Monocytosis is associated with hemolysis in sickle cell disease Naritsara Wongtong 1,2, Susan Jones 1, Yu Deng3, Jianwen Cai 3, Kenneth I. Ataga 1 1
Division of Hematology/Oncology, University of North Carolina, Chapel Hill, USA, 2Division of Hematology, Panyananthaphikkhu Chonpratan Medical Centre, Srinakharinwirot University, Pak Kret, Thailand, 3 Department of Biostatistics, University of North Carolina, Chapel Hill, USA Background: The clinical relevance of monocytosis in sickle cell disease (SCD) is uncertain. The purpose of this study was to explore the association of monocyte counts with clinical and laboratory variables in SCD. Design and methods: This cross-sectional study was performed using a cohort of adult patients with SCD and control subjects without SCD. Complete blood counts, markers of hemolysis, coagulation activation, endothelial injury, and other laboratory studies were obtained in patients with SCD. Clinical complications were ascertained at the time of evaluation and tricuspid regurgitant jet velocity was determined by Doppler echocardiography. Results: One hundred and fifty-seven patients with SCD and 24 healthy, African-American, control subjects were evaluated. The absolute monocyte count was increased in SCD patients compared with healthy controls (0.6 × 109/l vs. 0.4 × 109/l, P = 0.0025), with higher counts in HbSS/HbSβ0 thalassemia compared with HbSC/HbSβ+ thalassemia. In univariate analyses, absolute monocyte count was correlated with reticulocyte count, absolute neutrophil count, indirect bilirubin, lactate dehydrogenase, and inversely correlated with hemoglobin in SCD patients. Furthermore, monocyte count was correlated with soluble vascular cell adhesion molecule-1 (VCAM-1) in HbSS/HbSβ0 thalassemia patients. No significant associations were observed between absolute monocyte count and evaluated clinical complications. In multivariable analyses, reticulocyte count and absolute neutrophil count were significantly associated with absolute monocyte count. Conclusions: Monocytosis is associated with hemolysis and inflammation in SCD. We suggest that hemolysis and the resultant erythropoietic response contribute to monocyte activation. Keywords: Sickle cell disease, Monocytes, Hemolysis, Inflammation, Endothelial activation, Clinical complications
Introduction Sickle cell disease (SCD) is an inherited disorder characterized by the presence of chronic hemolysis, vaso-occlusive complications, a chronic vasculopathy, and organ damage. SCD is frequently referred to as an inflammatory state.1,2 Patients exhibit chronic elevation of leukocyte counts,3 abnormal activation of granulocytes,4–6 monocytes,5,7,8 and platelets5,9–11 even in the non-crisis, ‘steady state’. Circulating endothelial cells are abnormally activated, pro-adhesive, procoagulant and show evidence of oxidative stress.12,13 Finally, patients exhibit chronically elevated levels of inflammatory mediators,14 acute phase reactants1,15 and markers of endothelial injury1,16–18 even in the non-crisis, ‘steady state’. Baseline leukocyte count is a strong independent risk factor for disease severity in SCD. Leukocytosis Correspondence to: Kenneth I. Ataga, Division of Hematology/Oncology, University of North Carolina at Chapel Hill, Physicians’ Office Bldg., 3rd Floor, CB# 7305, 170 Manning Drive, Chapel Hill, NC 27599-7305, USA. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845415Y.0000000011
is a risk factor for increased mortality,19 acute chest syndrome,20 hemorrhagic stroke,21 and vaso-occlusive crises.22,23 In this cross-sectional study, we compared the absolute monocyte counts in SCD patients and healthy, African-American control subjects. Furthermore, we evaluated the association of absolute monocyte count with clinical complications and laboratory measures of hemolysis, markers of coagulation activation, inflammation and soluble vascular cell adhesion molecule-1 (VCAM-1), as a marker of endothelial injury, in patients with SCD.
Design and methods Patients and study design The study patients represent a cohort followed at the Sickle Cell Clinic at the University of North Carolina (UNC), Chapel Hill. The data were collected as part of a study to investigate the pathophysiology and natural history of pulmonary hypertension in SCD. Consecutive SCD patients seen in the clinic for routine follow-up, who agreed to participate, were
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Monocytosis in sickle cell disease
evaluated. Patients with SCD were evaluated while in the non-crisis, ‘steady state’; had not experienced an episode of acute chest syndrome in the 4 weeks preceding enrollment; and had no clinical evidence of congestive heart failure. The control subjects self-identified as African Americans or of African descent, had no known medical conditions, were not taking any medications, and were recruited by advertisement. The study was approved by the Institutional Review Board at UNC, Chapel Hill and all subjects gave written informed consent to participate.
