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Table 6. TCD risk classification based on STOP Trial criteria

TCD Class Number of cases Percentage

Standard risk 84 84

Conditional risk 11 11

High (abnormal) risk 3 3

Inadequate 2 2

Total 100 100

Up to 3% of the studied patients were found to have high risk velocities, 11%

had intermediate risk while 84% had standard risk. Two percent (2%) of patients, however, were categorised as inadequate. The percentage of patients with TAMMV of 170cm/sec or above (conditional or high risk), was 14%. Figure 14 shows a bar diagram depicting these findings.

Figure 14. Stroke risk classification based on TAMMV 84

11

3 2

0 10 20 30 40 50 60 70 80 90

Percentage of patients (%)

Stroke risk classification based on TAMMV

Standard Risk Conditional Risk High Risk Inadequate

Figure 15. TCD velocities (TAMMV) in the four major intracranial blood vessels

In order to determine the correlation between haematological parameters and TCD velocities, a simple linear univariate regression analysis was made with the haematological parameters as independent variables and the TAMMV in the left and right MCAs. Results of univariate analyses are depicted in Table 7.

0 20 40 60 80 100 120 140 160

MCA ACA PCA tICA

TAMMV (cm/sec)

Right Left

Table 7. Summary results of univariate regression with haematological parameters as independent variables and TAMMV as dependent variable

Independent variable Level of significance (p-value) Comment

Right MCA

TAMMV as

dependent variable

Left MCA TAMMV as the dependent variable

Age (months) 0.562 0.167 Not significant

Sex 0.416 0.182 Not significant

Haematocrit (%) <0.001 <0.001 Significant

Hb (g/dL) <0.001 <0.001 Significant

Total WBC (x10⁹/L) 0.001 0.008 Significant

ANC (x10⁹/L) <0.001 <0.001 Significant

PLT (x10⁹/L) <0.001 0.004 Significant

Retic count (%) <0.001 0.012 Significant

HbF level (%) <0.001 <0.001 Significant

Serum LDH (U/L) <0.001 <0.001 Significant

Serum total bilirubin (mmol/L)

<0.001 <0.001 Significant

Direct bilirubin (mmol/L) 0.002 0.007 Significant

There was statistically significant positive correlation between the TAMMV and total WBC count (R² = 0.112, p-value = 0.001, b1 = 1.986), ANC (R² = 0.277, p-value < 0.001, b1 = 4.439), platelet count (R² = 0.195, p-value < 0.001, b1 = 0.078), reticulocyte count (R² = 0.137, p-value < 0.001, b1 = 2.655), serum LDH (R² = 0.513, p-value < 0.001, b1 = 0.018) and serum total bilirubin (R² = 0.176, p-value < 0.001, b1 = 0.267), while TAMMV was negatively correlated with haematocrit (R² = 0.224, p-value < 0.001, b1 = -2.906) and HbF level (R² = 0.529, p-value < 0.001, b1 = -4.189). There is no significant correlation between TAMMV and age (R² = 0.004, p-value < 0.562, b1 = -0.402) and sex (R² = 0.007, p-value < 0.416, b1 = 4.539) of the patient.

Figure 13 shows a curve fit for univariate correlation between TAMMV in the right and left MCA and PCV. The rest of graphs and model summaries are included in Appendix V.

A B

Figure 16. Curve fit for univariate correlation between TAMMV in the (A) right and (B) left MCA (cm/sec) and PCV (%) showing negative correlation

The linear regression equation for the univariate model is as follows:

Y = b1 (X) + C

Where Y = the dependent variable b1= intercept

X = the independent variable

C = Constant

R² is the coefficient of determination, a measure indicating the goodness of fit of the model, as the proportion of total variation outcomes explained by the model.

5 DISCUSSION

Transcranial Doppler ultrasonography (TCD) has emerged as a powerful tool for assessing stroke risk in patients with SCD. Its usefulness stems from its flexibility, non-invasive nature, the use of relatively simple equipment, being inexpensive and, with the appropriate training, relatively easy to perform. With its debut into clinical practice, primary prevention of stroke in these children became possible, thereby reducing the burden of one of the most feared and debilitating consequences of SCD, with a potential of improving the quality of life of patients living with the disease. To our knowledge, this is the first descriptive report of TCD abnormalities and correlation with haematological characteristics in children with SCA from northern part of Nigeria.

