|Year : 2017 | Volume
| Issue : 3 | Page : 69-74
Assessment of plasma fibrinogen and vitamin D levels among participants with different haemoglobin phenotypes in a Nigerian tertiary health care facility
Patrick O Manafa1, Chide E Okocha2, John C Aneke2, Nancy C Ibeh1, Glory U Uduma1
1 Department of Medical Laboratory Science, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Nigeria
2 Department of Haematology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State, Nigeria
|Date of Web Publication||2-Apr-2018|
John C Aneke
Nnamdi Azikiwe University Teaching Hospital, Nnewi, PMB 5025, Anambra State
Background: Hyperfibrinogenaemia and vitamin D deficiency have been implicated in the pathogenesis and severity of sickle cell anaemia (SCA).
Objective: To determine the levels of fibrinogen and vitamin D in persons with different haemoglobin phenotypes and correlate these levels with disease severity.
Patients and Methods: Ninety adult volunteers were recruited, comprising 30 each of SCA (HbSS), heterozygous and normal (HbAA) haemoglobin type. Each volunteer had 6 mL of venous blood collected; 1.8 and 2 mL were used for plasma fibrinogen and serum vitamin D assay, respectively, while the remaining 2.2 mL was used for haemoglobin phenotype and full blood count determination. The disease severity for patients with SCA was determined using an objective scoring system. Data were analyzed using the Statistical Programme for Social Sciences version 20 software (SPSS Inc., Chicago, IL, USA); the level of significance was at P < 0.05.
Results: The median plasma fibrinogen and serum vitamin D levels in patients with HbSS vs. HbAA volunteers were 295.00 mg/dL (Q1–Q3; 230.00–342.25 mg/dL) and 27.35 ng/mL (Q1–Q3; 19.75–36.20 ng/mL), respectively. The levels of plasma fibrinogen and serum vitamin D were not significantly different in patients with HbSS vs. HbAA volunteers (P values = 0.32 and 0.47, respectively) and were not significantly correlated with disease severity.
Conclusion: Plasma fibrinogen and serum vitamin D levels do not predict the disease severity in steady-state SCA.
Keywords: Disease severity, fibrinogen, sickle cell anaemia, vitamin D
|How to cite this article:|
Manafa PO, Okocha CE, Aneke JC, Ibeh NC, Uduma GU. Assessment of plasma fibrinogen and vitamin D levels among participants with different haemoglobin phenotypes in a Nigerian tertiary health care facility. Sub-Saharan Afr J Med 2017;4:69-74
|How to cite this URL:|
Manafa PO, Okocha CE, Aneke JC, Ibeh NC, Uduma GU. Assessment of plasma fibrinogen and vitamin D levels among participants with different haemoglobin phenotypes in a Nigerian tertiary health care facility. Sub-Saharan Afr J Med [serial online] 2017 [cited 2019 Oct 17];4:69-74. Available from: http://www.ssajm.org/text.asp?2017/4/3/69/228972
| Introduction|| |
Sickle cell anaemia (SCA) is a single gene disorder which leads to a systemic clinical condition characterized by chronic anaemia, acute painful episodes, organ infarction, chronic organ damage and significant reduction in life expectancy. Sickled haemoglobin is a structural variant of normal adult haemoglobin, caused by a mutation in the β-globin gene that leads to the substitution of valine for glutamic acid at position 6 of the β-globin subunit of the haemoglobin molecule. Sickled red cells interact with the vascular endothelium, leading to microvascular occlusion, ischaemia/reperfusion and increased expression of inflammatory cytokines and adhesion molecules. Additionally, a number of complications such as anaemia, hypoxia, cholelithiasis, fatigue, exercise intolerability, hypercoagulability and vasculopathy could occur in patients with SCA due to chronic red cell haemolysis. Nearly every component of haemostasis is altered in patients with SCA; both acute and chronic inflammations are increasingly implicated in its pathophysiology., Vaso-occlusion (causing tissue infarction) appears to be the major inflammatory stimulus and has been linked to the development of a number of sickle-related complications, including vasculopathy. Many markers of inflammation are elevated in sickle cell disease (SCD) and could potentially be employed as the biomarkers of disease severity.,, Fibrinogen is an Acute Phase Reactant; raised levels have, therefore, been observed in relation to a variety of physiological and inflammatory conditions., The role of hyperfibrinogenaemia in vivo in increasing viscosity leading to vaso-occlusive crisis in SCA has not been studied in our environment. However, it has been suggested that the rise in fibrinogen (as in pregnancy and in acute infections) may actually play a significant role in the initiation of crisis in patients with SCA.