Study measurements Measurement of laboratory variables Complete blood counts, including white blood cell counts, absolute neutrophil counts, absolute monocyte counts, hemoglobin, platelet counts, and reticulocyte counts, were measured using the Advia 2120 Hematology Analyzer (Siemens Healthcare Diagnostics, Deerfield, IL, USA). Commercially available enzyme-linked immunosorbent assay kits were used to measure human soluble VCAM-1, D-dimer and thrombin antithrombin complexes (TAT) (R&D Systems, Minneapolis, MN, USA). Other laboratory tests, including hemoglobin electrophoresis, serum creatinine, lactate dehydrogenase, total bilirubin, direct bilirubin, and indirect bilirubin, were performed at the McClendon Clinical Laboratory at UNC Hospitals.
SCD-related clinical complications Clinical complications in SCD patients were ascertained at the time of evaluation using accepted definitions.24 Tricuspid regurgitant jet velocity (TRV) was measured by Doppler echocardiography as previously described.16 The estimated pulmonary artery systolic pressure (PASP) was calculated using the modified Bernoulli equation, and pulmonary hypertension was suspected if the estimated PASP value, adjusted for age, sex, and body mass index exceeded the upper limits of normal in the reference ranges.25 All the echocardiograms were interpreted by a cardiologist blinded to all patient data.
Statistical analyses For continuous variables, Shapiro–Wilk’s test was used to test the normality assumption. Two-sample t-test was used for variables in which the normality assumption was not violated while the Kolmogorov–Smirnov test was used otherwise. The association of monocyte counts with continuous variables was explored using Spearman rank correlations with 95% confidence intervals (CIs). Median and inter-quartile range (25th and 75th percentiles) values of monocyte counts were presented by categories of categorical variables and the
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Komogorov–Smirnov test was conducted for two group comparisons. Multiple regression analysis, using the bootstrap method with 10 000 replications to estimate the P-value and 95% CI,26 was conducted to investigate the association of monocyte count with clinical and laboratory variables. A backward selection procedure was used for variable selection. The final model included only those variables which were statistically significant at 0.05 level. Reported P-values are unadjusted for multiple comparisons. All analyses were performed using SAS (version 9.3, SAS Institute, Inc. Cary, NC, USA).
Results Demographics and laboratory characteristics Demographic and laboratory characteristics of all the study subjects are shown in Table 1. One hundred and fifty-seven patients with SCD (SS: 116; SC: 18; Sβ0 thalassemia: 12; Sβ+ thalassemia: 10; SD: 1) and 24 healthy, African-American, control subjects (AA: 22; AC: 2) were evaluated. The absolute monocyte counts in SCD patients were significantly higher than in control subjects (0.6 × 109/l vs. 0.4 × 109/l, P = 0.0025). As expected, SCD patients had significantly higher WBC counts, platelet counts, reticulocyte counts, hemoglobin F, lactate dehydrogenase, and total and indirect bilirubin compared with control subjects, while hemoglobin was significantly lower in SCD patients compared with control subjects. When SCD was stratified based on presumed severity, absolute monocyte count was significantly higher in HbSS/HbSβ0 thalassemia patients compared with HbSC/HbSβ+ thalassemia patients (0.64 × 109/l ± 0.58 vs. 0.41 × 109/l ± 0.29, P = 0.0024).
Association of absolute monocyte count with laboratory and clinical variables in patients with sickle cell disease In SCD patients, the absolute monocyte count was significantly correlated with reticulocyte count (Spearman’s correlation coefficient r = 0.46, P < 0.0001), absolute neutrophil count (r = 0.37, P < 0.0001), indirect bilirubin (r = 0.33, P < 0.0001), lactate dehydrogenase (r = 0.46, P < 0.0001), and inversely correlated with hemoglobin (r = −0.27, P = 0.0003). No significant correlations were observed between absolute monocyte count and TAT, D-dimer, or soluble VCAM-1 (Table 2). No significant correlations were observed between absolute monocyte count and number of acute pain episodes in the past year or TRV. In addition, no significant associations were seen between absolute monocyte count and suspected pulmonary hypertension, history of stroke, history of acute chest syndrome, or history of leg ulcers.
