Comparative sonographic assessment of the spleen in individuals with and without sickle cell anaemia in

Background: Sickle cell anaemia (HbSS) is a major public health problem in Sub-Saharan Africa and commonly affects the spleen, with associated increased morbidity and mortality. This study aimed to evaluate the ultrasonographic findings of the spleen in patients with HbSS in steady state and to compare findings with those of individuals with normal adult haemoglobin (HbAA). Methods: This was a cross-sectional comparative study. Haemoglobin phenotypes were determined using haemoglobin electrophoresis. The spleen was scanned using transabdominal ultrasonography. Splenic size and parenchymal echotexture were assessed. Data was analysed with SPSS software, version 20. Results: One-hundred-and-twenty participants were recruited for the study, made up of 60 patients with HbSS and 60 age-and-sex-matched individuals with HbAA. They were made up of 54 (45%) males and 66 (55%) females, aged 2 to 32years, with mean age of 15.78 ±7.2 years for HbSS and 15.80 ±7.5years for HbAA participants. Thirteen (21.7%) patients with HbSS had autosplenectomy, 7 (11.7%) had splenomegaly, 6 (10%) had shrunken spleens, while 34 (56.7%) had normal sized spleens. All the participants with HbAA had normal splenic size with age. Of the 47 patients with HbSS whose spleen were visualized, 20 (42.6%) had abnormal splenic echotexture, whereas all the 60 (100%) participants with HbAA, had normal echotexture. There was significant difference in splenic echotexture in patients with HbSS compared to those with HbAA (p <0.05). Conclusion: A number of patients with HbSS have abnormal splenic size and echotexture compared to those with HbAA. There should be adequate follow up of patients with HbSS to prevent possible resultant splenic complications.


Introduction
The spleen, which is the largest lymphoid organ in the body plays an important role in the body's immune response, quality control for senescent or 1,2 altered erythrocytes.Sickle cell anaemia (SCA, HbSS) is one of the wide range of pathologic 3 disorders that commonly affects the spleen.Sickle cell anaemia is a hereditary disorder of haemoglobin caused by point mutation of the haemoglobin gene with resultant substitution of th valine for glutamic acid at the 6 position of the beta globin chain with resultant abnormal haemoglobin (HbS).This leads to abnormally sickle-shaped and rigid red blood cells under hypoxic condition, with Sickle cell anaemia constitutes a major public health problem in Nigeria, a country with the top sickle cell endemic country in Africa as well as the country with the highest burden of sickle cell anaemia in the 5,6 world.Homozygous inheritance of Haempglobin S is known as sickle cell anaemia and heterozygous inheritance of Haemoglobin S with other abnormal 6 haemoglobin, all constitute sickle cell disease.One of the most frequently affected organ in sickle 7,8 cell anaemia is the spleen.This is as a result of the intrinsically tortuous and sluggish microcirculation in the spleen which enhances prolonged vascular transit time, hence favouring red blood cell sickling with repeated vascular occlusions and multiple 4 , 7 , 9 s p l e n i c i n f a r c t i o n s .
T h e s p l e e n i s morphologically normal at birth in sickle cell anaemia, but as patient's age advances, it experiences a sequence of parenchymal and dimensional alterations, as a result of repeated and 2,7 progressive injury.The spleen usually enlarges during infancy and childhood, then shrinks gradually as the patient increases in age, due to repeated episodes of vaso-occlusion and 7 , 1 0 i n f a r c t i o n .T h i s e v e n t u a l l y l e a d s t o autosplenectomy which usually occurs between the 10 ages of 8 and 10 years.However, this is not always the case, as splenomegaly, in some cases, can persist 7,10 into adulthood.and is commonly seen in patients 4,6 with SCA in Africa.Several studies, have reported racial, regional and ethnic variation in splenic changes in patients with SCA.When compared with sickle cell anaemic patients with autosplenectomy, the presence of persistent splenomegaly in adult may indicate a different severity of disease as patients with autosplenectomy are more likely to show evidence of greater immune dysfunction due 11 to absence of the spleen.Occasionally, patients with persistent splenomegaly may develop signs of hypersplenism, acute splenic sequestration and splenic abscess, which are associated with 7,10 morbidity and mortality.As a result, continuous splenic monitoring and early detection of splenic pathologies using ultrasonography will enable caregivers of HbSS patients to adopt appropriate treatment and preventive measures.Interestingly, sonographic findings of the spleen in HbSS are so characteristic and knowledge of the ultrasonographic findings of the spleen in HbSS is essential as the radiologist may be the first to draw attention of the craegivers to a case of HbSS in an unscreened individual when abdominal ultrasonography for other diseases are being carried 12 out.Ultrasonography is a more reliable and accurate method for measuring the actual splenic 13,14 size as well as assessing the splenic appearances.Ultrasonography is a simple, affordable, noninvasive, non-ionizing, repeatable, easily accessible and readily available technique of imaging the spleen.The aim of this study was to compare the various spectra of ultrasonographic findings of the spleen in patients with HbSS in steady state with that of ageand sex-matched individuals with haemoglobin AA.

