European Journal of Cardiovascular Medicine

Hemoglobinopathies are defined as qualitative defects in the structure of globin chains resulting in production of abnormal hemoglobin molecules. It is estimated that 7% of the world population are carriers of a severe haemoglobin disorder and 300-500 children are born with a severe haemoglobin disorder each year.

Sickle cell syndromes includes a group of congenital hemolytic anemias associated with the presence of Haemoglobin S. HbS so called because of the sickle shape it imparts to deoxygenated red cells is responsible for a wide spectrum of disorders. The sickle mutation substitutes thymine for adenine in the 6th codon of the beta gene [GAG –GTG], thereby encoding VALINE instead of GLUTAMIC ACID in the sixth position of the beta gene.

Sickle cell anemia was only barely known to exist in Kerala till the1980s, with occasional cases having been diagnosed in Calicut medical college among the Adivasi and Chetti

communities in Wayanad. M Feroze and KP Aravindan conducted screening studies in Wayanad district which has the biggest Adivasi population in Kerala. The study published in National medical journal of India found that the gene frequency of Haemoglobin S ranged from 0.019 in Kattunayakan to 0.196 in Wayanadan Chettis.[1] The survival of patients with sickle cell anemia seems to be higher in Kerala as compared to other states.

It appears that even small improvements in primary health care available to the population (as in Kerala) are sufficient to achieve this effect.[2]

A number of studies document that sickle cell anemia is associated with lower systemic blood pressures than controls and with a lower prevalence of systemic hypertension. [3-9] Potential mechanisms for the lower blood pressures in SCD patients include i] obstruction of the vasa recta in the kidney and repeated ischemia to the renal medulla, resulting in a distal renal tubular concentrating defect and hyposthenuria, [10] ii] lower body mass index in patients with SCD, [6] and iv] lower arterial stiffness. [11] The finding that patients with sickle cell disease have low blood pressure seems to counter recent pathophysiological studies suggesting that these patients develop endothelial dysfunction and systemic vasculopathy associated with reductions in no bioavailability. [11–18] However, blood pressures in SCD are higher than in thalassemia patients with a similar degree of anemia and there is an association between cerebrovascular accident and higher blood pressures in SCD even in a range of systolic and diastolic pressures that would be considered normal by conventional standards. [4,5] These studies raise the possibility that the normal range for blood pressure in patients with SCD should be lower than in healthy controls.

The purpose of the present study was to determine if relative systemic hypertension is

associated with renal function and coagulation abnormalities and adverse outcomes in sickle cell anemia.

The present study was conducted in patients who are diagnosed as sickle cell anemia in the various sickle cell clinics under the sickle cell project of the Department of Pathology,

Government Medical College, Kozhikode after getting clearance from the Institutional

Ethical committee. The study was a descriptive cross-sectional study for a period of 1.5 years from January 2017 to June 2018. Patients with comorbidities and pregnant females were excluded from the study.100 patients were included in the study with sample size calculated using the formula

N=4pq/d2

N: number of patients, p: prevalence (44%), q: (100-p), d: 10%

The patients were counseled regarding the testing for kidney function and thromboembolic complications and informed consent was taken in their mother tongue. The cases were confirmed by High Performance Liquid Chromatography in BioRad D10. In every case personal data and medical history was taken. Clinical examination was done and 5 ml venous blood was taken for laboratory investigations. Personal data was taken including name, age, gender, community, educational status, and address were collected. Medical history covered year of diagnosis, treatment with hydroxyurea, number of crises per year and admissions during preceding year. Blood pressure was recorded. The following laboratory tests were carried out:-

High performance liquid chromatography, Complete blood count by automated hematological analyzer -Sysmex 3 part XP100, Blood urea, Serum creatinine, Prothrombin time (PT), Activated partial thromboplastin time (APTT) in fully automated coagulometerDestiny Plus T coag.

