|Year : 2019 | Volume
| Issue : 2 | Page : 45-50
Pattern of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in sickle cell anemia patients at steady state and vaso-occlusive crisis
Adekunle Emmanuel Alagbe1, John Ayodele Olaniyi2
1 Department of Pathology, Clinix Healthcare Limited, Amuwo Odofin, Lagos, Nigeria
2 Department of Hematology, College of Medicine, University of Ibadan; Department of Hematology, University College Hospital, Ibadan, Oyo, Nigeria
|Date of Web Publication||10-Jul-2019|
Dr. Adekunle Emmanuel Alagbe
Department of Pathology, Clinix Healthcare Limited, 6 Ago Palace Way, Amuwo Odofin, Lagos
Source of Support: None, Conflict of Interest: None
BACKGROUND: Neutrophil-lymphocyte ratio (NLR) and Platelet-Lymphocyte ratio (PLR) are emerging markers of systemic inflammation in chronic disorders, including hemoglobinopathies.
OBJECTIVE: This study determined the pattern of NLR and PLR in sickle cell anemia (SCA) patients during steady state and vasoocclusive crisis (VOC).
MATERIALS AND METHODS: A cross-sectional study composed of 60 adult SCA patients (30 during VOC and 30 during steady state). Thirty apparently healthy hemoglobin A (HbA) age- and sex-matched controls were included in the study. Complete blood count (CBC) was done for each participant; NLR was calculated as absolute neutrophil count (ANC) divided by the absolute lymphocyte count (ALC), and PLR was calculated as platelet count divided by ALC.
STATISTICAL ANALYSIS: Data were analyzed using SPSS (version 22), and the results were considered statistically significant if value of P < 0.05.
RESULTS: Total white-cell count, ANC, ALC, platelet count, and NLR were significantly higher in SCA patients than those of HbA controls. Total white-cell count, ANC, and NLR were significantly higher in the VOC than the steady-state group. PLR was not statistically different between the steady state and the VOC groups. In both groups of SCA patients, NLR was positively correlated with PLR, while percentage fetal hemoglobin was negatively correlated with percentage hemoglobin S. Hematocrit was negatively correlated with ANC and ALC in SCA patients.
CONCLUSION: This study showed a significantly higher NLR and PLR in SCA than the control group. These are reflective of the roles of neutrophils, platelets, and lymphocytes in the constitutive inflammatory process. These ratios could be adopted for use in SCA patients because these markers are cheap, stable, and easily derivable from CBC.
Keywords: Fetal hemoglobin, neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, sickle cell anemia, vaso-occlusive crisis
|How to cite this article:|
Alagbe AE, Olaniyi JA. Pattern of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in sickle cell anemia patients at steady state and vaso-occlusive crisis. J Appl Hematol 2019;10:45-50
|How to cite this URL:|
Alagbe AE, Olaniyi JA. Pattern of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in sickle cell anemia patients at steady state and vaso-occlusive crisis. J Appl Hematol [serial online] 2019 [cited 2019 Nov 21];10:45-50. Available from: http://www.jahjournal.org/text.asp?2019/10/2/45/262541
| Introduction|| |
Vaso-occlusive crisis (VOC) is the most common presentation in sickle cell anemia (SCA) patients and may involve any tissue or system. VOC is a complex process and an important cause of morbidity and mortality in SCA patients that is influenced by numerous factors, including deformability of erythrocytes, leukocyte and platelet counts, inflammatory mediators, endothelial cell adhesiveness, and others.,,,
SCA is an inherited autosomal recessive disorder that results from a single amino acid substitution in the gene encoding the β-globin subunit of hemoglobin. The resultant sickle hemoglobin has valine instead of glutamic acid at the 6th position of the beta-globin subunit. This mutation leads to an alteration in the properties of the hemoglobin tetramer, and thus, predisposes the erythrocytes to polymerization. Polymerization of deoxygenated sickle hemoglobin leads to decreased deformability of red blood cells (RBCs). Through a complex interplay of events among blood cells with increased adhesiveness, these altered erythrocytes can obstruct the vasculature and produce episodes of pain, hemolysis, organ injury, and early mortality.