Consistent with the intention of the study design to evaluate the TCD characteristics of the most vulnerable group for stoke in SCD, the patients’ ages ranged between 2 and 16 years. Ischemic strokes due to SCD have been found to be uncommon before the age of 2, while the incidence after 16 years appears unremarkable.²⁵ The cerebral vasculopathy that results in ischemia and infarction is thought to develop gradually over time and appears to manifest as stroke from the third year of life, partly explaining the rarity of such events before the age of 2. Elevated HbF levels may offer additional protection against severe manifestations in infancy.¹⁰² It is, however, not known exactly why incidence of stroke diminishes in the third decade of life among these patients.

The study patients were selected based on previous diagnosis of SCA using Hb electrophoresis on cellulose acetate membrane at alkaline pH, confirmed by sickle solubility test. The diagnosis was further validated by Hb quantitation using HPLC. Patients with other forms of haemoglobinopathies such as HbSC were excluded from the study. No patient was encountered with haematological features suggestive of HbSβ-thal phenotype based on blood counts, blood film, red cell indices or HPLC during the study. Although patients with HbSβ⁰ -thalassaemia generally have stroke risk and TCD abnormalities similar to those with homozygous sickle cell disease²¹⁰, these patients were excluded partly because of lack of available facilities to conclusively make the diagnosis.

Although the demographic composition of the studied patients was expectedly dominated by the Hausa and Fulani ethnic groups as the most dominant tribes in northern Nigeria, the appearance in the study sample of other ethnic groups such as Yoruba, Igbo, Nupe, Igala and others, attests to the cosmopolitan nature of the studied population.

This study found most patients to have been diagnosed with SCA at infancy, the median age at diagnosis being 12 months (range, 3 to 156 months). This is lower than reports from Lagos and Ibadan with 27 and 24 months respectively and much lower than reports from Europe.^221–223 In Zaria, North-western Nigeria, Mamman et al found a mean age at diagnosis of 3.8years and 3.6years for transfused and untransfused SCA patients respectively.²²⁴ This has been attributed to superstitious beliefs, especially the Abiku phenomenon prevalent in

many African societies. Age at diagnosis is an index of severity in SCD, with earlier age at diagnosis predictive of severer clinical phenotypes, generally associated with more recurrent VOCs and occurrence of adverse complications later in life.^225–227 In this study, age at diagnosis was determined historically from parents/care-givers using a questionnaire.

The average lifetime hospitalisation found in this study of 2.49 per patient per year is higher than 1.52 reported from North America.²²⁸ It was also observed that more than half of the studied patients had an average of 1 to 5 hospitalisations in their lifetime. About 2% of these patients had lifetime hospital admissions exceeding 10. Frequency of hospitalisation has been used by various researchers as a surrogate for disease severity and quality of life in SCD^229–231 The difference in number of hospitalisations between our patients and those in America might be attributable to better socioeconomic conditions, better application of preventive measures or better accessibility to and utilisation of quality healthcare in developed countries. Higher prevalence of infectious diseases like malaria and pneumonia as well as ambient environmental conditions as precipitants of sickle cell crises in this environment might also partly contribute to this observed difference. The significance of this finding is that, SCD poses an added challenge to our financially deprived health system¹⁴² which is burdened by limited infrastructure, human resources and budgetary allocation. Although frequent hospitalisations often translates into poor quality of life, more recently, some authorities have advocated the use of “rehospitalisation

within 30 days of discharge” as a better indicator of quality of life, (and 14-day rehospitalisation as that of quality of care related to hospitalisation), than number of hospitalisations alone.228,232,233 Length of hospital stay (LOS) is also commonly used for the same purpose. This study is cross-sectional; both 30-day rehospitalisation and LOS will be better quantified under a prospective study design.

Frequency of acute painful episodes is another established and widely used marker of disease severity in SCD. In this study, acute painful episode was defined as any episode of pain that warrants a visit to a hospital for treatment.

This definition was used because SCD is a chronic disorder and there is evidence to suggest that sufferers experience some degree of pain on almost daily basis without presenting to the hospital.²³⁴ It is generally presumed that many patients manage mild to moderate pain episodes at home such that any visit to a healthcare facility because of pain would be construed as being ‘severe enough’.