Study design and study site
This was a cross-sectional comparative study conducted from May to October 2020 at Alex Ekwueme Federal University Teaching Hospital, Abakaliki, (AEFUTHA) Ebonyi state, South East Nigeria.

Study population
Study population was made up of patients with HbSS in steady state who attended Sickle Cell Clinic of AEFUTHA during the study period and a control group of age-and sex-matched individuals with HbAA recruited from among staff, medical students, patients' relatives and children that came for immunization.

Inclusion and exclusion criteria
Inclusion criteria for study group were: known HbSS patients, aged 0 to 60 years, and in steady 4 state.Steady state was described as a time when there had been no recent episodes of acute illness, or discomfort for at least 4 weeks and no blood 4 transfusions in three months prior to recruitment.Exclusion criteria for the study group included: known HbSS patients who had undergone surgical splenectomy, patients in crisis or with any form of acute illness, patients over the age of 60years, patients with any chronic illness not related to sickle cell anaemia (example: patients with any congenital malformation, diabetes mellitus, and chronic liver disease), patients on Hydroxyurea therapy, patients who had bone marrow transplantation and patients who are pregnant.The inclusion criteria for the control group were subjects with hemoglobin AA within the age range of 0 and 60 years.The exclusion criteria for HbAA participants were those with any chronic illness or systemic disease, those who have had surgical splenectomy, or pregnant.

Sample Size
This was calculated using the formula for cross 15 sectional study (Cochran's formula ) , and a HbSS 16 prevalence rate of 3.5% from a previous study.A sample size of 52 was calculated using the formula: Where, N is the required sample size, Z is the standardized normal deviation usually set at 1.96, which corresponds to 95% confidence level, P is the prevalence rate of the condition and D is the desired level of precision set at 0.05.However, 60 participants were enrolled for each group, making a total of 120 participants.

Sampling Technique
Participants were selected using the simple random sampling technique.

Ethical issues
Ethical clearance for this study was obtained from the Health Research and Ethics Committee of Alex Ekwueme Federal University Teaching Hospital A b a k a l i k i ( R e g i s t r a t i o n n u m b e r FETHA/REC/VOL 2/2019/274), prior to the commencement of the study.Informed written consent was obtained from participants who were 18 years and above while child assent was obtained from participants who were below 18years but not less than 7 years, once parental consent was obtained.