Although the mutation in erythrocyte is the etiology of SCA, various studies have shown a pivotal role of neutrophils in the disease. Both in vivo and in vitro models have noted the dynamic interactions between the endothelial wall and flowing blood cells, and among the flowing cells, of which the irreversibly sickled RBCs appeared to be the most adherent to neutrophils. Neutrophilia, as seen in SCA, would worsen such interactions involving neutrophils. In addition, recruitment of the adherent leukocytes to activated endothelium would enhance the progress of VOC., VOC could be worsened by the presence of activated platelets seen in SCA patients. Platelet count is higher and platelet activation is enhanced in SCA patients during steady-state conditions and platelet count is further elevated in VOC.,, The severity of VOC is worsened due to platelet activation, which leads to enhanced adhesion of sickle erythrocytes to endothelium. In addition, platelets form aggregates with monocytes, neutrophils, and erythrocytes, thereby worsening VOC by obstruction of microvasculature.,
Recently, the neutrophil-lymphocyte ratio (NLR) and PLR ratios have been suggested to be indicators of inflammation in various diseases, including SCA, atherosclerosis, and venous thromboembolism.,, These lymphocyte ratios (LRs) have rarely been investigated in SCA during VOC. We aimed at studying the pattern of NLR and PLR in SCA patients during steady state and VOC and compared with those of hemoglobin A (HbA) control individuals.
| Materials and Methods|| |
This cross-sectional study consisted of 90 adult individuals enrolled and grouped into three groups: VOC group made up of 30 SCA patients enrolled consecutively at presentation during the acute painful crisis at the hematology day care unit of University College Hospital, Ibadan, South West Nigeria. VOC was defined as the occurrence of acute pain in the extremities, back, chest (ribs and sternum) that led to a hospital presentation and could not be explained except by SCA. Steady state (steady) group made up of 30 SCA patients enrolled during routine follow-up visit. Steady state was defined as stable health state in SCA patients who did not have pain or any other crisis, and no blood transfusions in the previous 2 months;, and control (HbA) group composed of 30 HbA individuals who were workers in the study hospital. The control participants were healthy (HbA) age- and sex-matched adults without previous clinical evidence of hemoglobinopathies. The hemoglobin phenotypes of all the participants were confirmed using high-performance liquid chromatography (HPLC). The individuals with concurrent overt infection, other acute complications than VOC, pregnancy, and other hemoglobinopathies were excluded from the study.
The questionnaires were filled from patients' notes and by interview. The questionnaire contained sections on bio-data and past medical. The Institution's Ethics Review Committee gave approval for the study and all participants gave informed consent.
Subsequently, venous blood sample was collected from all the participants and dispensed into two BD® ethylenediaminetetraacetic acid vacutainers. One for analysis of the hematological parameters, and the second tube for analysis of percentage hemoglobin quantification.
Complete blood count was performed using Sysmex XS1000i (Sysmex Corporation, Kobe, Japan), a fully automated 5part counter present at the Hematology Department of the hospital. NLR was calculated as absolute neutrophil count divided by absolute lymphocyte count (ALC), and plateletlymphocyte ratio (PLR) was calculated as platelet count divided by ALC.
Percentage hemoglobin (percentage fetal hemoglobin and percentage hemoglobin A) quantification
Percentage hemoglobin (percentage fetal hemoglobin [%HbF] and percentage HbA) quantification was performed using HPLC (BioRad variant II β-Thalassaemia Short Program®, Hercules, CA) available at the Genetic and Bioethics Research Laboratory, Institute of Advanced Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria.
