges from the first to the third quartile. Mann–Whitney U test. *P < .05. **P < .01. MLR = monocyte-to-lymphocyte; MMVD = myxomatous mitral valve disease; NLR = neutrophil to lymphocyte ratio; PLR = platelet-to-lymphocyte ratio.

Citation: American Journal of Veterinary Research 84, 6; 10.2460/ajvr.23.01.0012

The NLR of the MMVD stage C and D groups (4.99 [3.69–7.29]) was significantly higher than that of the healthy group (3.05 [1.82–3.37], P < .001), MMVD stage B1 (3.15 [2.15–3.86], P < .001), and MMVD stage B2 groups (3.22 [2.45–3.85], P < .001). The MLR of the MMVD stage C and D groups (0.56 [0.36–0.74]) was significantly higher than that of the healthy (0.21 [0.14–0.32], P < .001), MMVD stage B1 (0.26 [0.20–0.36], P < .001), and MMVD stage B2 groups (0.30 [0.19–0.37], P < .001). There was no difference in PLR among the healthy, MMVD B1, MMVD B2, and MMVD C and D groups (P = .341; Figure 2).

Figure 2
Figure 2

Scatterplot comparing the NLR (A), MLR (B), and PLR (C) of the healthy groups (n = 22), MMVD B1 (n = 42), MMVD B2 (n = 39), and MMVD C, D group (n = 25). The horizontal bars show the medians and interquartile ranges from the first to the third quartile. Kruskal–Wallis test. ***P < .001. MLR = monocyte-to-lymphocyte; MMVD = myxomatous mitral valve disease; NLR = neutrophil to lymphocyte ratio; PLR = platelet-to-lymphocyte ratio.

Citation: American Journal of Veterinary Research 84, 6; 10.2460/ajvr.23.01.0012

Correlation of NLR, MLR, and PLR with radiography and echocardiography indices

Correlations of NLR, MLR, and PLR with VHS, LA:Ao, LVIDdN, EF, FS, E velocity, and A velocity were evaluated in dogs, respectively. After the analyses, we identified the correlations of NLR and MLR with VHS and LA:Ao, respectively, as shown (Figure 3). However, there were no correlations among other variables.

Figure 3
Figure 3

Correlation between (A) the NLR and VHS (n = 106, P < .001, r = 0.324), (B) the NLR and LA:Ao (n = 106, P = .009, rs = 0.253), (C) the MLR and VHS (n = 106, P < .001, r = 0.333), and (D) the MLR and LA:Ao (n = 106, P = .011, rs = 0.248) in dogs with MMVD. The dotted lines represent the 95% confidence intervals. Pearson’s correlation or Spearman’s rank test. Ao = aorta; LA = left atrium; MLR = monocyte-to-lymphocyte; NLR = neutrophil to lymphocyte ratio; VHS = vertebral heart score.

Citation: American Journal of Veterinary Research 84, 6; 10.2460/ajvr.23.01.0012

Based on these results, the correlations between NLR and VHS (P < .001, r = 0.324) and between NLR and LA:Ao (P = .009, rs = 0.253) were identified. However, there were no correlations between NLR and LVIDdN (P = .115, r = 0.155), EF (P = .092, rs = 0.167), FS (P = .097, r = 0.164), E velocity (P = .054, rs = 0.282), or A velocity (P = .591, r = 0.054).

The correlations between MLR and VHS (P < .001, r = 0.333) and between MLR and LA:Ao (P = .011, rs = 0.248) were identified. However, there were no correlations between MLR and LVIDdN (P = .156, rs = −0.139), EF (P = .196, rs = 0.128), FS (P = .063, r = 0.184), E velocity (P = .398, rs = −0.084), or A velocity (P = .301, r = 0.103).

There were no correlations of PLR with VHS (P = .433, r = −0.078), LA:Ao (P = .369, r = 0.089), LVIDdN (P = .374, r = −0.088), EF (P = .456, r = 0.074), FS (P = .266, r = 0.111), E velocity (P = .668, rs = 0.043), and A velocity (P = .498, r = −0.068), respectively.