Data collection
Pre-tested, semi-structured, intervieweradministered, questionnaires were used to collect data on socio-demographic characteristics, drug history, previous medical and surgical history.Standiometer was used to measure participants' height to the nearest centimeter, while weighing scale was used to measure their weights to the closest kilogram.Body mass index (BMI) was 2 computed as follows: BMI = Weight (kg)/ Height 2 (m ).The haemoglobin genotype of controls was determined by haemoglobin electrophoresis using electrophoretic tank (Gallenkomp, England).withcellulose acetate paper at pH 8.6.The test group were HbSS patients already diagnosed of SCA and were registered at the sickle cell center of the hospital.Abdominal scan of the participants were done using Sonoscape model S40R (Sonoscape Medical Corp, China), ultrasound scanner, equipped with a 2-5 MegaHertz multifrequency curved array transducer.No preparation or sedation was used.The splenic echopattern, as well as any lesions identified within it, were documented.The coronal plane section was utilized to rule out any splenic lesions.All measurements were made on sections through the splenic hilum in order to create a c o n s t a n t r e f e r e n c e p o i n t f o r r e p e a t i n g 14 measurements.The transverse, longitudinal and antero-posterior dimensions of the spleen were measured.Longitudinal dimension was measured between the most superomedial and the most inferior poles of the spleen on longitudinal section (figure 1).The transverse dimension was measured between the most medial and lateral aspects of the spleen at the level of the hilum on longitudinal section (in a plane perpendicular to the longitudinal dimension) as shown in figure 1.The anteroposterior dimension was measured at the maximum anteroposterior distance on transverse section at the level of the hilum as illustrated in figure 2. Measurements were made during quiet breathing in children, while in adults it was done on h o l d i n g t h e i r b r e a t h .T h r e e s e q u e n t i a l measurements were obtained in the nearest centimeter and the mean calculated.This is to ensure minimum intra-operator variation as well as greater reliability and accuracy of measurements.All dimensional measurements were expressed in centimeters.Splenomegaly was documented when the long axis 17,18 of the organ is greater than 13cm in adults.While in paediatric age group, splenomegaly was documented when the long axis of the spleen is greater than the normal splenic size for age and sex 19 as described by Megremis.Mild splenomegaly is

Table 1: Socio-demographic characteristics of the study participant, N =120
when the longitudinal measurement exceeds the upper limit of normal by less than 10%, moderate splenomegaly is when the measurement is 10-20% greater than normal and marked splenomegaly is when the measurement is more than 20% greater The parenchymal echopattern of the spleen was evaluated ultrasonographically and compared with the echopattern of the liver and that of the cortex of the adjacent left kidney.The normal parenchyma of the spleen has homogeneous echopattern with reflectivity that is similar or slightly brighter than the normal liver and slightly brighter than the 23 normal adjacent left kidney.Splenic echopattern was recorded as any of the following: normal homogenous parenchyma, diffuse homogenously hyperechoic parenchyma, diffuse homogenously hypoechoic parenchyma, heterogeneously hyperechoic parenchyma, heterogeneously hypoechoic parenchyma, echogenic shadowing foci (calcification littered) 24 parenchyma or presence of hypoechic nodules.

Data analysis
This was done using the International Business Machine Statistical Package for Social Sciences (IBM-SPSS) for windows, version 20.0.Normality of distribution of data was done using Shapiro-Wilk test.Normally distributed data: age, longitudinal and anteroposterior dimensions of the spleen were analyzed using parametric statistics (t-test) while the non-normally distributed data: the transverse dimension of spleen, splenic index and splenic volume were analyzed using non-parametric statistics (Mann-Whitney U-test).Comparison of the splenic size between the HbSS and HbAA participants was done using an independent t-test (for longitudinal and anteroposterior dimensions), and a Mann-Whitney U-test (for the transverse dimension, splenic index and splenic volume).The results were summarized as mean and standard deviation for normally distributed data and median and interquartile range for non-normally distributed data.However, categorical variables were summarized using frequencies and percentages.All test were two-tailed at 95% interval; p-values less than 0.05 was considered statistically significant.Results were presented in tables and figures.

Results
A total number of 120 participants were recruited into this study, with one half (60) being patients with HbSS, while the other half (60) were age-and sexmatched participants with HbAA.The age of the participants ranged from 2 -32 years in both groups.The socio-demographic characteristics of the study population revealed that the two study groups (HbSS and HbAA participants) were similar in age (p = 0.99) and gender (p =0.90).Forty five percent (45%) of the participants in both groups were males, while 55% were females (table 1).The sex and age distribution, and splenic size in patients with HbSS, showed that there was no significant differences in the distribution of the various categories of splenic size with gender and a g e ( p = 0 . 1 9 a n d 0 .6 9 , r e s p e c t i v e l y ) .Autosplenectomy, however, was recorded in 13  3).Majority (57.5%) of patients with HbSS had normal homogenous splenic echopattern.diffuse homogeneously hyperechoic splenic echopattern, was the most common abnormal splenic echopattern (23.4%) (Table 4) (figure 3 and figure 4).Mean values of all the dimensions (longitudinal,   transverse, and anteroposterior dimensions) of the splenic size, splenic index and splenic volume were significantly smaller in patients with HbSS when compared with persons with HbAA (table 5).

