Ray-Tracing

History taking and physical examination: Symptoms and signs of cardiac dysfunction may occur in SCA patients even without frank evidence of heart failure and efforts should be made to detect them.^2,5 Such early signs include prominent neck veins pulsation, 3^rd heart sound, loud P2, murmurs which may be found in SCA patients before the cardinal signs of heart failure such as tachycardia, tachypnoea or hepatomegaly develop.^2,5

Because of high pulse rate in SCA patients, especially those with very low haemoglobin, heart failure is often over-diagnosed and it is unreliable to grade heart failure in these children using available scoring systems like Ross score which classifies heart failure based on clinical features of tachypnoea, tachycardia, dyspnoea on exertion and hepatomegaly.^2,49

Exercise tolerance test (ETT): This is a recognized objective clinical assessment of cardiac impairment.¹⁶ ETT is usually a six minutes’ walk test which is simple, inexpensive, reproducible and safe, because patient is self-limited during exercise.¹⁶ It indicates severity of functional impairment and is defined as inability to walk more than 300m in six minutes but has limited use in very young children who may not be able to walk .¹⁶ The sensitivity of clinical assessment alone (76.8%) is significantly less than that of echocardiography (83.3%) in detecting cardiac abnormalities in SCA patients.¹² Attempts to improve sensitivity of clinical assessment by the introduction of Ross scoring system in children may not be useful in SCA patients due to the presence of hepatomegaly and relative tachycardia even in steady state.^12,49. The USA Social Security

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Administration has recognized the ETT as indicative of significant cardiac impairment and qualification for disability benefit to patients with chronic heart diseases.⁵⁰ The significant association between high serum NT-proBNP and disease severity in patients with chronic LV dysfunction^51,52 was also the reason for the adoption of NT-proBNP levels as part of disease severity criteria by the Social Security Administration.⁵⁰

Common Physical findings in SCA patients in steady state

Common steady state findings, usually due to hyperdynamic circulation or underlying cardiovascular impairment may include the following:

i. Neck veins pulsation.²

ii. Blood pressure: Blood pressure in the steady state is lower than normal and fails to show the age-related rise common in normal populations.⁵³

iii. Left parasternal heave.²

iv. Loud 1^st and 2^nd heart sounds in keeping with hyperdynamic state with 3^rd heart sound sometimes.²

v. Loud pulmonary component if pulmonary hypertension is present.²

vi. Murmurs: Common in SCA but frequently absent in patients with high Hb levels.⁵⁴ vii. Cardiomegaly usually develops before the age of five and produces visible

precordial apical impulse.^2,54

2. Radiology

Cardiomegaly is evident on chest x-ray in 86% of SCA patients. ⁵⁵ But interpretation of cardiomegaly on chest x-ray is limited by type of view (the heart appears larger in antero-posterior views), poor positioning especially in children and inability to differentiate chamber dilatation from hypertrophy.²

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3. Electrocardiography: ECG changes are common but non-specific and include prolonged PR interval, sinus arrhythmia, tachycardia, abnormal septal waves seen in other types of chronic anemic states¹¹ and increased P-wave dispersion suggestive of LV diastolic dysfunction.⁵⁶ Akinola and Balogun⁵⁷ studied ECG changes in SCA patients in Nigeria and found abnormalities in 16 (72.7%) of them of which left ventricular hypertrophy (LVH) was commonest (N=14; 63.8%). No feature of ischaemic heart disease was found although the sample size was small, involving only 22 patients and the findings may not be representative of the large population with SCA.⁵⁷

Aluko⁵⁸ also analysed electrocardiographs of 78 Nigerian children with SCA and found more of LVH (48%) than other changes which suggests that LV diastolic dysfunction predominates.

4. Echocardiography

Echocardiographic evidence of cardiac dysfunction in SCA patients in steady state has been reported in many studies and was demonstrated conclusively in the Cooperative Study by Covit et al.^2,38,59 These abnormalities occur mostly on the left side of the heart and include LV hypertrophy, increased left ventricular mass and high left ventricular dimension with LV diastolic dysfunction.²

5. Serum cardiac markers and cardiac function.

Although serum cardiac markers including troponin and creatine kinase have been in use in the past two decades for diagnosis of cardiac diseases, they are limited by comparatively rapid clearance rate from the blood.⁵⁸ Currently, there is growing interest in the use of newer longer-lasting biomarkers to identify and monitor individuals who are at risk of developing cardiac disease and who could benefit