AUC of the NLR and MLR in MMVD dogs

The ROC curve was used to determine the diagnostic roles of the NLR and MLR in the occurrence of MMVD and CHF in dogs.

ROC curves for diagnosing MMVD were drawn by comparing the healthy and MMVD groups (Figure 4). The corresponding optimal cutoff values were as follows: (1) NLR > 3.454 (sensitivity 51.89%; specificity 81.82%; AUC curve 0.66; 95% confidence interval, CI [0.54–0.77]; P = .020), and (2) MLR > 0.209 (sensitivity 77.36%; specificity 54.55%; AUC curve 0.70; 95% CI [0.57–0.82; P = .004).

Figure 4
Figure 4

Receiver operating characteristic (ROC) curve analysis of predicting (A) the NLR and (B) MLR between the healthy group and MMVD group. The AUCs were 0.66 (95% CI [0.54–0.77]) (A) and 0.70 (95% CI [0.57–0.82]) (B), respectively. The point of intersection in all ROC curves represents the optimal cutoff value (sensitivity and specificity) of 3.454 (51.89% [95% CI {41.97% to 61.70%} and 81.82% [95% CI {59.72% to 94.81%}]) (A), and 0.209 (77.36% [95% CI {68.21% to 84.92%} and 54.55% [95% CI {32.21% to 75.61%}]) (B), respectively. AUC = area under the receiver operating characteristic curve; CI = confidence interval; MLR = monocyte-to-lymphocyte; MMVD = myxomatous mitral valve disease; NLR = neutrophil to lymphocyte ratio; ROC = receiver operating characteristic.

Citation: American Journal of Veterinary Research 84, 6; 10.2460/ajvr.23.01.0012

The ROC curves for diagnosing the onset of CHF in dogs with MMVD were obtained by comparing the MMVD group without clinical symptoms (MMVD stage B) and the group of dogs with clinical symptoms (MMVD stages C and D; Figure 5). The corresponding optimal cutoff values were as follows: (1) NLR > 4.296 (sensitivity 68.00%; specificity 83.95%; AUC curve 0.84; 95% CI [0.76–0.92; P < .001), and (2) MLR > 0.322 (sensitivity 96.00%; specificity 66.67%; AUC curve 0.89; 95% CI [0.82–0.95]; P < .001).

Figure 5
Figure 5

Receiver operating characteristic (ROC) curve analysis of predicting (A) the NLR and (B) MLR between MMVD B and MMVD C, D group dogs. The AUCs were 0.84 (95% CI [0.76–0.92] (A) and 0.89 (95% CI [0.82–0.95]) (B), respectively. The point of intersection in all ROC curves represents the optimal cutoff value (sensitivity and specificity) of 4.296 (68% [95% CI {46.50% to 85.05%} and 83.95% [95% CI {74.12% to 91.17%}]) (A), and 0.322 (96% [95% CI {79.65% to 99.90%} and 67.9% [95% CI {56.6% to 77.85%}]) (B), respectively. AUC = area under the receiver operating characteristic curve; CI = confidence interval; MLR = monocyte-to-lymphocyte; MMVD = myxomatous mitral valve disease; NLR = neutrophil to lymphocyte ratio; ROC = receiver operating characteristic.

Citation: American Journal of Veterinary Research 84, 6; 10.2460/ajvr.23.01.0012

Treatment response

A significant decrease (P < .001) in NLR was confirmed post-treatment (3.34 ± 1.54) compared with pre-CHF treatment (5.74 ± 2.11). A significant decrease (P < .001) in MLR was confirmed post-treatment (0.32 ± 0.11) compared with pre-CHF treatment (0.60 ± 0.26). There was no difference in the PLR (P = .191) pre-treatment (172.4 [155.1–248.6]) and post-treatment (197.5 [173.1–308.2]).