Data were analyzed using SPSS version 22.0 (Statistical Package for Social Sciences, Inc., Chicago, USA). The descriptive data were presented as means ± standard deviation except otherwise stated. Frequencies are presented in tables and graphs. Student's t-test was used to compare means of the independent variables. Spearman's rho analysis was performed for correlation of the hematological parameters and percentage of hemoglobins in SCA patients. The results were considered statistically significant if value of P < 0.05.
| Results|| |
Hematological parameters for hemoglobin A controls and sickle cell anemia patients
Demographic characteristics of the study participants, as well as the hematological parameters, percentage hemoglobins, and lymphocyte ratios, are summarized in [Table 1] and [Table 2]. Ninety adults were evaluated in this study. Sixty previously diagnosed with SCA by alkaline electrophoresis comprised 27 males (45%) and 33 females (55%). Of the 30 apparently healthy HbA controls, 12 were male and 18 were female. There was significantly elevated total white blood cell count in the VOC group compared to the steady-state group (15.88 ± 4.61 × 103/μL vs. 11.27 ± 3.17 × 103/mm3; P = 0.000).
|Table 1: Hematological parameters of hemoglobin A controls and sickle cell anemia patients|
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|Table 2: Hematological parameters of sickle cell anemia patients during steady state and vaso-occlusive crisis|
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Correlations of hematological parameters, leukocyte ratios, and percentage of hemoglobin in patients with sickle cell anemia
Correlation analysis was performed in the SCA patients during steady state and VOC. There are positive correlations between NLR and PLR (r = 0.32, P = 0.015) [Figure 1]. As expected, there was a negative correlation between the percentage hemoglobin F and S (r = −0.87, P < 0.0001), [Figure 2], while hematocrit was negatively correlated with absolute neutrophils count and ALC [Figure 3] and [Figure 4], respectively]. In addition, %HbF was negatively correlated with total white cell count (r = −0.29, P = 0.047) but not significantly associated with NLR (r = −0.03, P = 0.856) and PLR (r = −0.09, P = 0.567).
|Figure 1: Correlation between platelet-lymphocyte ratio and neutrophil-lymphocyte ratio in sickle cell anemia patients. Neutrophil-lymphocyte ratio and platelet-lymphocyte ratio are positively correlated|
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|Figure 2: Correlation between percentage hemoglobin fetal and percentage hemoglobin S level in sickle cell anemia patients. Percentage hemoglobin fetal and percentage hemoglobin S are negatively correlated in the sickle cell anemia patients|
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|Figure 3: Correlation between hematocrit (%) and absolute neutrophil count in sickle cell anemia patients. Absolute neutrophil count and hematocrit are negatively correlated in the sickle cell anemia patients|
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|Figure 4: Correlation between hematocrit and absolute lymphocyte count in sickle cell anemia patients. Absolute lymphocyte count and hematocrit are negatively correlated in the sickle cell anemia patients|
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| Discussion|| |
Leukocytes play an important role in the pathophysiology of SCA by adhering to blood vessel walls and obstructing the lumen, forming aggregates with other blood cells, thereby worsening vaso-occlusion, stimulating the vascular endothelium to increase its expression of ligands for adhesion molecules on blood cells, and causing tissue damage and inflammatory reaction, which predispose to vaso-occlusion. Studies have shown that severity of SCA is worsened by leukocytosis., Neutrophils are the most abundant leukocytes in the circulation and are important in acute inflammation, and neutrophilia, as well as thrombocytosis, are associated with increased severity of vaso occlusion in SCA patients. NLR and PLR have been postulated as markers of inflammation, and studies have associated elevated NLR and PLR with clinical outcomes in cardiovascular disease, cancers, as well as renal disease in SCA.,, In the present study, NLR was significantly higher in SCA patients than HbA controls and in the VOC group than the steady-state group. Contrarily, the platelet-lymphocyte ratio was not statistically different among these groups. However, NLR was positively correlated with PLR, and hematocrit was negatively correlated with absolute neutrophil count (ANC) and ALC in SCA patients. Our findings are similar to those of previous studies, in which NLR was found to be significantly elevated in SCA and other disease conditions.,,, They found a higher NLR and/or PLR in patients with SCA, diabetes mellitus, and systemic lupus erythematosus than controls. These findings show that the rate of rise of neutrophils during these disease conditions is higher than the rate of rise of lymphocytes.