OPERATIONAL DEFINITION

Blood pressure - Normal systemic blood pressure (<120 mm Hg systolic and <70 mm Hg diastolic), relative hypertension (SBP 120–139 mm Hg or DBP 70–89 mm Hg), or hypertension (SBP 140 mm Hg or DBP 90 mm Hg) based on the baseline blood pressures determined at study entry. Patients with systemic blood pressures > 140 mm Hg systolic or >90 mm Hg diastolic.[6] Serum Creatinine - Elevated serum creatinine concentration was taken to be 1.0 mg/dL. We chose this serum creatinine value as indicative of early renal involvement because among the cohort of hemoglobin SS patients < 40 years of age in the large CSSCD cohort, the 75^th percentiles for serum creatinine values were at or below 1.0 mg/dl. [19] Blood urea- 20-40 mg/dl is normal range.[19] Prothrombin time- normal range is 10-20 sec.[19 Activated thromboplastin time- 30-45 is normal.[19]

All the data collected were entered in Microsoft Excel sheet and was analyzed using SPSS

statistical software for Windows. Association between the various risk factors and outcomes were done by using chi square test and the level of significance was estimated with 95% confidence interval.

The following observations were noted:

• Age The age of the patients studied ranged from 2.50 years to 60 years with a mean age of 25.88 years and standard deviation of 14.24 years. Majority of our cases (~50%) were in the age group 10-29 years of age. (b) Gender Of the 100 patients studied, 56 (56%) patients were males and 44 (44%) patients were females. (c) Tribewise distribution of cases Of the 100 patients studied, majority 88 patients [88%] belonged to the Wayanadan tribal population and only 23 [23%] were non tribals including 11 Wayanadan Chettis. Among the tribal populations, most 42 [42%] patients were from the Paniya tribe, 23 [23%] cases were Kurumas, 6[6%] cases each belonging to Adiyas and the Katunayaka tribe. (d) Treatment History Of the 100 patients studied, 65 [65%] were on regular hydroxyurea therapy and the rest 35 [35%] were not on hydroxyurea.
• Fifteen of these cases were newly diagnosed and were promptly started on hydroxyurea regimen.

The remaining cases were provided counseling for the same.

In this study 56 patients [56%] required hospitalization in the past one year and the rest 44 of the patients had an apparently uneventful year. (e) Assessment of blood pressure

The systolic blood pressure of the study population varied from 80-190 mm of Hg with a mean of 113 mm of Hg and standard deviation of 21.78. Of the 100 patients studied, 60

[60%] had normal systolic blood pressure, 23 [23%] cases had relative hypertension and 17 [17%] cases were hypertensive.

The diastolic blood pressure of the study population varied from 50-100 mm of Hg with a mean of 72.56 mm of Hg and standard deviation of 12.22 mm Hg. Of the 100 patients

studied, 27 [27%] had normal diastolic blood pressure, 56 [56%] cases had relative hypertension and 17 [17%] cases were hypertensive. Of the 100 patients studied, 27 [27%] had normal systemic blood pressure, 55 [55%] cases had relative hypertension and 18 [18%] cases were hypertensive. (f) Grading of Anemia, The 100 patients involved in the study were divided into five groups, based on hemoglobin levels on their complete blood counts, according to the WHO classification of anemia viz. less than 4gm/dl, 4-6.9 gm/dl, 7-8.9 gm/dl, 9-10.9 gm/dl and >11 gm/dl. Forty-two patients [42%] had received blood transfusion in the past one year for anemia. Of the 100 people studied, the hemoglobin levels ranged from 2.7- 13.3 gm/dl with a mean hemoglobin level of 8.4 gm/dl and standard deviation of 2.2. 4 patients had very severe anemia, 21 [21%] patients had severe anemia, 27 [27%] patients had moderate anemia, 37 [37%] patients had only mild anemia and the rest 11 [11%] patients had hemoglobin levels within normal limits. (g) Distribution of Fetal Hemoglobin

