In the present study, we show that elevated NLR was associated with one-year all-cause and cardiac-related mortality in patients with CLI but was not associated with one-year MACE or MALE. The associations between the NLR and our study outcomes can be clinically explained by the functions of neutrophils and lymphocytes in the human immune system. The percentage of white blood cells accounted for by neutrophils increases as the immune response activates against bacterial infection in CLI patients with sepsis; a higher neutrophil ratio indicates a stronger inflammatory response and is associated with a poor prognosis in patients with LEAD14. Lymphocytes play a role in adaptive immunity, and a lower lymphocyte ratio was found to indicate a lower survival probability in previous studies of patients with sepsis15. In our study, patients with a lower lymphocytes ratio were relatively immunocompromised and predisposed to all-cause mortality resulting from CLI-induced sepsis. In addition, experimental evidence has shown that lymphocytes activate and modify ischemia-reperfusion injury and wound healing16. As mentioned above, the NLR is an inflammatory biomarker and a biomarker of poor wound healing in patients with CLI that is associated with an increased risk of all-cause mortality. Statistically, the neutrophil ratio was positively associated and the lymphocyte ratio was inversely associated with all-cause mortality. As it considers both the neutrophil and the lymphocyte ratios with regard for all-cause mortality, the NLR is a more powerful predictor of all-cause mortality than using wither the neutrophil or lymphocyte ratio alone.
A strength of our study is that we report comprehensive outcomes, including MACE. All-cause mortality has been reported in many studies12,17−20, cardiac morality in only a few studies12,18, and amputation in only a few studies12,17,19,20; however, no study has previously reported MACE and MALE in patients with CLI or stable LEAD with regard for the NLR. The best cut-off for NLR, as determined by receiver operating characteristic curves, was eight in the present study, but this cut-off for NLR is not consistent with previous studies, in which it has also not been universal and ranges from three to five12,13,17−20. Spark et al.12 previously reported that NLR ≥ 5.25 vs. NLR < 5.25 was associated with all-cause mortality, and an NLR cut-off of 5.25 was further validated in the study by Chan et al.18 Notably, the study by Spark et al.12 enrolled patients with Rutherford stages IV and V but not Rutherford stage VI, and Chan et al.18 enrolled only one patient (less than 1%) with Rutherford stage VI. In the study by Erturk et al.13, the NLR cut-off was even lower, at three, because more than sixty percent of the patients were at Rutherford stages III and IV. The major differences between our and other studies are related to the study populations; compared to previous investigators who enrolled patients with peripheral artery diseases and weaker inflammatory responses, we enrolled CLI patients with stronger inflammatory responses13,19. In other words, the neutrophil ratio in our patients indicates a stronger inflammatory response, whereas patients in the other study had weaker inflammation12. In studies that enrolled patients with either CLI or stable LEAD19, the cut-off for NLR may be reduced by the patients with stable LEAD, and the resulting lower NLR threshold may be unable to adequately stratify CLI patients with higher cardiovascular risks. We conducted the present study by enrolling only CLI patients while excluding patients with stable LEAD to make our study population homogeneous, and we therefore suggest that an NLR cut-off of eight could be used in CLI patients to evaluate the risk of all-cause mortality in future studies.
The NLR value increased with higher Rutherford stages in the present study, consistent with a previous study by Bath et al.19 Another study also showed that disease severity and the values of high-sensitivity C-reactive protein significantly increased across tertiles of NLR in patients with lower limb arteriosclerosis obliterans20. NLR, as a biomarker of the inflammatory response and disease severity, has been proposed to contribute to mortality in CLI patients, but NLR as a continuous variable was not associated with MALE in the present study. The NLR was also associated with the complexity of CLI-related arteries, and atherothrombosis has also been shown to be an additional risk factor for MALE in patients with CLI21,22. Because CLI patients had increased platelet activation at baseline than was found in healthy subjects23, the platelet to lymphocyte ratio may be a better predictor of incident MALEs in CLI patients17.
