Neutrophil Counts and Initial Presentation of 12 Cardiovascular Diseases

Background Neutrophil counts are a ubiquitous measure of inflammation, but previous studies on their association with cardiovascular disease (CVD) were limited by small numbers of patients or a narrow range of endpoints. Objectives This study investigated associations of clinically recorded neutrophil counts with initial presentation for a range of CVDs. Methods We used linked primary care, hospitalization, disease registry, and mortality data in England. We included people 30 years or older with complete blood counts performed in usual clinical care and no history of CVD. We used Cox models to estimate cause-specific hazard ratios (HRs) for 12 CVDs, adjusted for cardiovascular risk factors and acute conditions affecting neutrophil counts (such as infections and cancer). Results Among 775,231 individuals in the cohort, 154,179 had complete blood counts performed under acute conditions and 621,052 when they were stable. Over a median 3.8 years of follow-up, 55,004 individuals developed CVD. Adjusted HRs comparing neutrophil counts 6 to 7 versus 2 to 3 × 109/l (both within the ‘normal’ range) showed strong associations with heart failure (HR: 2.04; 95% confidence interval [CI]: 1.82 to 2.29), peripheral arterial disease (HR: 1.95; 95% CI: 1.72 to 2.21), unheralded coronary death (HR: 1.78; 95% CI: 1.51 to 2.10), abdominal aortic aneurysm (HR: 1.72; 95% CI: 1.34 to 2.21), and nonfatal myocardial infarction (HR: 1.58; 95% CI: 1.42 to 1.76). These associations were linear, with greater risk even among individuals with neutrophil counts of 3 to 4 versus 2 to 3 × 109/l. There was a weak association with ischemic stroke (HR: 1.36; 95% CI: 1.17 to 1.57), but no association with stable angina or intracerebral hemorrhage. Conclusions Neutrophil counts were strongly associated with the incidence of some CVDs, but not others, even within the normal range, consistent with underlying disease mechanisms differing across CVDs. (White Blood Cell Counts and Onset of Cardiovascular Diseases: a CALIBER Study [CALIBER]; NCT02014610)

T he most numerous type of white blood cell, neutrophils, play a major role in inflammation. Neutrophil count is used routinely as a biomarker of acute infection and inflammation, but not in cardiology. Chronic inflammation contributes to atherosclerosis and cardiovascular diseases (CVD) (1,2) but, compared with other inflammatory biomarkers such as C-reactive protein (3) and interleukin-6 (4), neutrophil counts have been little studied in relation to long-term CVD risk, even though they are available at scale in clinically collected electronic health record data.
Previous studies have shown that high neutrophil counts are associated with an higher incidence of coronary disease (5), heart failure (HF) (6), and stroke (7) (Online Table 1). However, these studies were too small to examine thresholds or shape of the association. No study used a clinically recorded measure of neutrophil count, which is important to understand the relevance of findings to usual practice, or studied associations with peripheral vascular diseases.
This study investigated the association of neutrophil counts with initial presentation of 12 CVDs in a large, population-based cohort from a linked electronic health record database: the CALIBER program (Clinical Research Using Linked Bespoke Studies and Electronic Health Records) (8). CALIBER has been extensively validated, replicating known prospective associations of CVDs with sex (9), smoking (10), blood pressure (11), socioeconomic deprivation (12), and type 2 diabetes (13).