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from preventive measures.⁶⁰ B-type natriuretic peptide (BNP) and the N-terminal fragment (NT-proBNP) of its pro-hormone are already well established as diagnostic and risk markers for heart failure.⁶¹ Troponin and Creatine kinase have also been shown not to be sensitive in detecting cardiac dysfunction in SCA patients because cardiac ischaemic injury which is more accurately detected by these two markers is rare in SCA.^2,62 NT-ProBNP is currently recommended for the assessment of cardiac function and diagnosis of heart failure by the European Society of Cardiology while cardiac troponin is retained for diagnosis of heart attack which is a sudden ischaemic event.⁶³ Although troponin is specific to the heart, its use for the diagnosis of chronic cardiac dysfunction as seen in SCA is unreliable because serum troponin levels normalize after 2 weeks of ischaemic injury.⁶⁴Creatine Kinase, apart from being much less specific to the heart, normalizes after 48-72 hours of cardiac injury making it unsuitable for diagnosis of the progressive and long-standing LV dysfunction seen in SCA.¹⁷ The discovery of NT-proBNP which, not only correlates with degree of LV dysfunction, but is released by ventricular wall stretch as seen in SCA patients is expected to popularize the use of this marker to assess LV function in SCA patients who have insidious LV dysfunction and cardiomegaly.^{2, 16, 65}

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Serum NT-proBNP and Clinical features of Cardiac Dysfunction

Previous studies have assessed the association between serum NT-proBNP and clinical features of cardiac disease but this marker has shown strong correlation with exercise tolerance test but not with any other single clinical parameter.^64,65

In a study that measured NT-proBNP levels in patients with chronic LV dysfunction, Tsutamoto et al⁵¹ found that elevated NT-proBNP levels predicted diminished exercise tolerance and poor prognosis in patients with left ventricular failure. In a study by Ekure et al⁵² involving 28 patients with congestive cardiac failure, no other clinical parameter except modified Ross score was a significant determinant of NT-proBNP levels (OR=1.341;p<0.001).

In a study involving 133 adult SCA patients in Nigeria aged 18-52 years, elevated NT-proBNP level of above 160ng/l was associated with severe functional impairment and inability to walk more than 300m in six minutes (95% confidence interval 1.5-32.6;

P=0.005) .¹⁶

BASIC BIOCHEMISTRY AND PHYSIOLOGY OF BNP RELATED PEPTIDES The human BNP gene is on chromosome 1 and encodes Pro-BNP which is the pro-hormone with 108 amino acids.²² Furin, a proteolytic enzyme splits Pro-BNP into the active BNP and inactive N-terminal part of the pro-hormone called NT-ProBNP.⁶⁶ The stimulus for synthesis and secretion of ProBNP is cardiac wall stress and stretch.²²

ProBNP is produced constitutively in both the atria and ventricles but in conditions associated with chronic myocyte stretch as seen in chronic heart failure states, there is up-regulation of ventricular proBNP synthesis.⁶⁷ Slight increase in BNP production also occurs after myocardial infarction possibly due to local stretch mechanisms around areas of old infarction.⁶⁸

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Active BNP binds to natriuretic peptide receptor type A (just like ANP) to increase cGMP and cause diuresis, vasodilatation, inhibition of rennin - angiotensin system and reduction in blood pressure.²² It is yet unclear whether the N-terminal portion (NT–ProBNP) has biological effect.²² BNP-related peptides are resistant to neutral endopeptidases which readily degrade ANP.⁶⁹ In addition, clearance of BNP is mostly by binding to an elimination receptor called natriuretic peptide receptor type C while renal excretion is the major means of clearance for NT-ProBNP.²² This may explain why the half-life of NT-ProBNP is much longer than that of BNP; 120 minutes and 20 minutes respectively.⁷⁰

NT–ProBNP and BNP are produced in equal amounts in healthy people but serum NT-ProBNP level becomes higher than that of BNP in individuals with heart failure.⁷¹ The cause of this concentration shift is unclear but it makes NT-proBNP more sensitive and more reliable than BNP in detecting ventricular dysfunction and heart failure.²² Suggested explanations for this shift are alteration of degradation, longer half-life of NT-ProBNP and reduced renal blood flow in heart failure which reduces NT-proBNP excretion.²²