Like other researchers, this study buttresses that SCA is a chronic inflammatory disease as evidenced by a higher NLR during steady state and VOC.,, Leukocytes, which are both increased and activated in SCA, aid the transition from steady state to VOC. In the presence of the appropriate VOC-inducing agents, leukocytes enhance adhesiveness of the endothelial cells by the secretion of pro-inflammatory mediators and expression of corresponding adhesion molecules, thereby enhancing vaso-occlusion and the pain process. There is increased hematopoietic activity in addition to transition of the marginating leukocytes to the circulating pool just before the onset of or during VOC. This could be responsible for the marked leukocytosis noted in the VOC group compared to the steady-state group. Leukocytosis increases the propensity of leukocytes to adhere to the postcapillary venular endothelium, thus, reducing the vessel lumen. This occlusion is worsened by sickled and unsickled RBCs, platelets, and leukocytes, which all have increased tendency to adhere to the vessel wall and to other cells in these patients.,, Paradoxically, leukocytosis in these patients may be a form of defense response against overwhelming infectious agents to which SCA patients are susceptible sequel to functional asplenia that results from recurrent splenic infarction. Functional asplenia predisposes the patients to overwhelming infections, particularly encapsulated organisms such as Streptococcus pneumonia and Hemophilus influenzae.,,,
A high NLR in SCA patients is a risk factor for developing VOC as well as a risk of other end-organ damage. Similar to John et al., this study found a higher NLR in SCA patients than HbA controls. Contrarily, the latter study failed to demonstrate a significant relationship between higher NLR and sickle cell nephropathy probably because their cohort was made of SCA patients in the steady state. Consistent with other studies, significantly elevated platelet count in the VOC and steady-state group may have resulted from a rebound thrombocytosis. Rebound thrombocytosis may accompany hemolytic anemias and anemia of chronic disease because of the compensatory feedback effect of anemia on erythropoietin production. This is due to a structural homology between erythropoietin and thrombopoietin.,, In addition, hyposplenism in SCA leads to the exclusion of the pooling effect of the spleen on platelets, thus, contributing significantly to the elevated platelet count observed. These platelets are not only elevated in patients with SCA but also they are activated because of the sickle cell-induced milieu. Blunted response to erythropoietin; very short RBC life span because of the presence of abnormal hemoglobin S leading to continuous hemolysis; and recurrent infection also contribute to anemia in these patients, hence, worsening thrombocytosis., The percentage hemoglobin S in each red cell, though not significantly different between the steady-state and VOC groups, is proportional to the degree of polymerization of deoxygenated sickle hemoglobin and inversely proportional to the HbF level.,,,,, However, %HbF was not significantly associated with PLR or NLR.
Positive correlation of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in SCA patients supports the contribution of neutrophils and platelets to chronic inflammatory state in SCA patients. These ratios could be useful cheap and stable markers of inflammation. The use of these LRs may be useful and better than using ANC or platelet count solitarily in assessing the degree of inflammation in SCA patients. This is because these ratios are somewhat more stable and not easily influenced by physiological, pathological, or stressful events known to influence neutrophil and platelet counts and activities.