Wongtong et al.
Monocytosis in sickle cell disease
Table 1 Demographic and laboratory characteristics of study subjects
Variable
Number
Age Gender (male) Genotype (SS) Genotype (SC) Genotype (Sβ0) Genotype (Sβ+) Genotype (SD) Genotype (AA) Genotype (AC) Body mass index White blood Cell (×109/l) Hemoglobin (g/dl) Platelet count (×109/l) Reticulocyte count (%) Haemoglobin F (%) Absolute neutrophil count (×109/l) Absolute monocyte count (×109/l) Lactate dehydrogenase (U/l) Total bilirubin (mg/dl) Direct bilirubin (mg/dl) Indirect biliribin (mg/dl) Creatinine (mg/dl)
157 157 157
Sickle cell disease mean (SD) or n (%) 36.8 ± 12.3 66 (36.5%) 116 (64.1%) 18 (10.0%) 12 (6.6%) 10 (5.5%) 1 (0.6%) – – 25.9 ± 6.2 9.4 ± 2.8 9.2 ± 1.7 406.0 ± 152.1 7.3 ± 4.7 8.2 ± 7.5 5.2 ± 2.1 0.6 ± 0.5 984.6 ± 497.2 2.5 ± 2.4 0.2 ± 0.2 2.4 ± 2.3 0.9 ± 0.6
– – 157 157 157 155 155 157 157 157 154 154 154 154 157
Number 24 24 24
24 24 24 24 24 24 24 24 23 23 23 22 24
Healthy controls mean (SD) or n (%)
P-value
35.3 ± 12.4 5 (20.8%) – – – – – 22 (12.2%) 2 (1.1%) 32.3 ± 8.6 7.5 ± 2.2 12.6 ± 1.1 290.1 ± 75.9 1.7 ± 0.5 0.7 ± 1.1 4.6 ± 1.8 0.4 ± 0.1 464.6 ± 66.6 0.4 ± 0.2 0.1 ± 0.0 0.3 ± 0.2 0.8 ± 0.2
0.8553 0.03 –
0.0026 0.0017*
Division of Hematology/Oncology, University of North Carolina, Chapel Hill, USA, 2Division of Hematology, Panyananthaphikkhu Chonpratan Medical Centre, Srinakharinwirot University, Pak Kret, Thailand, 3 Department of Biostatistics, University of North Carolina, Chapel Hill, USA Background: The clinical relevance of monocytosis in sickle cell disease (SCD) is uncertain. The purpose of this study was to explore the association of monocyte counts with clinical and laboratory variables in SCD. Design and methods: This cross-sectional study was performed using a cohort of adult patients with SCD and control subjects without SCD. Complete blood counts, markers of hemolysis, coagulation activation, endothelial injury, and other laboratory studies were obtained in patients with SCD. Clinical complications were ascertained at the time of evaluation and tricuspid regurgitant jet velocity was determined by Doppler echocardiography. Results: One hundred and fifty-seven patients with SCD and 24 healthy, African-American, control subjects were evaluated. The absolute monocyte count was increased in SCD patients compared with healthy controls (0.6 × 109/l vs. 0.4 × 109/l, P = 0.0025), with higher counts in HbSS/HbSβ0 thalassemia compared with HbSC/HbSβ+ thalassemia. In univariate analyses, absolute monocyte count was correlated with reticulocyte count, absolute neutrophil count, indirect bilirubin, lactate dehydrogenase, and inversely correlated with hemoglobin in SCD patients. Furthermore, monocyte count was correlated with soluble vascular cell adhesion molecule-1 (VCAM-1) in HbSS/HbSβ0 thalassemia patients. No significant associations were observed between absolute monocyte count and evaluated clinical complications. In multivariable analyses, reticulocyte count and absolute neutrophil count were significantly associated with absolute monocyte count. Conclusions: Monocytosis is associated with hemolysis and inflammation in SCD. We suggest that hemolysis and the resultant erythropoietic response contribute to monocyte activation. Keywords: Sickle cell disease, Monocytes, Hemolysis, Inflammation, Endothelial activation, Clinical complications
Introduction Sickle cell disease (SCD) is an inherited disorder characterized by the presence of chronic hemolysis, vaso-occlusive complications, a chronic vasculopathy, and organ damage. SCD is frequently referred to as an inflammatory state.1,2 Patients exhibit chronic elevation of leukocyte counts,3 abnormal activation of granulocytes,4–6 monocytes,5,7,8 and platelets5,9–11 even in the non-crisis, ‘steady state’. Circulating endothelial cells are abnormally activated, pro-adhesive, procoagulant and show evidence of oxidative stress.12,13 Finally, patients exhibit chronically elevated levels of inflammatory mediators,14 acute phase reactants1,15 and markers of endothelial injury1,16–18 even in the non-crisis, ‘steady state’. Baseline leukocyte count is a strong independent risk factor for disease severity in SCD. Leukocytosis Correspondence to: Kenneth I. Ataga, Division of Hematology/Oncology, University of North Carolina at Chapel Hill, Physicians’ Office Bldg., 3rd Floor, CB# 7305, 170 Manning Drive, Chapel Hill, NC 27599-7305, USA. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845415Y.0000000011