FACTORS AFFECTING SERUM LEVELS OF NT-PROBNP

Apart from cardiac disease, the following factors may affect serum NT-proBNP levels.

i. Age: NT-proBNP levels are highest at birth, decrease rapidly in the first week of life followed by a more gradual fall to adult values at 18 years of age.⁷³ There is elevation after 50 years of age which is likely due to age-related decline in renal function.⁷⁰ Girls have slightly higher levels at between 10-14 years than boys but the upper limit of normal remain the same irrespective of gender.^53,73 Using 95th percentile as upper limit of normal, Nir et al⁷³ reviewed data from NT-proBNP levels of 690 children from birth to 18 years of age and found the upper limit of normal to be as follows:

22 o First 2 days of life: 12,000 ng/L o Days 3 to 11: 6,000 ng/L o 1 month to11 months: 650 ng/L o 12 months to 23 months: 400 ng/L o 2 to 5 years: 300 ng/L

o 6 to 18 years: 160 ng/L.

ii. Renal impairment: Significant kidney disease results in elevation of NT-proBNP because this marker is excreted mostly via the kidneys and this elevation becomes significant when estimated glomerular filtration rate (eGFR) is less than 60 ml/min/1.73 m² or patient has chronic kidney disease (CKD) stage ≥III.⁶⁰

iii. Obesity: Also reduces levels of natriuretic peptides especially BNP and much less NT-ProBNP due to abundant clearance receptors expressed on adipocytes which remove natriuretic peptides from circulation.⁷⁴ This may explain slightly lower levels of natriuretic peptides in pubertal girls than boys because average BMI is higher in girls than boys at puberty.⁷⁴

iv. Drugs: Beta blockers have been reported to increase serum levels of NT-proBNP while angiotensin converting enzyme (ACE) inhibitors and frusemide reduce the levels; thus their effects should be considered when interpreting serum NT-proBNP assay.⁷⁵ The mechanism of how these drugs alter NT-proBNP levels is poorly understood but it has been attributed to their ability to cause direct myocardial remodeling or alter renal clearance of NT-proBNP.^75,60

v. Infections: HIV and chronic lung infections have also been associated with elevated serum NT-proBNP levels. This may be because HIV as well as chronic lung infection could cause pulmonary artery hypertension and significant right-sided heart disease which increase serum NT-proBNP levels.^16,46,47

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vi. Markers of increased haemolysis: Aliyu et al¹⁶ showed correlation between elevated serum NT-proBNP levels and markers of increasing haemolysis such as decreasing haemoglobin, increasing serum bilirubin and increasing lactate dehydrogenase (LDH) levels. This study also showed that low haemoglobin concentration serves as a reflection of both the degree of anaemia and degree of haemolysis.¹⁶ Elevated serum NT-proBNP was also associated with low haemoglobin and LDH levels in these patients.¹⁶ LDH has five known isoforms released from many tissues but isoforms LDH-1 and LDH-2 released by erythrocytes during haemolysis make up about 50% of total LDH thereby making total serum LDH very sensitive in detecting increasing haemolysis in SCA .^16,76,77

vii. Iron Overload: Iron overload and myocardial haemosiderosis are associated with LV dysfunction and high serum NT-proBNP seen in haemolytic anaemias caused by thalassaemia but this is rare in SCA because SCA patients receive much fewer total life-time transfusions.^42,44 Reports have also shown that most Nigerian children with SCA had normal serum iron and ferritin levels but frequent or chronic transfusion therapy may cause iron overload and elevated serum ferritin which has been associated with elevated NT-proBNP.^3,5,15 Adamkiewicz et al⁷⁸ however reported that serum ferritin was unreliable as a measure of iron load or degree of haemolysis in SCA because SCA is an inflammatory disease and serum ferritin is significantly elevated if there is inflammation. This was supported by liver biopsy studies done by Karam et al⁷⁹ that showed a poor correlation between liver iron concentration (LIC) and serum ferritin (r=0.46).⁷⁹ The sensitivity and specificity of serum ferritin as high as ≥2500 ng/ml in predicting high liver iron content (≥7 mg/g) were only 62.5% and 77.8% respectively.⁷⁹ Total life-time transfusions however had correlation with liver iron content (0.41) which was