Among other factors that may influence the degree of inflammation in SCA is the hematocrit. A negative correlation between the hematocrit and ANC or platelet count in SCA patients suggests that a high hematocrit may be protective in SCA patients and may reduce the severity of VOC. However, previous studies have shown that high hematocrit will lead to increased viscosity and VOC becomes worse. Therefore, it is logical to suggest that there is a critical hematocrit “cut off” that will worsen inflammation in such patients; hence, there is a need for further research to determine the critical high hematocrit that is deleterious in SCA patients.
| Conclusion|| |
NLR is higher in SCA patients compared with HbA controls and positively correlates with PLR in SCA during the steady state and VOC. These are reflective of the roles of neutrophils, platelets, and lymphocytes in the constitutive inflammatory process. These LRs are cheap, stable, and simple derivatives of blood count that could be adopted as the markers of inflammation in SCA patients.
We would like to appreciate the contribution and support given by Professor A.G. Falusi, and the staff of Genetic and Bioethics Research Laboratory, Institute of Advanced Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria. We also acknowledge the contribution by all Resident Doctors (Laboratory Medicine) and SCA patients at the Hematology department, University College Hospital, Ibadan.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Platt OS, Thorington BD, Brambilla DJ, Milner PF, Rosse WF, Vichinsky E, et al.
Pain in sickle cell disease. Rates and risk factors. N Engl J Med 1991;325:11-6.
Manwani D, Frenette PS. Vaso-occlusion in sickle cell disease: Pathophysiology and novel targeted therapies. Blood 2013;122:3892-8.
Turhan A, Weiss LA, Mohandas N, Coller BS, Frenette PS. Primary role for adherent leukocytes in sickle cell vascular occlusion: A new paradigm. Proc Natl Acad Sci U S A 2002;99:3047-51.
Zhang D, Xu C, Manwani D, Frenette PS. Neutrophils, platelets, and inflammatory pathways at the nexus of sickle cell disease pathophysiology. Blood 2016;127:801-9.
Alagbe AE, Justo Junior AS, Ruas LP, Tonassé WV, Santana RM, Batista TH, et al.
Interleukin-27 and interleukin-37 are elevated in sickle cell anemia patients and inhibit in vitro
secretion of interleukin-8 in neutrophils and monocytes. Cytokine 2018;107:85-92.
Rees DC, Williams TN, Gladwin MT. Sickle-cell disease. Lancet 2010;376:2018-31.
Sparkenbaugh E, Pawlinski R. Interplay between coagulation and vascular inflammation in sickle cell disease. Br J Haematol 2013;162:3-14.
Okpala I. The intriguing contribution of white blood cells to sickle cell disease – A red cell disorder. Blood Rev 2004;18:65-73.
Proença-Ferreira R, Brugnerotto AF, Garrido VT, Dominical VM, Vital DM, Ribeiro Mde F, et al.
Endothelial activation by platelets from sickle cell anemia patients. PLoS One 2014;9:e89012.
Emokpae M, Abdu A, Gwaram B. Neutrophil-to-lymphocyte, platelet-to-lymphocyte ratios and their association with atherogenic index of plasma in sickle cell nephropathy. J Appl Hematol 2016;7:24-9. [Full text]
John CA, Adegbola OA, Emmanuel CO, Christian EO, Nancy CI, Muheez DA. Neutrophil-to-lymphocyte ratio in sickle cell anaemia patients with nephopathy. Br J Med Res 2015;10:1-6.
Artoni A, Abbattista M, Bucciarelli P, Gianniello F, Scalambrino E, Pappalardo E, et al.
Platelet to lymphocyte ratio and neutrophil to lymphocyte ratio as risk factors for venous thrombosis. Clin Appl Thromb Hemost 2018;24:808-14.
Keikhaei B, Mohseni AR, Norouzirad R, Alinejadi M, Ghanbari S, Shiravi F, et al.
Altered levels of pro-inflammatory cytokines in sickle cell disease patients during vaso-occlusive crises and the steady state condition. Eur Cytokine Netw 2013;24:45-52.
Osadnik T, Wasilewski J, Lekston A, Strzelczyk J, Kurek A, Gonera M, et al.