Discussion

In this study, significantly higher NLR and MLR were observed in the MMVD group than in the healthy group. In addition, dogs with MMVD with clinical symptoms (eg, MMVD C and D) had higher NLR and MLR than those with subclinical MMVD. These results are consistent with a previous study that showed an increase in the number of neutrophils and monocytes in dogs with MMVD that developed CHF compared with normal dogs.3 However, the diagnostic accuracy of NLR and MLR for MMVD was relatively low. In dogs with CHF and MMVD, the NLR and MLR were significantly decreased after CHF treatment. This study revealed the applicability of NLR and MLR in combination with other tests as adjuvant indicators for CHF diagnosis and treatment response in dogs with MMVD.

A CBC is the most basic blood test, and the NLR is a well-known marker of inflammatory and neoplastic diseases in humans.7,11 Recently, NLR has been studied in dogs with pancreatitis, meningoencephalitis of unknown cause, and inflammatory bowel disease, and a significant difference was confirmed when compared with the healthy group.2224

In humans, neutrophilia has been identified in patients with heart disease and clinical symptoms.4 Lymphopenia is associated with a poor prognosis in heart failure patients.5 Monocyte accumulation in the ischemic myocardium initiates inflammatory responses, including the production of pro-inflammatory cytokines.25,26 This deteriorating effect of monocyte infiltration in myocardial tissue was supported by a strong association between peripheral monocytosis and elevated left ventricular dysfunction.27 Similarly, relative monocytosis has also been reported in dogs with CHF.3 Further, increased numbers of monocyte-derived endothelial progenitor cells were identified in human HF patients,28 suggesting angiogenesis could occur due to elevated left-sided filling pressure.29 Additionally, the NLR was observed to be significantly elevated in human patients with CHF compared with patients without clinical symptoms, and as the NLR increased, the average survival time decreased.11,30

One of the reasons for the increase in the NLR and MLR in dogs with CHF considered in this study is a stress leukogram since a stressful situation occurs as a clinical symptom such as dyspnea. Acute stressors activate the autonomic nervous system (ANS) and hypothalamic-pituitary-adrenal cortex (HPA) axes as a physiological stress response.31 The upregulation of ANS causes an increase in circulating catecholamines.32 Activating the HPA axis results in cortisol release into the bloodstream a few minutes after perceiving the stressor.33 Cortisol release into the bloodstream can result in neutrophilia, lymphopenia, eosinopenia, and monocytosis, which increase the NLR and MLR.34

The increase in NLR and MLR in the CHF group observed in this study may be due to stress; however, the possibility of an increase due to inflammation remains. Systemic inflammatory markers include WBC and its subtypes, and C–reactive protein (CRP) levels, which are increased in human patients with CHF.35,36 Dogs with CHF had higher levels of the inflammatory markers WBC, CRP, TNF-α, and monocyte chemoattractant protein-1 than in the healthy dogs’ group.37,38 N-terminal pro-B-type natriuretic peptide was positively correlated with TNF-α and monocyte counts in CHF dogs.39 An increase in band-neutrophils was recently identified in dogs with CHF, suggesting that CHF may be associated with inflammation.3 These findings imply that inflammation may increase due to CHF. In CHF, pro-inflammatory cytokines such as TNF- α and IL-1 may increase adhesion molecule expression, which promotes leukocyte recruitment in the cardiac microcirculation.40 The trapped leukocytes may contribute to cardiac tissue injury. Therefore, the greater presence of neutrophils and monocyte in the circulation, supported by the increase in NLR and MLR, could be related to their greater action in the cardiac tissues in dogs with CHF. However, the present study did not assess inflammatory factors or compare the CHF situation with other inflammatory conditions. With subsequent further studies, the authors believe that the mechanisms underlying the elevation of these inflammatory markers, including NLR and MLR, in dogs with CHF should be investigated.

This study confirmed positive correlations between NLR and MLR for VHS and LA:Ao, respectively. MMVD induces blood flow regurgitation due to degenerative changes in the valve, leading to volume overload in the left atrium and left ventricle.8 VHS is an index that confirms the heart’s overall size, and LA:Ao is an index that confirms the size of the left atrium. However, the indices of systolic (eg, EF and FS) and diastolic functions (eg, E velocity and A velocity) were not correlated with NLR and MLR, respectively. It is considered that increased inflammation or stress is associated with structural changes in the heart but not related to ventricular function in dogs with MMVD. However, the exact reason for these unexpected results is not known.