Discussion
The age range of subjects that participated in this study was between 2 and 32 years.No HbSS patients below the age of 2 years presented to the sickle cell clinic during the period of this study.This may be due to delay in clinical manifestation of the disease seen in infancy, probably caused by the persistence of significant amount of foetal haemoglobin (HbF) seen in this age group, which inhibits polymerization of haemoglobin-S (HbS), resulting in delayed clinical presentation of the disease.25 As the child grows, the HbF declines with commensurate rise in HbS, which results in 25 clinical manifestation of the disease.Another reason for delayed presentation may be parental lack of adequate knowledge of the disease and its manifestation as well as lack of routine newborn screening for sickle cell anaemia, for early detection 26 of the disease in our environment.The highest age recorded in the index study was 32 years.This may be attributed to the considerable reduced average life expectancy in patients with HbSS in our environment due to organ damage, 4,27 especially in those with severe disease.Another reason may be due to poor compliance to medical visits by adult patients.This may be attributed to financial impact of the disease on the patients, as most of them pay out of their pockets, and may prefer to present to the hospital only when they have severe symptoms, in order to reduce cost of care.The age range of patients with HbSS who participated in this index study, is similar to those of 21 28 Luntsi et al and al , who documented the rates of autosplenectomy at 11% and 6.6%, respectively.This may be attributed to differences in clime and haplotype, as well as co-inheritance of alpha thalassaemia and high fetal haemoglobin in subjects with HbSS from 22 these regions .Out of the 13 patients with autosplenectomy in this study, 2(15.4%) were aged 10years and below, while 11(84.6%)were above the age of 10years.This is similar to the findings of Inah and colleagues who found autosplenectomy to be much higher in individual greater than 10 years of age.In this study, all the patients with HbSS who had autosplenectomy were aged 8 years and above, which is in consonance with the generally believed natural progression of sickle cell anaemia.The higher rate of autosplenectomy seen in this age group has been attributed to the progressive reduction in size of the spleen in patients with HbSS, due to recurrent episodes of vaso-occlusion and infarction that happen over time, and finally, 10,37 resulted to autosplenectomy.S h r u n k e n s p l e e n i s a n o t h e r s p l e n i c ultrasonographic finding seen in patients with HbSS in this study.The presence of shrunken spleen in patients with HbSS has been attributed to the repetitive occlusion of the splenic vessels by the sickle red blood cells, followed by splenic ischaemia, which progressively leads to splenic 7 atrophy.In this study, shrunken spleen was seen in 6(10%) of patients with HbSS.This is similar to the findings from a study done in Boston, United States 24 by Gale et al which found shrunken spleen in 14% of patients.This may suggest that shrunken spleen is a common splenic ultrasonographic finding in 18 patients with HbSS.However, Inah and colleagues found a lower rate of 2.5%.The lower rate of shrunken spleen found in the latter research may be due to variation in sampling method.size in 65% of patients with HbSS.Asnani et al postulated that the presence of the spleen in patients with HbSS especially in adult patients may represent a different severity in the disease when compared with those who had autosplenectomy, as the latter was as a result of repeated attacks of vasoocclusion and resulting infarction, while the presence of the spleen is due to fewer vasoocclusive crises indicating milder disease.The rate of normal splenic size in patients with HbSS from this study is high compared to the value of 9.8% and This study also showed that the mean longitudinal and anteroposterior dimensions, as well as the median transverse dimension, splenic volume and splenic index, were significantly smaller in patients with HbSS compared to those of HbAA participants (p<0.05).These findings showed that splenic size in patients with HbSS tends to progressively reduce, due to repeated vaso-occlusion and infarction.The finding of this study is in contrast with previous SCA tends to increase as age advances.Ezeike, reported no statistically significant difference between the splenic length and splenic transverse diameter of patients with HbSS and those with HbAA .He also found that the mean splenic volume and anterior posterior dimension of the spleen, were significantly higher in patients with HbSS than those with HbAA, contrary to the findings of this present study, for which no clear reason may be adduced for this rather unexpected variation.
Contrasting finding was also reported by Olatunji et HbSS patients compared with the control.The contrasting observation may be because majority of the participants in their study population were less than 20years of age.