METHODS
We used the same study cohort as our study on the association of eosinophil and lymphocyte counts with incidence of CVD (14). The study population was drawn from the CALIBER program (8) . CALIBER includes about 4% of the population of England (15) and is representative in terms of age, sex, ethnicity, and mortality (8).
The study period was January 1998 to March 2010, and individuals were eligible for inclusion when they were at least 30 years of age and had been registered for at least 1 year in a practice that met research data recording standards. The study start date (index date) for each participant was the date of the first complete blood count recorded in the primary care record while the participant was eligible. Persons with a prior history of CVD and women with a pregnancy record within 6 months of the start of the study were excluded.
Approval was granted by the Independent Scientific Advisory Committee of the Medicines and Healthcare Products Regulatory Agency (protocol 12_153R) and the MINAP Academic Group.
The main exposure was the neutrophil count (part of the complete blood count) as recorded in primary care. We investigated the neutrophil count initially as a categorical variable to avoid assuming a particular shape for association with CVD. We wished to specifically look at associations with 'normal' as well as extreme neutrophil counts; there is no consensus definition for the normal range, but many laboratories quote the range of 2 to 7 Â 10 9 /l (16 In secondary analyses, we explored associations between onset of CVD and the mean of the first 2 stable measurements of neutrophil count taken since the start of eligibility. We extracted demographic For continuous covariates, we used the most recent value in the year before or up to 1 day after the complete blood count measurement. We also extracted the first measurement after this time window and the last measurement before the time window, along with the timing of these measurements relative to the index date, to use as auxiliary variables for multiple imputation. We also used comorbidity information from hospitalization records.  STATISTICAL ANALYSIS. We examined associations of CVD with neutrophil counts initially as a categorical variable. If the shape of the association was found to be linear, we also performed analyses with neutrophil count as a continuous variable. We generated cumulative incidence curves by category of neutrophil count under a competing risks framework. We used Cox proportional hazards models to estimate cause-specific hazards for each of the 14   For cardiovascular presentations among people without prior cardiovascular disease (CVD), crude cumulative incidence curves are shown for the highest and lowest categories of neutrophil count within the normal range. An artefact of imprecise coding rather than a clinical diagnosis, 'nonspecific coronary disease' was combined with unstable angina. Similarly, nonspecific stroke was combined with ischemic stroke. The plots show that, for myocardial infarction, heart failure, ischemic stroke, peripheral arterial disease (PAD), and abdominal aortic aneurysm, the incidence was greater among people with higher neutrophil counts.    Table 3). People with neutrophil counts of 6 to 7 Â 10 9 /l (at the upper end of the normal range) had a higher incidence of nonfatal MI, unheralded coronary death, HF, PAD, and abdominal aortic aneurysm compared with people with neutrophil counts of 2 to 3 Â 10 9 /l ( Figure 1). The risk difference appeared to be greatest for the first few months (Online Table 4). Individuals with higher neutrophil counts were relatively more likely to present with unheralded coronary death, HF, or PAD than stable angina (Online Table 5).
There were strong, specific associations between neutrophil counts and different initial presentations of CVD ( Figure 2). Adjusted HRs comparing neutrophil counts 6 to 7 versus 2 to 3 Â 10 9 /l showed strong as-  (Figure 2).
The associations were stronger in models adjusted only for age and sex (Online Figure 2).
There was a strong association of neutrophil count with noncardiovascular death when comparing neutrophil counts of 6 to 7 versus 2 to 3 Â 10 9 /l (HR: 2.01; 95% CI: 1.91 to 2.11), with a higher proportion of deaths due to pneumonia or chronic obstructive pulmonary disease (Online Table 4). There was also an association of higher neutrophil count with the composite of CVD (Online Figure 3) and all-cause mortality (Online Figure 4). Neutrophil counts of <2 Â 10 9 /l were associated with greater risk of noncardiovascular death (compared with 2 to 3 Â 10 9 /l: HR: 1.52; 95% CI: 1.41 to 1.63), but were not associated with greater risk of any presentation of CVD (Online Figure 3).
Because the associations between neutrophil counts and cardiovascular presentations were monotonic and linear, we treated neutrophil count as a linear variable in subsequent modeling. We found stronger associations within the normal range (Online Figure 5), but no interaction with smoking  Figure 1. Neutrophil Counts in Cardiovascular Disease status (Online Figure 6). Additional adjustment for eosinophil and lymphocyte counts did not alter the estimates (Online Figure 7).
There was some evidence that associations between neutrophil counts and cardiovascular presentations were stronger for stable compared with acute measurements, particularly for PAD and HF (Figure 3).
Associations were further strengthened when we used the mean of 2 consecutive stable neutrophil counts, which were available in 393,543 patients and were taken at a median of 1.4 years apart (IQR: 0.6 to 2.7 years) (Figure 3). There was considerable variability but minimal trend over time between repeat measurements of stable neutrophil counts; the SD of differences between 2 consecutive measurements was 1.67 Â 10 9 /l, the correlation coefficient was 0.568, and the mean rate of change was a decrease of 0.014 per year (95% CI: 0.011 to 0.017) (Online Figure 8).
We found that hazards were nonproportional for some of the endpoints. We therefore split the followup time by 6 months, and found that neutrophil counts were more strongly associated with HF, unheralded coronary death, and ischemic stroke in the first 6 months (Online Figure 9). Associations with coronary endpoints, HF, and PAD were stronger among younger patients (Online Figure 10). The association between neutrophil count and initial presentation with HF was stronger in men than women (HR per 10 9 /l higher neutrophil count: 1.10 vs. 1.07; p ¼ 0.001). There was an association between neutrophil count and initial presentation with transient ischemic attack in women but not men (HR: 1.05 vs. 1.00; p ¼ 0.007) (Online Figure 11).

DISCUSSION
Neutrophil counts within the range clinicians currently consider normal had strong linear associations with some, but not all, CVDs in a populationbased cohort. We found a greater cumulative incidence of unheralded coronary death, nonfatal MI, HF, PAD, and abdominal aortic aneurysm in patients      (19).
We also observed associations of higher neutrophil count with noncardiovascular and overall mortality, suggesting that chronic inflammation has noncardiovascular adverse effects that warrant further study.
We examined associations with other leukocyte subtypes and found that monocyte counts had a similar pattern of associations with initial presentations of CVD to neutrophils, but the associations were not as strong (Online Figure 12). Eosinophil and lymphocyte counts have a different pattern of association with initial presentations of CVD (14), but additional adjustment for these leukocyte subtypes did not alter the results for neutrophils (Online Figure 7), suggesting that these associations are independent. in neutrophils, reduced the incidence of acute coronary syndrome and stroke in a trial among patients with stable coronary disease (23). Trials are underway to investigate whether anti-inflammatory agents such as methotrexate (24) or canakinumab (a human monoclonal antibody against interleukin-1-beta) (25) can prevent CVD events in high-risk individuals.
Smoking causes an elevation of neutrophil counts and is associated more strongly with MI than stable angina (10) Neutrophil Counts in Cardiovascular Disease