Approximately half (48%) of the patients interviewed in this study have pain episodes between 1 and 3 per annum. This is in sharp contrast to the pain rates reported in the Cooperative Study of Sickle Cell Disease (CSSCD) where up to 42% of patients in the same age range with our patients had between 0 and 1 episodes per year.⁸⁵ Curiously, up to 2% of patients in the present study were found to have annual pain frequency in excess of 10 episodes in contrast to 0%

among the CSSCD cohorts of the same age range. It thus appears that patients in this study experience more frequent painful episodes than their counterparts

elsewhere. This finding further corroborates the earlier observation of more frequent hospitalisations in these patients, given that VOC is documented to be the most common cause of hospital admissions among patients with SCD.²³⁵ Indeed, this study might have underestimated the pain frequency in these patients since it relied upon questionnaire interviews on past events which might be beset by bias of recall. A prospective study design using pain diary would probably capture such information more accurately.

The study also showed that blood transfusion is quite common among these patients, as almost half (49%) of all the studied subjects have had at least one blood transfusion in their lifetime. The average of about 2 transfusions per patient suggests that blood transfusion is a fairly common practice in children with SCA in this part of the world. Considering the upper age limit of 16 years in this study, this frequency translates roughly into having a probability of at least 2 transfusions before the child celebrates 16^th birthday. This figure is quite alarming and places children at increased risk of alloimmunisation and TTIs at an early age. This is in addition to putting further constraints on already meagre blood supply in a resource-limited setting. Known indications for blood transfusion in SCD range from simple “top-up” transfusion in severe anaemia due to malaria, hyperhaemolysis, aplastic and other forms of sickle cell-related anaemic crises. Other indications include exchange blood transfusion (EBT) in the treatment of ACS, acute CVA as well as chronic transfusion in primary and secondary stroke prevention among others.²³⁶ While blood transfusion is

therapeutically beneficial and sometimes lifesaving in many situations, it is not without immediate complications and long-term sequelae. Proper and judicious use of blood in these children will go a long way in ameliorating such complications as well as reduce encumbrances on our health resources.

The main haematological findings in this study are generally consistent with previous reports of steady-state haematological parameters inpatients with SCA in Nigeria.^236,237 These results largely depict moderate anaemia, neutrophilia with absolute leucocytosis, reticulocytosis and platelet counts on the upper limits of normal. Anaemia in SCA is typically multifactorial; contributors range from chronic haemolysis, to secondary folate depletion (when the vitamin is not adequately supplemented), and anaemia from increased susceptibility to parasitic and bacterial infections among others. In tropical endemic countries like Nigeria, malaria is an important contributor to development of anaemia in these children.²³⁸ This is in addition to causes related to poor nutrition and high prevalence of helminthic infestations. Severity of anaemia in SCD is an established predictor of adverse clinical events including ACS, pain, stroke and death.²³⁸ Similarly, leucocytosis and neutrophilia are known manifestations of steady-state SCD resulting from multiple mechanisms including structural and functional asplenia/hyposplenism, chronic inflammation and bone marrow response to haemolysis.^239,240 The clinical implications and significance of leucocytosis and neutrophilia in the pathogenesis of SCD and its complications have long been demonstrated by numerous observational and mechanistic

studies. Elevated ANC in steady-state has been shown to strongly correlates with occurrence of ACS, silent and overt strokes, and early SCD-related death ^239&

citations therein. There is evidence to suggest that reduction in number of leucocytes by therapies such as EBT and hydroxyurea partly ameliorate the severe clinical manifestations of the disease. Leucocytes are therefore believed to be active participants in the evolution of symptoms and complications of SCD rather than innocent bystanders. In spite of this body of evidence, however, exactly why would leucocytes play such a critical role in the pathogenesis of a primarily red blood cell disorder caused by a SNP, remains a mystery.

Thrombocytosis is another feature of steady-state SCD and represents a combined manifestation of bone marrow response to haemolysis, autosplenectomy and chronic inflammation.²³⁹ Elevated platelet count contributes to the hypercoagulable state seen in SCD and correlates well with other markers of disease severity. The finding in this study of platelet counts in the upper limit of normal is consistent with existing knowledge of steady-state SCD.