Conclusion
This study showed significant difference in the splenic ultrasonographic findings between individuals with sickle cell anaemia and those with haemoglobin AA.Splenic size was significantly smaller in patients with sickle cell anaemia, compared with those with haemoglobin AA.Splenomegaly, autosplenectomy, and shrunken spleen, were found only in patients with HbSS.
Only patients with sickle cell anaemia had abnormal splenic echopattern.There should be follow up of HbSS patients with ultrasonography, for early detection and prevention of splenic complications.

18 than
upper limit of normal.Non visualization of 18 the spleen was documented as autosplenectomy.Shrunken spleen is when the long axis of the spleen 20,21 measures less than 5cm in adults and less than 7 50% of the mean splenic size for age in children.Splenic index (SI) was obtained by the following 2 formula: SI (cm ) = L (cm) x W (cm).Where L =longitudinal dimension and W = transverse 22 dimension.Splenic volume was calculated using the formula: 3 Spleen volume (cm ) = L (cm) x W (cm) x D (cm) x 0.523.Where, L = longitudinal dimension, W = transverse dimension, D = anterior-posterior dimension.

Table 2 :
Age and sex distribution with splenic size among patients with sickle cell anaemia Table 3: Age distribution and splenic measurement in patients with sickle cell anaemia in steady state Table 4: Age distribution of splenic echopattern in patients with sickle cell anaemia who have their spleen demonstrated ultrasonographically patients (21.7%), with the highest frequency seen in patients aged 18 and above (13.3%)(table 2).There were variations in splenic measurement in different age groups among patients with HbSS, w h o s e s p l e e n s w e r e v i s u a l i z e d ultrasonographically.The mean longitudinal dimension, anteroposterior dimension, transverse dimension, splenic volume and splenic index were highest in patients aged 15-17 years.The splenic volume and splenic index were lowest in those aged 2-4 years (table

Figure 1 :Figure 2 :
Figure 1: Transabdominal ultrasonogram showing the longitudinal dimension and transverse dimension of the spleen in longitudinal scan (transverse dimension measured in a plane perpendicular to the longitudinal dimension)

Figure 4 :
Figure 4: Image showing the presence of hypoechoic nodule in a patient with sickle cell anaemia (black arrow)

40 study
done in Nigeria by Akinlosotu et al, who found no significant difference in the median spleen size of patients with HbSS and those with HbAA.This may be due to variations in the make-up of 40 study population.Akinlosotu et al worked exclusively in children while this study composed of both children and adult.Finding of their study may be due to the fact that splenic abnormalities in41

42 al,
who found that the mean longitudinal and coronal dimensions of the spleen were higher in the Ibom Med.J. Vol.16 No.3.Sept.-Dec.,2023

, 2023 within
the age group of 11-17years, while 2(28.6%)were aged 18 years and above.This shows that the rate of splenomegaly in patients with HbSS does not follow a particular trend.However, this is contrary Ezeike et al in their studies, with age ranges 3-38years and 3-30years, respectively.Other studies done in different geographical locations included age groups at variance with that 29-34 of the present study, in children and adults.Analysis of our results showed that splenomegaly was found in 11.7% of the participants with HbSS.This result is similar to the findings of Ojo et al, in 34 Ile-Ife, Nigeria, who found splenomegaly in 15% of patients with HbSS.Result of this study is also in line with other studies done in Boston, United States 24 by Gale et al , in Northern Greece by Christakis et 35 22 al and in Saudi Arabia by Al-Salem et al , where the rates of splenomegaly found in patients with HbSS were 15.1%, 14% and 11.8% respectively.The rate of splenomegaly in this study is lower than 31 the findings of Abdullahi et al in their study in Kano, who recorded splenomegaly of 53 (35%) in subjects with HbSS.This may be related to the fact that in their study, only splenic index was used in assessing the splenic size which does not take into consideration the normal splenic size in relation www.ibommedicaljournal.org 280 Ibom Med.J. Vol.16 No.3.Sept.-Dec.
However, most splenic hypoechoic nodules seen in patients with HbSS have been reported to correspond to the normal functioning splenic tissues and tend to have a benign course with no need for further investigation for 39neoplastic transformation.