is a risk factor for increased mortality,19 acute chest syndrome,20 hemorrhagic stroke,21 and vaso-occlusive crises.22,23 In this cross-sectional study, we compared the absolute monocyte counts in SCD patients and healthy, African-American control subjects. Furthermore, we evaluated the association of absolute monocyte count with clinical complications and laboratory measures of hemolysis, markers of coagulation activation, inflammation and soluble vascular cell adhesion molecule-1 (VCAM-1), as a marker of endothelial injury, in patients with SCD.
Design and methods Patients and study design The study patients represent a cohort followed at the Sickle Cell Clinic at the University of North Carolina (UNC), Chapel Hill. The data were collected as part of a study to investigate the pathophysiology and natural history of pulmonary hypertension in SCD. Consecutive SCD patients seen in the clinic for routine follow-up, who agreed to participate, were
Hematology
2015
VOL.
20
NO.
10
593
Wongtong et al.
Monocytosis in sickle cell disease
evaluated. Patients with SCD were evaluated while in the non-crisis, ‘steady state’; had not experienced an episode of acute chest syndrome in the 4 weeks preceding enrollment; and had no clinical evidence of congestive heart failure. The control subjects self-identified as African Americans or of African descent, had no known medical conditions, were not taking any medications, and were recruited by advertisement. The study was approved by the Institutional Review Board at UNC, Chapel Hill and all subjects gave written informed consent to participate.
Study measurements Measurement of laboratory variables Complete blood counts, including white blood cell counts, absolute neutrophil counts, absolute monocyte counts, hemoglobin, platelet counts, and reticulocyte counts, were measured using the Advia 2120 Hematology Analyzer (Siemens Healthcare Diagnostics, Deerfield, IL, USA). Commercially available enzyme-linked immunosorbent assay kits were used to measure human soluble VCAM-1, D-dimer and thrombin antithrombin complexes (TAT) (R&D Systems, Minneapolis, MN, USA). Other laboratory tests, including hemoglobin electrophoresis, serum creatinine, lactate dehydrogenase, total bilirubin, direct bilirubin, and indirect bilirubin, were performed at the McClendon Clinical Laboratory at UNC Hospitals.
SCD-related clinical complications Clinical complications in SCD patients were ascertained at the time of evaluation using accepted definitions.24 Tricuspid regurgitant jet velocity (TRV) was measured by Doppler echocardiography as previously described.16 The estimated pulmonary artery systolic pressure (PASP) was calculated using the modified Bernoulli equation, and pulmonary hypertension was suspected if the estimated PASP value, adjusted for age, sex, and body mass index exceeded the upper limits of normal in the reference ranges.25 All the echocardiograms were interpreted by a cardiologist blinded to all patient data.
Statistical analyses For continuous variables, Shapiro–Wilk’s test was used to test the normality assumption. Two-sample t-test was used for variables in which the normality assumption was not violated while the Kolmogorov–Smirnov test was used otherwise. The association of monocyte counts with continuous variables was explored using Spearman rank correlations with 95% confidence intervals (CIs). Median and inter-quartile range (25th and 75th percentiles) values of monocyte counts were presented by categories of categorical variables and the
594
Hematology
2015
VOL.
20
NO.