Ordinarily, the reticulocyte count in this study is elevated and is reflective of erythropoietic response to haemolysis.^2,218 But assessment of marrow efficacy to haemolysis is indicated by the reticulocyte index or corrected reticulocyte count which in this study is suggestive of a relative reticulocytopenia.²⁴¹The lower reticulocyte index observed in this study may be attributed to background malnutrition, subtle renal dysfunction with sub-optimal erythropoietin levels, inflammation or folate deficiency, among others.²⁴¹ In SCD, increased

steady-state reticulocytosis has been associated with VOC, hospitalisation, stroke and death.^242–244 It has been established that young RBCs released during the course of haemolysis (stress reticulocytes) exhibit increased complement of adhesion molecules on their surfaces leading to enhanced interaction with vascular endothelium. This triggers events that culminate in sickling, vasoocclusion and vasculopathy.²⁴⁴

Of particular importance is the influence of HbF levels on clinical severity of SCD. It has been well established that an elevated level of HbF is associated with fewer VOCs, fewer hospitalisations and longer survival in patients with SCD.

This finding is prompted by the observation of a generally milder clinical phenotype among patients who inherited haplotypes with higher HbF levels.

High HbF has also been speculated to reduce the occurrence of both silent cerebral infarctions and overt strokes in SCD.¹⁰¹ This study found a mean HbF of 7.08 ± 5.44%, a level largely consistent with the Benin haplotype which is the predominant haplotype encountered in Nigerian patients.^245,246 Sickle cell patients with this phenotype are known to have intermediate clinical severity, being more severe than the Saudi Arabian haplotype but generally milder than the Bantu and the Central African Republic (CAR) haplotypes. Increasing the HbF level is the main target of several established or experimental disease-modifying therapies in SCD including Hydroxyurea.

Recent insight into disease mechanisms of SCD is increasingly recognising the role of chronic intravascular haemolysis in the pathogenesis of some of its

complications. Serum LDH concentration has now emerged as an important marker of haemolysis in SCD and the quartile distribution of LDH is now frequently used to categorise patients into clinical subphenotypes such as chronic hyperhaemolysis (represented by the top quartile of LDH distribution) or viscosity/vasoocclusion (represented by the lower quartile) subphenotypes.

Taylor and colleagues²¹ have suggested that these clinical groups have distinctassociated spectrum of complications including ischemic stroke, and that steady-state serum LDH measurements might be used to predict those patients at risk of these presentations. This categorisation is important because it helps clinicians to anticipate complications and plan appropriate treatment.

Interestingly, this study found a mean steady-state LDH concentration of 1445.60

±

1070.80 U/L with almost half (up to 47%) of individuals having their LDH values falling above the upper quartile of 1938.25 U/L. This is significantly higher than both 356 U/L found in Saudi Arabia ²⁴⁷ and 875 U/L found in the UK

248. Within the top quartile of the LDH distribution in this study, the mean LDH value of 2709.51 ± 685.063 U/L found is significantly higher than both 610.6 U/L reported in the American National Institutes of Health (NIH) and 680.8 U/L in the CSSCD cohorts of the same category.²¹ This finding is significant and may be the result of confounding by LDH released from other sources notably the liver, since patients with preexisting hepatopathy were not excluded from this study. In hindsight, excluding these patients by assessing and removing those with concomitantly elevated serum alanine aminotransferase (ALT) would

significantly reduce this effect. Within the ambit of this limitation, however, the finding suggests that patients in this environment have significantly higher steady-state haemolytic rates than their counterparts elsewhere, and thus disprove our earlier hypothesis of a lower haemolytic rate in this environment as the reason for the observed lower prevalence of TCD abnormalities. Considering the concomitant finding of higher pain rates in these same patients (vide supra), it would therefore appear that patients with SCA in this environment probably have a unique phenotype, characterised by both high pain rates and high haemolysis rate suggesting a possible mixed vasoocclusive-haemolytic phenotype. It would be interesting to explore this phenomenon further in more elaborate studies involving much larger sample size than this, and controlling for extraneous sources of elevated LDH, in order to validate these observations.