The platelet-to-lymphocyte ratio as a predictor of all-cause mortality in patients with coronary artery disease undergoing elective percutaneous coronary intervention and stent implantation. J Saudi Heart Assoc 2015;27:144-51.
Templeton AJ, McNamara MG, Šeruga B, Vera-Badillo FE, Aneja P, Ocaña A, et al.
Prognostic role of neutrophil-to-lymphocyte ratio in solid tumors: A systematic review and meta-analysis. J Natl Cancer Inst 2014;106:dju124.
Lou M, Luo P, Tang R, Peng Y, Yu S, Huang W, et al.
Relationship between neutrophil-lymphocyte ratio and insulin resistance in newly diagnosed type 2 diabetes mellitus patients. BMC Endocr Disord 2015;15:9.
Wu Y, Chen Y, Yang X, Chen L, Yang Y. Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were associated with disease activity in patients with systemic lupus erythematosus. Int Immunopharmacol 2016;36:94-9.
Gonçalves MS, Queiroz IL, Cardoso SA, Zanetti A, Strapazoni AC, Adorno E, et al.
Interleukin 8 as a vaso-occlusive marker in Brazilian patients with sickle cell disease. Braz J Med Biol Res 2001;34:1309-13.
Frenette PS, Atweh GF. Sickle cell disease: Old discoveries, new concepts, and future promise. J Clin Invest 2007;117:850-8.
Anyaegbu CC, Okpala IE, Akren'Ova YA, Salimonu LS. Peripheral blood neutrophil count and candidacidal activity correlate with the clinical severity of sickle cell anaemia (SCA) Eur J Haematol 1998;60:267-8.
Olopoenia L, Frederick W, Greaves W, Adams R, Addo FE, Castro O, et al.
Pneumococcal sepsis and meningitis in adults with sickle cell disease. South Med J 1990;83:1002-4.
Borić MP, Donoso V, Fournier A, St Pierre S, Huidobro-Toro JP. Endothelin reduces microvascular blood flow by acting on arterioles and venules of the hamster cheek pouch. Eur J Pharmacol 1990;190:123-33.
Duits AJ, Schnog JB, Lard LR, Saleh AW, Rojer RA. Elevated IL-8 levels during sickle cell crisis. Eur J Haematol 1998;61:302-5.
Akinbami A, Dosunmu A, Adediran A, Oshinaike O, Adebola P, Arogundade O,et al
. Haematological values in homozygous sickle cell disease in steady state and haemoglobin phenotypes AA controls in Lagos, Nigeria. BMC Res Notes 2012;5:396.
Naik RP, Streiff MB, Lanzkron S. Sickle cell disease and venous thromboembolism: What the anticoagulation expert needs to know. J Thromb Thrombolysis 2013;35:352-8.
Sherwood JB, Goldwasser E, Chilcote R, Carmichael LD, Nagel RL. Sickle cell anemia patients have low erythropoietin levels for their degree of anemia. Blood 1986;67:46-9.
Rosse WF, Narla M, Petz LD, Steinberg MH. New views of sickle cell disease pathophysiology and treatment. ASH Education Program Book 2000;2000:2-17.
El-Hazmi MA, Al-Hazmi AM, Warsy AS. Sickle cell disease in Middle East Arab countries. Indian J Med Res 2011;134:597-610.
] [Full text]
Kotila TR, Fawole OI, Shokunbi WA. Haemoglobin F
and clinical severity of sickle cell anaemia among Nigerian adults. Afr J Med Med Sci 2000;29:229-31.
Akinsheye I, Alsultan A, Solovieff N, Ngo D, Baldwin CT, Sebastiani P, et al.
Fetal hemoglobin in sickle cell anemia. Blood 2011;118:19-27.
Marsh A, Vichinsky EP. Sickle cell disease. In Postgraduate Haematology, Ed Hoffbrand AV, Higgs DR, Keeling DM, Mehta AB. 7th
ed. Wiley, West Sussex, 2016:98-113.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]