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similar to the correlation with serum feritin (r=0.46).⁷⁹ This has been supported by Drasar et al ⁸⁰ who recently reported that 20 units of life-time transfusions was as good a screening tool for iron overload (LIC:2 mg/g) as elevated serum ferritin (≥1000g/l). This is similar to earlier report by Olivieri et al ⁴⁴ that up to 18 life-time transfusions were required to cause significant myocardial iron deposit that can impair cardiac function and probably elevate serum NT-proBNP levels.

viii. Pulmonary artery hypertension: Pulmonary artery hypertension (PAH) which can develop from repeated chest infections or NO scavenging following haemolysis in SCA patients has been associated with elevated serum NT-proBNP.^8,16 PAH is diagnosed when right ventricular pressure (RVP) calculated by adding an assumed right atrial pressure of 10mmHg to the regurgitant jet gradient across the tricuspid valve exceeds 30 mmHg or if tricuspid regurgitation velocity (TRV) is above 2.5 m/s.^16,48 In a study involving 133 adult Nigerians with SCA aged 18-52 years by Aliyu et al,¹⁶ elevated serum NT-proBNP above 160 ng/L showed a 78% positive predictive value for pulmonary hypertension. Machado et al⁸ also reported a strong correlation between between serum NT-proBNP levels and TRV in SCA patients in USA although in the Nigerian study by Aliyu et al¹⁶ NT-proBNP concentrations did not correlate significantly with TRV.

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PREVENTION AND MANAGEMENT OF CARDIAC DYSFUNCTION IN SICKLE CELL ANAEMIA.

Heart failure is a common complication of SCA and contributes to its morbidity and mortality.⁸

In a study by Lagunju and Omokhodion⁸¹ involving 100 children aged 8 years to 12 years in University College Hospital Ibadan, severe anaemia was the third most common cause of heart failure (28%) after respiratory tract infection and intrinsic heart disease.

Among these children with anaemic heart failure, SCA (17.9%) was the second most common cause after malaria (57.1%).⁸¹

Transfusion of SCA patients in anaemic crisis with heart failure is generally accepted but blood transfusion for chronic anaemia when in steady state has remained controversial.⁵⁴ However, Josephson et al⁸² after a critical review of the literature of transfusion in patients with SCA, recommended red blood cell transfusion as first line therapy in patients with cardiac compromise. They recommended direct transfusion for those with symptomatic severe anaemia but exchange blood transfusion to reduce HbS fraction for patients with packed cell volume above 30% to improve blood oxygen-carrying capacity and cardiac function.⁸²

Lester et al,³⁵ after a serial echocardiography of 64 SCA patients with cardiomegaly reported reversal of cardiomegaly in the children receiving hypertransfusion therapy.

Controlling any other underlying cause of the cardiac impairment, such as pulmonary hypertension, may also improve ventricular function as seen with correction of anaemia which is an important underlying factor for cardiac disease in SCA.^16,35

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RELEVANCE OF PROPOSED PROJECT TO PRACTICE OF PAEDIATRICS It is now known that the heart is even more commonly affected in SCA patients than the brain with about 82% of patients having significant cardiac impairment in adulthood.^10,14

Current researches have identified controllable factors contributing to LV dysfunction in these patients and that prevention/early detection and treatment of LV dysfunction would reduce morbidity and mortality from heart failure among patients with SCA.^34,35

However, while transcranial Doppler is currently used routinely to assess risk for cerebrovascular accident in SCA patients, no such screening test is used in many countries, including Nigeria, for detecting any form of cardiac dysfunction in the teeming population of SCA patients.^9,11

In spite of readily available echocardiography in Europe and USA, serum NT-proBNP concentration is currently being used as a strong marker of cardiac dysfunction and risk of death.^4,29 This is because NT-proBNP, as a point of care test, is a simple, reproducible, and easy to use cardiac marker which is also a very sensitive ‘rule-out’ marker to exclude presence of heart failure. Also, NT-proBNP assay does not require the expertise and high cost associated with echocardiography which nevertheless remains the gold standard.⁴ Many studies have established the use of NT-proBNP to screen for heart failure and that it may also detect asymptomatic LV dysfunction because of associated ventricular stretch which is the major stimulus required for its secretion. Because LV dysfunction (and stretch) usually precedes frank heart failure, NT-proBNP may be useful for screening patients at risk of heart failure like children with SCA.^2,16,63 Ventricular stretch, not myocardial ischaemic injury, is the main pathway for development of so-called sickle cell cardiomyopathy and NT-proBNP has potential to serve as a useful point-of-care screening test for early identification and interventions to improve quality of life and reduce