The goal is to slow the progression of MMVD through early detection, early treatment, and continuous management because MMVD has a relatively benign natural history, and dogs have long survival times when properly cared for. However, the onset of CHF can be life-threatening in dogs with MMVD. Therefore, early diagnosis of CHF is important in dogs with MMVD. Various diagnostic procedures, such as radiography and echocardiography, have been proposed to diagnose MMVD and CHF. On the other hand, in-depth inspection methods are expensive and time-consuming to undertake regularly. Therefore, this study examined the possibility of MMVD and CHF diagnosis using the NLR, MLR, and PLR. Based on the AUCs of NLR (0.66) and MLR (0.70) for distinguishing dogs with MMVD from healthy dogs, the diagnostic accuracy of these markers for MMVD was somewhat insufficient. However, to diagnose CHF in our study, we calculated the AUCs (NLR: 0.84; MLR: 0.89) of MMVD B and MMVD C+D. These results show that NLR and MLR had very good diagnostic accuracy.21 CHF is a syndrome that results from the progression of MMVD, and dogs experiencing respiratory distress can die from pulmonary edema within a short time. Consequently, prompt drug treatment and oxygen supply are necessary. Therefore, NLR and MLR may be potential diagnostic biomarkers for CHF screening in dogs with MMVD.

In this study, diuretics, angiotensin-converting enzyme inhibitors, and pimobendan were prescribed for each dog with MMVD according to the ACVIM consensus.8 To monitor treatment response, we compared the NLR, MLR, and PLR before and after treatment. Post-treatment was measured at a regular check-up within 3 days and up to 1 month (15.38 ± 10.16 days) after discharge, and it was confirmed that NLR and MLR decreased after treatment compared with before treatment. Therefore, monitoring NLR and MLR along with clinical signs and imaging monitoring can provide information on treatment response and help evaluate clinical symptom improvement.

This study had some limitations. First, it should be premised that stressful situations and other inflammations should be excluded from diagnosis and treatment monitoring when using NLR and MLR in dogs with MMVD. In this study, exclusion was sufficiently conducted to rule out this possibility; however, there may have been unexpected effects. Second, additional research is needed to distinguish whether a dog with respiratory distress has CHF or inflammatory respiratory diseases, such as bacterial pneumonia. In a previous study, NLR was identified in a group of pneumonia (aspiration pneumonia [n = 36], pneumonia or bronchopneumonia [n = 13]), and the median NLR was 16.7, with a range of 2.4–47.9.41 Although NLR was not measured under the same conditions, a greater increase in the NLR in dogs with pneumonia was observed compared with the NLR of dogs with CHF in the present study. However, research on other diseases that can cause dyspnea is still lacking, and no study has directly compared the NLR and MLR in bacterial pneumonia and CHF. Therefore, additional research is needed to compare the NLR and MLR of CHF and bacterial pneumonia or other respiratory diseases (eg, bacterial tracheobronchitis, chronic bronchitis, and eosinophilic bronchopneumopathy). Another limitation is that we could not ensure that all cell counts were manually verified because of the retrospective nature of this study. We manually verify the CBCs and morphologic evaluation of the cellular components if any messages provided by our automated hematology cell counter indicate that the accuracy of cellular evaluation in a blood sample may be compromised. Thus, unnoticed errors in our automated hematology cell counter cannot be completely excluded.

In conclusion, increased NLR and MLR were observed in the group with clinical symptoms compared with those without clinical symptoms in dogs with MMVD. NLR and MLR have the potential to be used as adjunctive indicators of diagnostic and clinical treatment responses in dogs with CHF. Further prospective investigations are needed to determine the clinical usefulness of the NLR and MLR in dogs with MMVD.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT; No. NRF-2021R1F1A1061799). This work was presented as an abstract at the 2023 ACVIM Forum, Philadelphia, Pennsylvania. USA.

The authors have no conflicts of interest to declare.

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Supplementary Materials

Contributor Notes

Contributed equally to this work.