The 100 patients involved in the study were divided into three groups based on their fetal hemoglobin levels viz. less than or equal to 10gm/dl, 11-20 gm/dl, and 21-30 gm/dl. Of the 100 people studied, the fetal hemoglobin levels ranged from 0- 28 gm/dl with a mean hemoglobin level of 13.4 gm/dl and standard deviation of 7. 35 [35%] patients had

fetal hemoglobin <10 gm/dl, 46 [46%] patients had fetal hemoglobin 11-20 gm/dl and the remaining 19 [19%] patients had fetal hemoglobin of 21-30 gm/dl. (h) Assessment of Kidney Function tests & coagulation function Out of the total 100 cases, 15 (15%) people had elevated creatinine levels [>1.0 mg/dL] and nine (9%) people had elevated urea levels [>40mg/dl]. Coagulation evaluation showed 9 (9%) people had elevated PT levels and nine (9%) people had elevated aPTT levels.

(Table /figure 1: Assessment of kidney and coagulation functions)

The above findings were compiled and the following inferences made:

The study showed statistically significant [P value=0] high incidence of crisis in the preceding year in patients receiving hydroxyurea treatment (Chi square = 16.44 [df=1]).

(Table/figure 2: Association of hydroxyurea with the number of crisis in the preceding year)

The study showed statistically significant [P value=0.002] higher levels of fetal hemoglobin in patients receiving hydroxyurea than in the patients not receiving treatment (Chi square = 12.458 [df=2]). (Table/ figure 3: Association of hydroxyurea with levels of fetal hemoglobin)

TABLES AND LEGENDS

Table 1: Assessment of kidney and coagulation functions. Assessment of kidney and coagulation functions showing mean/standard deviation and range of urea, creatinine, PT, APTT in the 100 cases.

Table 2: Association of hydroxyurea with the number of crisis in the preceding year. Association of hydroxyurea treatment with the number of crisis in the preceding year with P value, Chi square and degree of freedom.

Table 3: Association of hydroxyurea with levels of fetal hemoglobin. Association of hydroxyurea treatment with levels of fetal hemoglobin with P value, Chi square and degree of freedom.

Fifty six patients [56%] required hospitalization in the past one year for various sickle cell disease associated crisis. The types of complications documented in most studies are

skewed toward those associated with visible signs or physical complaints, such as pain crisis in the present study.[20] This study failed to show any statistical significance [P value= 0.060] between age and the number of crisis in the previous year.

Hydroxyurea and levels of fetal hemoglobin

The study showed statistically significant [P value=0] higher incidence of crisis and the need for blood transfusions in the preceding year in patients receiving hydroxyurea than

in the patients not receiving treatment, contrary to its widely accepted efficiency as the most effective disease modifying therapy capable of reducing the total duration and need of hospital stay for both adults and children with sickle cell anemia. [21] This spurious association could be attributed to the fact that people with more severe disease report to the sickle clinics resulting in sampling errors, the relatively high number of newly diagnosed cases that had just been initiated on hydroxyurea therapy and also the lack of follow up required for corroboration of the various outcomes in the study. Hydroxyurea has been shown to potently increase HbF levels, which is its main mechanism of action. Our study also proved this positive association with statistical significance [P value=0.002]. But our study failed to demonstrate any statistically significant association between the HbF levels and the various outcomes, possibly because of the small sample size.

Blood pressure

No statistically significant association between hemoglobin levels and blood pressure was found. Our observation is surprising. A prior study that demonstrated a positive association between hemoglobin levels and BP measurements speculated that the underlying physiology is related to anemia-associated systemic vasodilation and subsequently decreased systemic vascular resistance. [1]

Thus, increased hemoglobin levels may act as an independent risk factor for hypertension. Additional monitoring and assessment may be indicated for children with BP measurements above the 95^th percentile including ambulatory BP monitoring and evaluating the presence of

physiologic nocturnal drops in BP. These cases may be more susceptible to complications associated with relative high BP. The study could not establish the previously suggested fact that relative hypertension and crisis are associated [P value=0.112].