10
Komogorov–Smirnov test was conducted for two group comparisons. Multiple regression analysis, using the bootstrap method with 10 000 replications to estimate the P-value and 95% CI,26 was conducted to investigate the association of monocyte count with clinical and laboratory variables. A backward selection procedure was used for variable selection. The final model included only those variables which were statistically significant at 0.05 level. Reported P-values are unadjusted for multiple comparisons. All analyses were performed using SAS (version 9.3, SAS Institute, Inc. Cary, NC, USA).
Results Demographics and laboratory characteristics Demographic and laboratory characteristics of all the study subjects are shown in Table 1. One hundred and fifty-seven patients with SCD (SS: 116; SC: 18; Sβ0 thalassemia: 12; Sβ+ thalassemia: 10; SD: 1) and 24 healthy, African-American, control subjects (AA: 22; AC: 2) were evaluated. The absolute monocyte counts in SCD patients were significantly higher than in control subjects (0.6 × 109/l vs. 0.4 × 109/l, P = 0.0025). As expected, SCD patients had significantly higher WBC counts, platelet counts, reticulocyte counts, hemoglobin F, lactate dehydrogenase, and total and indirect bilirubin compared with control subjects, while hemoglobin was significantly lower in SCD patients compared with control subjects. When SCD was stratified based on presumed severity, absolute monocyte count was significantly higher in HbSS/HbSβ0 thalassemia patients compared with HbSC/HbSβ+ thalassemia patients (0.64 × 109/l ± 0.58 vs. 0.41 × 109/l ± 0.29, P = 0.0024).
Association of absolute monocyte count with laboratory and clinical variables in patients with sickle cell disease In SCD patients, the absolute monocyte count was significantly correlated with reticulocyte count (Spearman’s correlation coefficient r = 0.46, P < 0.0001), absolute neutrophil count (r = 0.37, P < 0.0001), indirect bilirubin (r = 0.33, P < 0.0001), lactate dehydrogenase (r = 0.46, P < 0.0001), and inversely correlated with hemoglobin (r = −0.27, P = 0.0003). No significant correlations were observed between absolute monocyte count and TAT, D-dimer, or soluble VCAM-1 (Table 2). No significant correlations were observed between absolute monocyte count and number of acute pain episodes in the past year or TRV. In addition, no significant associations were seen between absolute monocyte count and suspected pulmonary hypertension, history of stroke, history of acute chest syndrome, or history of leg ulcers.
Wongtong et al.
Monocytosis in sickle cell disease
Table 1 Demographic and laboratory characteristics of study subjects
Variable
Number
Age Gender (male) Genotype (SS) Genotype (SC) Genotype (Sβ0) Genotype (Sβ+) Genotype (SD) Genotype (AA) Genotype (AC) Body mass index White blood Cell (×109/l) Hemoglobin (g/dl) Platelet count (×109/l) Reticulocyte count (%) Haemoglobin F (%) Absolute neutrophil count (×109/l) Absolute monocyte count (×109/l) Lactate dehydrogenase (U/l) Total bilirubin (mg/dl) Direct bilirubin (mg/dl) Indirect biliribin (mg/dl) Creatinine (mg/dl)
157 157 157
Sickle cell disease mean (SD) or n (%) 36.8 ± 12.3 66 (36.5%) 116 (64.1%) 18 (10.0%) 12 (6.6%) 10 (5.5%) 1 (0.6%) – – 25.9 ± 6.2 9.4 ± 2.8 9.2 ± 1.7 406.0 ± 152.1 7.3 ± 4.7 8.2 ± 7.5 5.2 ± 2.1 0.6 ± 0.5 984.6 ± 497.2 2.5 ± 2.4 0.2 ± 0.2 2.4 ± 2.3 0.9 ± 0.6
– – 157 157 157 155 155 157 157 157 154 154 154 154 157
Number 24 24 24
24 24 24 24 24 24 24 24 23 23 23 22 24
Healthy controls mean (SD) or n (%)
P-value
35.3 ± 12.4 5 (20.8%) – – – – – 22 (12.2%) 2 (1.1%) 32.3 ± 8.6 7.5 ± 2.2 12.6 ± 1.1 290.1 ± 75.9 1.7 ± 0.5 0.7 ± 1.1 4.6 ± 1.8 0.4 ± 0.1 464.6 ± 66.6 0.4 ± 0.2 0.1 ± 0.0 0.3 ± 0.2 0.8 ± 0.2
0.8553 0.03 –
0.0026 0.0017*