Corresponding author: Dr. Hakhyun Kim (kimh@chungbuk.ac.kr)
  • Figure 1

    Scatterplot comparing the NLR (A), MLR (B), and PLR (C) of the healthy group (n = 22) and MMVD group (n = 106). The horizontal bars show the medians and interquartile ranges from the first to the third quartile. Mann–Whitney U test. *P < .05. **P < .01. MLR = monocyte-to-lymphocyte; MMVD = myxomatous mitral valve disease; NLR = neutrophil to lymphocyte ratio; PLR = platelet-to-lymphocyte ratio.

  • Figure 2

    Scatterplot comparing the NLR (A), MLR (B), and PLR (C) of the healthy groups (n = 22), MMVD B1 (n = 42), MMVD B2 (n = 39), and MMVD C, D group (n = 25). The horizontal bars show the medians and interquartile ranges from the first to the third quartile. Kruskal–Wallis test. ***P < .001. MLR = monocyte-to-lymphocyte; MMVD = myxomatous mitral valve disease; NLR = neutrophil to lymphocyte ratio; PLR = platelet-to-lymphocyte ratio.

  • Figure 3

    Correlation between (A) the NLR and VHS (n = 106, P < .001, r = 0.324), (B) the NLR and LA:Ao (n = 106, P = .009, rs = 0.253), (C) the MLR and VHS (n = 106, P < .001, r = 0.333), and (D) the MLR and LA:Ao (n = 106, P = .011, rs = 0.248) in dogs with MMVD. The dotted lines represent the 95% confidence intervals. Pearson’s correlation or Spearman’s rank test. Ao = aorta; LA = left atrium; MLR = monocyte-to-lymphocyte; NLR = neutrophil to lymphocyte ratio; VHS = vertebral heart score.

  • Figure 4

    Receiver operating characteristic (ROC) curve analysis of predicting (A) the NLR and (B) MLR between the healthy group and MMVD group. The AUCs were 0.66 (95% CI [0.54–0.77]) (A) and 0.70 (95% CI [0.57–0.82]) (B), respectively. The point of intersection in all ROC curves represents the optimal cutoff value (sensitivity and specificity) of 3.454 (51.89% [95% CI {41.97% to 61.70%} and 81.82% [95% CI {59.72% to 94.81%}]) (A), and 0.209 (77.36% [95% CI {68.21% to 84.92%} and 54.55% [95% CI {32.21% to 75.61%}]) (B), respectively. AUC = area under the receiver operating characteristic curve; CI = confidence interval; MLR = monocyte-to-lymphocyte; MMVD = myxomatous mitral valve disease; NLR = neutrophil to lymphocyte ratio; ROC = receiver operating characteristic.

  • Figure 5

    Receiver operating characteristic (ROC) curve analysis of predicting (A) the NLR and (B) MLR between MMVD B and MMVD C, D group dogs. The AUCs were 0.84 (95% CI [0.76–0.92] (A) and 0.89 (95% CI [0.82–0.95]) (B), respectively. The point of intersection in all ROC curves represents the optimal cutoff value (sensitivity and specificity) of 4.296 (68% [95% CI {46.50% to 85.05%} and 83.95% [95% CI {74.12% to 91.17%}]) (A), and 0.322 (96% [95% CI {79.65% to 99.90%} and 67.9% [95% CI {56.6% to 77.85%}]) (B), respectively. AUC = area under the receiver operating characteristic curve; CI = confidence interval; MLR = monocyte-to-lymphocyte; MMVD = myxomatous mitral valve disease; NLR = neutrophil to lymphocyte ratio; ROC = receiver operating characteristic.

  • 1.

    Horne BD, Anderson JL, John JM, et al. Which white blood cell subtypes predict increased cardiovascular risk? J Am Coll Cardiol. 2005;45(10):16381643. doi:10.1016/j.jacc.2005.02.054

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 2.

    Piantedosi D, Musco N, Palatucci AT, et al. Pro-inflammatory and immunological profile of dogs with myxomatous mitral valve disease. Vet Sci. 2022;9(7):326. doi:10.3390/vetsci9070326

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3.