The study could not establish that hypertension is associated with renal insufficiency and coagulation abnormalities, possibly due to our lack of follow up of the hypertensives and the large number of younger patients in our study group.[4,22,23].

According to studies of Victor R  Gordeuk et al 44% had Systolic BP of 120–139 mm Hg or Diastolic BP of 70–89 mm Hg (classified as relative hypertension) and 10% had Systolic BP ≥140 mm Hg or Diastolic BP ≥90 mm Hg (classified as hypertension) where as in this study23 [23%] cases had relative hypertension and 17[17%] cases were hypertensive which is very much comparable.(19)

Renal function tests

Out of the total 100 cases, 15 (15%) people had elevated creatinine levels [>1.0 mg/dL] and nine (9%) people had elevated urea levels [>40mg/dl] . This is similar to findings of al Naama et al but who found no significant difference in the level of both analytes.[24,25] . In contrast to comparison with Victor R Gordeuk et al this study showed no significant correlation of systemic hypertension and raised levels of Urea and creatinine .

These findings could be due to low sample size. .[19] There are also other studies proving renal impairment in patients with sickle  cell anaemia which is more compared to sickle cell trait like Enoch odame Anto et al , Amin S Banaga et al .They even suggested significant morbidity and mortality in sickle cell anaemia patients due to end stage renal disease .(32,33)

Surprisingly however, this study failed to establish any other significant association between hydroxyurea treatment or levels of fetal hemoglobin with the renal function tests or any age dependent variations. This emphasizes the fact that hydroxyurea is not effective in all patients with sickle cell disease (SCD), and those who fail to improve while on hydroxyurea therapy may represent a subgroup of patients, possibly with more

severe disease and organ damage. Low Hb levels before hydroxyurea therapy could be indicator of poor outcomes in patients with SCD on hydroxyurea. Alternative approaches that should be considered in this group of patients include the institution of hydroxyurea therapy at earlier ages, administration of higher hydroxyurea doses, and combination therapies particularly with erythropoietin, because a potentiating effect has been described between the two agents.[26,27] Systolic BP is a strong predictor of renal dysfunction; however, we found fewer than expected instances of renal insufficiency associated with hypertension [P value >0.005].[28]

Coagulation functions

Increased PT(9%) and APTT(6%) levels in patients with sickle cell haemoglobinopathy are in concordance with the findings of David sackey et al; who also reported either elevated PT or both PT and APTT levels in HbSS patients in their studies.[30] .Similar other studies include Shiek awoda et al , which showed a significant reduction in abnormal coagulation profile by treatment with hydroxyurea or omega 3 fatty acids.(31)

There are certain possible explanations for the significantly increased PT and APTT values observed in our study. The first explanation could be hepatocytic dysfunction frequently observed in sickle cell disease. Decreased synthesis of clotting factors and vitamin K deficiency have also been reported in HbSS patients.[29] Factor concentration is not measured in the present study, neither liver function test was performed in all the patients. Future research is required to study the clinical significance of increased PT and APTT seen in these patients.

Limitations:

Low sample size , short period of followup are the major drawbacks of this study.furtherdetailed is suggested for better understanding.

The survival of the patients with sickle cell anemia seems to be higher in Kerala as ompared to the other states due to early and aggressive management by multidisciplinary teams. Routine check up can help to alleviate the general well being of the sickle cell anemia patients. If crisis is not managed in time, very fast progression to end organ damage and even sudden death can occur.

The relatively high prevalence of relative hypertension, renal function tests and coagulation abnormality in sickle cell anemia patients suggests that they should be monitored regularly for early detection and prevention of adverse effects on disease outcome.

Acknowledgement

Department of Pathology, Government Medical College, Kozhikode, Kerala, India & staff of the various sickle cell clinics under the sickle cell project for their whole hearted

and timely technical help in the completion of this work.

Declaration Of Interest: There is no conflict of interest.

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