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mortality in children with SCA.¹⁶Interventions that have shown efficacy are hydroxycarbamide (hydroxyurea), exchange blood transfusion, and rarely iron chelation therapy.^29,82

A related study done in Northern Nigeria showed that NT-proBNP concentrations correlated strongly with functional impairment without any significant correlation with pulmonary artery hypertension (assessed by tricuspid regurgitation velocity) although pulmonary hypertension is known to elevate serum NT-proBNP levels.¹⁶ This may mean that serum NT-proBNP level is more sensitive to ventricular dysfunction than pulmonary hypertension and there is need to investigate further. The study also relied solely on E/A ratio which is less reliable than E/E′ ratio in detecting diastolic dysfunction in hyperdynamic states like SCA.^16,35 This study used both E/A and E/E′ ratios to assess LV diastolic function.

Thus, this study sought to determine if serum NT-proBNP was significantly higher in asymptomatic SCA patients with LV dysfunction; and whether serum NT-proBNP increased with worsening echocardiographic indicators of LV dysfunction in SCA patients in stable state. The results from this study may indicate whether NT-proBNP will be useful to screen for and detect ventricular dysfunction in SCA patients before they develop frank heart failure.

Although wide-spread use of serum NT-proBNP assay may be limited by cost in resource poor countries, it could still be a relatively more affordable biomarker for cardiac complications and mortality in SCA than echocardiography.¹⁶ NT-proBNP ELISA kit for 96 samples costs about five hundred US dollars and the assay methods are simple without need for specialized training while the cheapest form of echocardiography, the hand-held echocardiographic machine, costs tens of thousands of US dollars and requires special

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training of echocardiographers.^16,72,75 In a way similar to the ‘NT-proBNP ticket for echo’

recommended by Cowie et al,⁷⁵ the use of a relatively affordable marker like NT-proBNP to first screen and identify patients who require echocardiography, will reduce the national cost of screening all four million SCA patients in Nigeria with echocardiogram.

This will also increase national coverage and detection of SCA patients who require additional treatments to improve cardiac function, quality of life and also reduce morbidity/mortality in SCA patients in Nigeria and Sub-Saharan Africa which have the largest burden of SCA but limited expertise and equipment for echocardigraphy.⁵

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AIMS AND SPECIFIC OBJECTIVES General Aim

The aim of the study was to assess the relationship between serum levels of N-terminal pro-brain natriuretic peptide and left ventricular function indices from echocardiography in children with SCA in steady state.

The Specific Objectives were to:

1. determine the proportion of children with SCA who have elevated serum NT-proBNP.

2. assess left ventricular function using ejection fraction and fractional shortening for systolic function; and E/A and E/E′ ratios for diastolic function.

3. correlate serum levels of NT-proBNP with ejection fraction, fractional shortening, E/A ratio and E/E′ ratio.

4. determine the association of serum NT-proBNP with pulmonary hypertension, body mass index, haemoglobin concentration, HIV status, serum unconjugated bilirubin, serum creatinine and lactate dehydrogenase.

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SUBJECTS AND METHODS Study Location

The study was conducted at the Paediatric Haematology clinic, Cardiovascular Laboratory and AIDS Prevention Initiative in Nigeria (APIN) Facility of the Lagos University Teaching Hospital, Idi-Araba, a tertiary health institution located in Surulere Local Government Area of Lagos State, South-West Nigeria.

The haematology clinic is run once a week by two consultant Paediatricians with about eight resident doctors. On a typical clinic day, about two children newly diagnosed with SCA and 20 previously diagnosed patients between 6 - 17 years of age attend the clinic.

In a six-month period about 100 patients (old and newly registered) aged 6-17 years of age attend the clinic when the repeat appointment visits are excluded. Commonest duration of next appointment for stable SCA patients in LUTH was three months.

Routinely, in the Haematology Clinic, the patient’s history is taken, physical examinations are carried out; and investigations including complete blood count, electrolytes, urea and creatinine, haemoglobin fractions, peripheral blood film, Hb electrophoresis and transcranial Doppler scan are requested. Also, daily folic acid and proguanil are routinely given to all SCA patients while oral penicillin V and pneumococcal vaccine are prescribed for children below the age of eight years.

Study Population

Sickle cell anaemia patients in steady state aged 6-17 years attending Sickle Cell Clinic in LUTH.