Vitamin D (25-hydroxyvitamin D) deficiency has been implicated in adverse skeletal and extra-skeletal manifestations. Moreover, individuals living with SCA have been reported to have a high prevalence of vitamin D deficiency., In addition to its effects on bone health, vitamin D deficiency has been variously linked with cardiovascular disease, asthma, nephropathy and chronic pain. The role of vitamin D in patients with SCA is still unclear.
| Patients and methods|| |
This was a cross-sectional study, conducted from 1 October 2014 to 30 September 2015, at the Haematology Department of Nnamdi Azikiwe University Teaching Hospital, Nnewi. Ninety volunteers were recruited for this study, 30 each of sickle cell anaemia (HbSS) in steady state, heterozygous sickle cell and normal (HbAA) individuals, who served as the control group. The steady-state selection criteria for the HbSS were based on being clinically stable (with the absence of any form of crises or any symptom of ill health) for at least 3 weeks and no history of blood transfusion in the preceding 3 months prior to the study.
Volunteers on anticoagulant therapy and contraceptives, pregnancy, lactation and history of vitamin D supplementation were excluded.
The ethical approval was obtained from the ethics and research committee of the Nnamdi Azikiwe University Teaching Hospital, Nnewi, and informed written consent was obtained from each participant prior to recruitment.
Each participant had 6 mL of venous blood collected following the standard protocols for phlebotomy and dispensed into three different specimen containers; the needles were immediately discarded (without capping) into designated sharp boxes. For fibrinogen estimation, 1.8 mL of the blood was dispensed into a tube containing 0.2 mL of 3.2% trisodium citrate (ratio 9:1). The mixture was centrifuged at 1500×g for 15 min to obtain platelet-poor plasma and stored at −20°C for batch analysis at weekly intervals. For vitamin D estimation, 2 mL of the venous blood was dispensed into a plain container, allowed to stand for 30 min to clot and centrifuged at 5000 rpm for 5 min to obtain serum; this was stored at −20°C for batch analysis at weekly intervals. The remaining blood sample (2.2 mL) was dispensed into ethylene diamine tetraacetic acid specimen containers for haemoglobin phenotype and full blood count determination, which were performed within 1 h of collection.
Fibrinogen and vitamin D levels were estimated using the thrombin clotting time fibrinogen assay and enzyme-linked immunosorbent assay (ELISA), respectively. The thrombin clotting time fibrinogen assay was based on the method originally described by Clauss. In the presence of high concentration of thrombin, the time required for clot formation in the sample plasma is inversely proportional to the fibrinogen concentration. The time taken for clot to form following the incubation of diluted plasma, diluted 1:10 in buffer (at 37°C) with phospholipid, thrombin and calcium was recorded. The fibrinogen concentration in the test plasma was deduced by comparing this time to a calibration curve, plotted on a log–log graph paper (using a reference plasma).
The vitamin D assay was based on the Calbiotech Vitamin D Kit, a solid phase ELISA based test, which works on the principle of competitive antibody binding.