Consistent with what has been reported by other similar studies³³, this study found the highest TAMMV to be on the MCA. This is followed by ACA, PCA and tICA in that order. The usual left to right variation in the velocity was also observed in all the intracranial vessels insonated except for the left ACA where the blood flow velocity was found to be higher than the right. The reason for this aberration is, however, not immediately clear. TAMMV measured in the arteries of the Circle of Willis has been shown to predict individuals at high risk of developing ischemic stroke among children with SCD, and its significance in prevention of stroke has been widely acknowledged. Based on the STOP trial criteria, the TAMMV can be used to categorise patients into three risk groups:

the standard risk, when the TAMMV in any of the insonated vessels is below 170cm/sec which confers a 2% risk of CVA to patients; conditional risk, when the velocity is between 170 and 199cm/sec carrying 7% risk of stroke, while high risk is seen when the TAMMV is 200m/sec or over, conferring 40% risk of stroke to sufferers.¹⁵¹ The significance of this observation is that first stroke can be prevented if children in the high risk category are subjected to chronic transfusion regimen or other similar modalities such as hydroxyurea therapy.

Adams and his colleagues²⁸ have observed that up to 90% of stroke might be prevented if this approach is taken, and their study provided level-1 evidence for the efficacy of prophylactic blood transfusion in primary stroke prevention in children at risk.

Although some reports have found the non-imaging TCD protocol that was used in the STOP trial to be reproducible with the conventional duplex imaging machine, some disparity has also been observed between the TAMMV as measured with the two techniques, with the later technique generally giving velocity readings up to 10% lower than those acquired using the former protocol.

This apparent difference has led to suggestions that lower threshold of velocities be assigned to stroke risk categories, and a 10% correction to be routinely applied to readings obtained with the imaging technique. Going by this, McCarville and his colleagues²⁴⁹ have proposed 180cm/sec or more to be considered abnormal velocity with the duplex (imaging) procedure, 153-179cm/sec as conditional, while <153cm/sec as standard risk whenever the

conventional imaging technique is used, thereby effectively modifying the STOP trial criteria. Padayachee et al²⁵⁰have, however, pointed out the danger in this approach, suggesting that since the two techniques closely correlate with each other with little overall systematic bias, routinely applying this correction to the STOP trial velocities would only increase the number of patients categorized as abnormal or conditional. This would lead to inappropriate selection of patients for chronic transfusion programs. They further suggested that the observed differences are largely attributable to physiological variations such as changes in pCO2 levels, and thus recommended that centres validate their TCD velocities for classification of stroke risk in order to avoid this potential dilemma. In the present study, the non-imaging technique was used to measure the TAMMV in the major arteries of the Circle of Willis which, essentially, is the same protocol used in the STOP trial, hence there was no need to apply any correction for comparison with similar studies.

This study has also further confirmed a lower prevalence of abnormal TCD velocities in Nigerian children with SCA compared with their counterparts in North America, with a finding of 3% prevalence of TAMMV ≥200cm/sec in the present study, being similar to the 4.7% found in Ibadan.³³ Both findings are, however, lower than the 9.3% found in STOP-trial.¹⁶⁹Although the finding in the present study is similar to the American BABY-HUG study where a prevalence of 2% abnormal TCD was found²⁵¹, the study subjects in the BABY-HUG were much younger (less than 2 years of age) while our sample size is much smaller.

A Kenyan study²⁵² of 105 children with SCA found 0% prevalence of abnormal TCD velocities. The reason for this low prevalence of abnormal TCD velocities in Nigerian children with SCA is not clear, and is intriguing especially given that we have higher prevalence of stroke in Nigeria than in America. Lagunju et al³³ had earlier speculated that it is quite possible that Nigerian patients with SCA may have relatively lower thresholds of TCD velocities at which they develop stroke compared to Americans, or that stroke might have already occurred in many patients with abnormal TCD velocities in this environment before the patients get screened. Although these hypotheses are quite plausible, appropriately and specifically designed long-term prospective studies are needed to verify the propositions.

In order to gain insight into the relationship between haematological parameters of these patients and cerebral blood flow velocities as captured by the TAMMV in the major cerebral blood vessels, a simple linear regression analysis was performed with the TAMMV in the right and left MCAs as the dependent variables, and haematological parameters as independent variables. The finding of significant correlation between the measured TAMMV and haematological parameters is in keeping with findings of other similar studies242,253,254 and with the existing knowledge of pathophysiology of neurological complications in sickle cell disease. This model was built using the TAMMV in the right and left MCAs because it was in those vessels the most significant abnormalities were found. Analysis of the relationship between TAMMV and the haematological

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