A weakness of our study is that we found that the prescription of medication for CLI patients was relatively low at baseline, including the use of statin and angiotensin-converting enzyme inhibitors or angiotensin receptor blockers. Additionally, the all-cause mortality rate was as high at approximately 25.4%, and the rate of MALE was 17.3%. A meta-analysis that included thirteen studies showed that the rates of all-cause mortality and MALE were both 22% in the natural history of untreated CLI patients24. Another retrospective cohort study showed that four-year mortality was significantly higher among the subgroups at 37.7%, 52.2%, and 63.5% in Rutherford stages IV, V, and VI, respectively; while the four-year rates of amputation were 12.1%, 35.3%, and 67.3% for Rutherford classification IV, V, and VI, respectively25. The REACH registry showed that statin use was significantly associated with a decreased risk of mortality in patients with artherothrombosis22. Although the present study was an all-comer study, the guideline-direct medical therapy to patients at the stage of symptomatic or asymptomatic LEAD could be enhanced to improve clinical outcomes before these patients present with CLI. Given that statins have anti-inflammatory properties and pleiotropic effects on reducing mortality and MALE, future studies should investigate the effect of statin intensity on changes in NLR. Second, the prescription rate of anti-platelet agents was sixty percent at baseline in the CLI patients, while the rate at discharge was 97%. The contemporary guidelines suggest that an anti-platelet, such as aspirin or clopidogrel, is strongly recommended in patients with LEAD26. Although we prescribed anti-platelet agents to CLI patients according to the affordable rule of the National Health Insurance program, mortality and MALE remained high. The high incidence of mortality and MALE in the CLI patients in the present and previous studies indicates that the use of only anti-platelet agents might not be good enough to prevent future cardiovascular events. A previous double randomized controlled trial enrolled patients with atherosclerotic cardiovascular diseases and showed that compared to aspirin alone, low-dose rivaroxaban plus aspirin reduced MALE with the trade-off of bleeding complications27. The VOYAGER study randomized patients with LEAD undergoing revascularization to a low-dose rivaroxaban plus aspirin group or an aspirin alone group and found that randomization to low-dose rivaroxaban plus aspirin was associated with the significantly lower incidence of MACE and MALE than randomization to aspirin alone27; the trade-off of an increased risk of major bleeding was also reported in the VOYAGER study. Regarding the net clinical benefit of reduced MALE and increased bleeding complications, we propose that NLR ≥ 8 may be useful for stratifying CLI patients with a high risk of mortality who may benefit from additional low-dose rivaroxaban in addition to aspirin.
Another limitation is that we investigated the association between NLR and several outcomes, but the best cut-off of NLR was not consistent across all study outcomes in our own study. NLR ≥ 8 vs. < 8 was associated with an increased risk of mortality but not MALE, whereas NLR ≥ 6 vs. NLR < 6 was associated with the incidence of MALE. Given that it would be difficult to apply multiple cut-off values in a clinical setting and difficult to validate them externally in other studies, we suggest that a cut-off of NLR ≥ 8 vs. < 8 may be adequate for identifying CLI patients at a risk of high mortality. Other predictors, such as the platelet-lymphocyte ratio, should also be investigated regarding the incidence of MALE in CLI patients. In addition, a systematic review and meta-analysis could be performed to determine the best cut-off value for NLR among patients with LEAD with various risks.
We previously reported that elevated serum uric acid was associated with the prevalence of metabolic syndrome, left ventricular hypertrophy, left ventricular diastolic dysfunction in various study populations such as apparently healthy individuals, patients with cardiometabolic abnormalities, and patients with ST-segment elevation myocardial infarction28–31. However, we did not find a significant association between hyperuricemia and MACE and MALE in the present study. The possible explanation may be that serum uric acid serves as weaker inflammation, and it, therefore, did not play a role in CLI patients with stronger inflammatory responses.
In conclusion, we show that CLI patients with a higher NLR have significantly higher incidences of all-cause and cardiac-related mortality and MALE at one year. Each increment in NLR was associated with an increased risk of all-cause and cardiac-related mortality but not MALE. NLR ≥ 8 vs. NLR < 8 can be used to predict mortality in CLI patients.