Haemoglobin electrophoresis was performed using the alkaline cellulose acetate paper haemoglobin electrophoresis method of Kohn, as modified by Erhabor et al. (with the Mupid®-exu Electrophoresis System, ADVANCE Co., Ltd, Tokyo, Japan), whereas full blood count was determined using the Sysmex automated haematology analyser (KX21N® Model, Sysmex Corporation, Kobe, Japan).
The disease severity scoring for patients with SCA was derived using the information on transfusion history and the presence of SCA-related complications. The details are as given in [Table 1].
Over all disease severity scores
- ≤3 = mild;
- >3–5 = moderate;
- >5 = severe.
Data analysis was performed using the Statistical Programme for Social Sciences version 20 software (SPSS Inc., Chicago, IL, USA), and the results of plasma fibrinogen and serum vitamin D levels were presented as medians interquartile range (IQR). The Mann–Whitney U test was used to compare the medians of plasma fibrinogen and serum vitamin C levels among patients with HbSS and HbAA controls; plasma and serum levels were compared with objective scores of disease severity using the Pearson’s linear regression for bivariate correlation; P was significant at <0.05.
| Results|| |
The median plasma fibrinogen level in patients with HbSS vs. HbAA volunteers was 295.00 mg/dL (Q1–Q3; 230.00–342.25 mg/dL). There was no statistically significant difference in plasma fibrinogen concentration between patients with HbSS and HbAA controls (P = 0.32).
The median serum vitamin D level in patients with HbSS vs. HbAA participants was 27.35 ng/mL (Q1–Q3; 19.75–36.20 ng/mL), and these were not significantly different among patients with HbSS and HbAA controls (P = 0.47).
Only 19 patients with HbSS had complete clinical and laboratory information that were required for disease severity calculation; seven, three, and nine of these had severe, moderate and mild diseases, respectively.
Plasma fibrinogen concentration and vitamin D levels did not show significant correlations with disease severity in HbSS (r = 0.09, P = 0.70; r = −0.21, P = 0.40, respectively, [Figure 1],[Figure 2],[Figure 3]).
|Figure 3: Correlation of fibrinogen levels with disease severity in homozygous sickle cell (HbSS) group. Key: Fibrinogen levels in mild disease = 276 mg/dL; Fibrinogen levels in moderate disease = 295 mg/dL; Fibrinogen levels in severe disease = 285 mg/dL|
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| Discussion|| |
Fibrinogen is an acute phase protein, which aside from promoting the formation of fibrin strands during physiologic haemostasis, equally functions as a major determinant of whole blood viscosity. Because of its effect on whole blood rheology, it has been proposed that raised fibrinogen concentration may be a contributory factor to red cell sludging as seen in sickle cell vaso-occlusive crises. Earlier reports by Tatkare, Buseri and Famodu had observed significant increase in plasma fibrinogen concentration among patients with SCA compared with HbAA controls.,, These findings were more marked in SCA in crises and were thought to be as a result of a combination of increased production (in response to the chronic haemolytic process of SCA) as well as the increased acute phase reaction of inflammation, which has been noted to be significant in SCA, particularly during crises.,
In contrast to the above reports, Soheir et al. and Mauryne et al. observed significantly lower plasma level of fibrinogen in patients with SCA in steady state when compared with controls. It is probable that subclinical hepatic injury (hepatopathy), which could occur in SCA, with a resultant decrease in the synthesis of clotting factors, might have contributed to this observation.