    Hamilton-Elliott J, Ambrose E, Christley R, Dukes-McEwan J. White blood cell differentials in dogs with congestive heart failure (CHF) in comparison to those in dogs without cardiac disease. J Small Anim Pract. 2018;59(6):364372. doi:10.1111/jsap.12809

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4.

    Arruda-Olson AM, Reeder GS, Bell MR, Weston SA, Roger VL. Neutrophilia predicts death and heart failure after myocardial infarction: a community-based study. Circ Cardiovasc Qual Outcomes. 2009;2(6):656662. doi:10.1161/CIRCOUTCOMES.108.831024

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5.

    Ommen SR, Hodge DO, Rodeheffer RJ, McGregor CG, Thomson SP, Gibbons RJ. Predictive power of the relative lymphocyte concentration in patients with advanced heart failure. Circulation. 1998;97(1):1922. doi:10.1161/01.cir.97.1.19

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6.

    Uthamalingam S, Patvardhan EA, Subramanian S, et al. Utility of the neutrophil to lymphocyte ratio in predicting long-term outcomes in acute decompensated heart failure. Am J Cardiol. 2011;107(3):433438. doi:10.1016/j.amjcard.2010.09.039

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7.

    Mirna M, Schmutzler L, Topf A, Hoppe UC, Lichtenauer M. Neutrophil-to-lymphocyte ratio and monocyte-to-lymphocyte ratio predict length of hospital stay in myocarditis. Sci Rep. 2021;11(1):18101. doi:10.1038/s41598-021-97678-6

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8.

    Keene BW, Atkins CE, Bonagura JD, et al. ACVIM consensus guidelines for the diagnosis and treatment of myxomatous mitral valve disease in dogs. J Vet Intern Med. 2019;33(3):11271140. doi:10.1111/jvim.15488

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9.

    Vezzosi T, Mannucci T, Pistoresi A, et al. Assessment of lung ultrasound B-lines in dogs with different stages of chronic valvular heart disease. J Vet Intern Med. 2017;31(3):700704. doi:10.1111/jvim.14692

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Fox PR, Oyama MA, Hezzell MJ, et al. Relationship of plasma N-terminal pro-brain natriuretic peptide concentrations to heart failure classification and cause of respiratory distress in dogs using a 2nd generation ELISA assay. J Vet Intern Med. 2015;29(1):171179. doi:10.1111/jvim.12472

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11.

    Cho JH, Cho HJ, Lee HY, et al. Neutrophil-lymphocyte ratio in patients with acute heart failure predicts in-hospital and long-term mortality. J Clin Med. 2020;9(2):557. doi:10.3390/jcm9020557

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12.

    Goldmann F, Bauer N, Moritz A. Evaluation of the IDEXX ProCyte Dx analyzer for dogs and cats compared to the Siemens Advia 2120 and manual differential. Comp Clin Pathol. 2014; 23:283296. doi:10.1007/s00580-012-1608-1

    • Search Google Scholar
    • Export Citation
  • 13.

    Buchanan JW, Bücheler J. Vertebral scale system to measure canine heart size in radiographs. J Am Vet Med Assoc. 1995;206(2):194199.

  • 14.

    Chetboul V, Tissier R. Echocardiographic assessment of canine degenerative mitral valve disease. J Vet Cardiol. 2012;14(1):127148. doi:10.1016/j.jvc.2011.11.005

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15.

    Thomas WP, Gaber CE, Jacobs GJ, et al. Recommendations for standards in transthoracic two-dimensional echocardiography in the dog and cat. Echocardiography Committee of the Specialty of Cardiology, American College of Veterinary Internal Medicine. J Vet Intern Med. 1993;7(4):247252. doi:10.1161/01.cir.58.6.1072

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Sahn DJ, DeMaria A, Kisslo J, Weyman A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation. 1978;58(6):10721083. doi:10.1161/01.cir.58.6.1072

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17.

    Cornell CC, Kittleson MD, Della Torre P, et al. Allometric scaling of M-mode cardiac measurements in normal adult dogs. J Vet Intern Med. 2004;18(3):311321. doi:10.1892/0891-6640(2004)18<311:asomcm>2.0.co;2

    • PubMed
    • Search Google Scholar
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