In the present study, the levels of plasma fibrinogen were not significantly different in patients with SCA compared to HbAA controls (P = 0.32). Tatkare et al. had earlier reported a significantly higher plasma fibrinogen levels in patients with SCA at the Mahatma Ghandi Institute of Medical Sciences, India, compared with HbAA controls (P < 0.001). The study concluded that the high plasma fibrinogen levels parallel intense inflammatory process observed in patients with SCA, particularly those in crisis. This reason for the discrepancy between the result this study and that Tatkare et al. is because all our study participants were recruited in steady state conditions, while the latter study involved patients in crisis. Additionally, we observed that plasma fibrinogen levels were not significantly correlated with disease severity in patients with SCA (r = 0.09, P = 0.70, [Figure 3]). This is in contrast with the observations of Tatkare and Emokpae, which showed significant correlations among plasma fibrinogen, severity of crises and end organ dysfunction (sickle cell nephropathy) in SCA., It is important to note that while Tatkare et al. studied patients with SCA in crises, Emopkae et al. selected patients with SCA who had established renal insufficiency; these observations might have accounted for the differences between the findings in this study and the above two reports.
In this study, no significant difference was observed in the mean serum level of vitamin D in patients with SCA compared with HbAA controls (P value = 0.47). The lack of statistical difference in the serum vitamin D levels of patients with HbSS and HbAA volunteers in our study may be predicated by a number of factors which have been noted to influence vitamin D metabolism, such as the degree of exposure to ultraviolet radiation, extent of skin pigmentation, adequacy of dietary intake and genetic variations in vitamin receptors.,,, This would have equally accounted for the non-significant correlation between serum vitamin D levels and disease severity in patients with HbSS (r = −0.21, P = 0.40, [Figure 4]). Our finding is in agreement with the report of Van der Dijs et al., which showed no significant difference between serum vitamin D levels in patients with SCA and controls in Curacao and concluded that these patients had no need for vitamin supplementation. In contrast, Mohammed et al. noted a tendency towards hypocalcaemia (in association with supranormal parathyroid hormone levels) among Saudi patients with SCA and argued that this could be due to the reduced intestinal absorption of vitamin D with resultant disturbance in calcium metabolism (and characteristic skeletal changes which are commonly seen in these patients). In another series, Garrido et al. and Ozen et al. independently reported vitamin D deficiency in 56.4% and 63.1% of the patients with SCA, respectively. These deficiency states may be accentuated by less exposure to ultraviolet radiation; previous report from Saudi Arabia among normal participants had emphasized that vitamin D deficiency was related to the habit of avoidance of direct sunlight in the hot desert climates.
|Figure 4: Correlation of vitamin D levels with disease severity in homozygous sickle cell (HbSS) group. Key: Vitamin D levels in mild disease = 29.7 ng/dL; Vitamin D levels in moderate disease = 30.9 ng/dL; Vitamin D levels in severe disease = 23.0 ng/dL|
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Akinlade et al. studied 60 patients with SCA in Ibadan, South-west Nigeria and reported that vitamin D levels were significantly lower in patients in vaso-occlusive crises, compared with those in steady states. The authors equally observed that vitamin D levels were significantly related to the presence of sickle-related liver dysfunction in study participants. The importance of vitamin D in the pathogenesis of sickle-related bone disease has been emphasized in the report of Osunkwo which showed complete resolution of chronic pain symptoms and improvement in bone density, with vitamin D supplementation, in a patient with sickle cell disease who had chronic pain, associated with profound vitamin D deficiency, osteoporosis and osteonecrosis. Vitamin D deficiency has, therefore, been confirmed to be an important factor in sickle cell related bone disease. Considering the foregoing, it is very likely that vitamin D supplementation could constitute an important therapeutic intervention in appropriately selected groups of patients with SCA, particularly those with symptomatic bone disease and suboptimal serum levels.
| Conclusion|| |
The patients with SCA in this study may have less intense inflammation and coagulation activation than previously reported from other studies. This may partly explain the absence of significant correlation between plasma fibrinogen levels and objective scores of disease severity in this study. Similarly, vitamin D supplementation may not be necessary in patients with steady state SCA, because they have optimal serum levels. It would be interesting to investigate these parameters in our patients with HbSS in crisis by performing further studies.
Limitation of the study
This study is limited by the small population of patients with steady state SCA that had complete parameter required for disease severity calculation as well as the non-inclusion of assay for serum calcium.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]