Prevalence of Sickle Cell Trait in the Southern Suburb of Beirut, Lebanon

Objective The objective of this study was to assess the prevalence, gender differences, and time trends of Sickle Cell Trait in the Southern Suburb of Beirut, Lebanon, as well as to highlight the importance of screening for Sickle Cell Trait carriers in this population. Another objective was to describe a new screening technique for Sickle Cell Trait carriers. Methods This was a retrospective cohort study carried out at a private laboratory in the Southern Suburb of Beirut, Lebanon between 2002 and 2014. The sickling test was carried out for each patient using two methods: the classical “sodium metabisulfite sickling test”, and the new “sickling test method” used in the private lab. As a confirmatory test, hemoglobin electrophoresis was run on a random sample of 223 cases which were found to be positive using the two sickling tests. Results A total of 899 cases were found to be positive for the sickle cell trait out of 184,105 subjects screened during the 12-year period, prevalence = 0.49% (95% CI: 0.46 – 0.52). Among the total sample, females were found to have higher prevalence, where no time trend over the studied period was noted. The haemoglobin electrophoresis method confirmed the results of this new sickling test technique among the random sample of the 223 cases. Conclusion We found that the prevalence of sickle cell trait is lower as compared to other Arab countries, higher in females, with no significant time trend. The sickle cell test was found to be an accurate, simple and cheap test that could be easily added as a requirement for the pre-marital testing to screen for Sickle Cell Trait carriers.


Introduction.
Sickle syndromes include several inherited diseases that cause red blood cells to sickle in vivo, where the most recognized ones are Sickle Cell Disease (SCD), Sickle Cell Trait (SCT), and Sickle Cell/Thalassemia (S/β thalassemia) 1 Phenotypically, only persons with double recessive genes of sickle cell homozygotes (SS) do manifest disease, while the heterozygotes (AS) are being referred to as Sickle Cell Trait carriers. 2 It is estimated that each year more than 330,000 children are born worldwide with haemoglobinopathies, 83% of which are sickle cell disorders. 3 Sickle Cell Disease is a disorder of haemoglobin synthesis that results from the substitution of glutamic acid at the sixth position of its β-globin chain by valine (HbS). 4 It is also known as sickle-cell anemia (SCA) and is characterized by an abnormality in the oxygencarrying haemoglobin molecule in red blood cells. 5 It is one of the most common haemoglobinopathies in Africa, the Middle East and India. Sickle cell disease is now found throughout the world, and its incidence has increased in Europe and North America because of the high rate of migration from areas in which the disease is prevalent. 3,6 In 2006, the World Health Organization (WHO) recognized sickle cell disease as a global public health problem.7 According to Diallo et al., Africa is the most highly affected continent with 200,000 newborn affected by sickle cell disease per year. 2 In the United States, sickle cell disease affects about 72,000 people. 8 Moreover, Piel et al. expected that the annual global number of newborns with sickle cell disease will increase from 305,800 patients in 2010 to 404,200 patients in 2050 (about one-third increase). 9 The clinical manifestations of the sickle cell disease are diverse where any organ system may be affected. These signs are commonly divided into vaso-occlusive (where the episodes of pain, acute chest syndrome, splenic infarction, stroke, and avascular necrosis of joints predominate) and hematologic (where severe anemia, leg ulcers, and pulmonary hypertension predominate). 10,11 Early diagnosis and treatment can ameliorate the course of these diseases and improve survival. More specifically, prophylactic penicillin, immunizations, comprehensive care, and parental education about serious sickle cell disease complications has been shown to significantly give a better survival rate and quality of life for sickle cell disease patients. 12,13 Sickle Cell Trait is present in approximately 300 million people worldwide, with the highest prevalence of approximately 30% to 40% in sub-Saharan Africa. 11 Despite the benign nature of the Sickle Cell Trait, several potentially serious complications have been described with it including, gross hematuria, increased urinary tract infection in women, complications of hyphema, splenic infarction with altitude hypoxia or exercise, and life-threatening complications of exercise. 14 Hematuria is the most common manifestation of sickle cell trait. 15 Spontaneous sickling can occur in the renal papilla (normally under low oxygen pressure) in patients with renal papillary necrosis; therefore, 5% of patients with sickle cell trait can suffer episodes of hematuria at some point during their lives. 16,17 Studies in America, England and Jamaica reported that the rates of urinary tract infection are higher for women with sickle cell trait in comparison to racially matched controls. 14 This finding is well established for asymptomatic bacteriuria of pregnancy, in which the rate is approximately doubled with sickle cell trait. 18 People with sickle cell trait are more susceptible to complications following treatment of hyphema. The slow flow of relatively hypoxic fluid in the chamber of the eye out of the filtration apparatus is a location in which both polymerization of hemoglobin S and obstruction of flow by rigid erythrocytes is likely. All this can result in glaucoma and secondary hemorrhage. 14 Splenic Infarction from sickle cell trait is favoured by exercise at high altitude but has occurred with altitude exposure at rest or during exercise at sea level. 11 The spleen is susceptible to vaso-occlusion related to hemoglobin S polymerization and red cell deformation. When subjects with hemoglobin S are exposed acutely to high altitude hypoxia, the spleen is the organ most consistently injured by microvascular obstruction. 19 Moreover, there has been growing concern about the possible association of Sickle Cell Trait and exerciserelated morbidity and mortality in healthy young college athletes due to the effects of extreme physical exertion, dehydration and relative hypoxia (typically at high altitudes). 1 The risks of microvascular complications in Sickle Cell Trait carriers in response to exercise are dependent on alterations in blood rheology and vascular adhesion processes. These abnormalities include higher red blood cell rigidity and higher blood viscosity in the sickle cell trait carriers compared with the non-carriers, particularly 24 and 48 hours after exercise. 20 Pre-marital screening to identify carriers of genetic disorders, especially those with recessively inherited diseases, has been shown to decrease the incidence of inherited Sickle Cell Disease. 21 25 This study was conducted to assess the prevalence of Sickle Cell Trait among a sample from a private laboratory (Modern Medical Center / Modern Laboratory) in the Southern Suburb of Beirut, as well as to assess any time trends over a 12-year period, and any differences in gender. Moreover, the importance of screening for Sickle Cell Trait carriers in this population is also highlighted. Another objective was to describe a new "sickling test method" for the screening of Sickle Cell Trait carriers, as well as to assess the correlation of the results with them of the "sodium metabisulfite sickling test", and to confirm findings with the hemoglobin electrophoresis technique (Sebia Capillarys).

Methods.
Study design and setting: This research was a retrospective cohort study using data from a private laboratory (Modern Medical Center / Modern Laboratory, established in 1963) in the Southern Suburb of Beirut, Lebanon from 2002 till 2014. Population served by this laboratory includes mainly subjects from a Southern suburb of Beirut, irrespective of their socio-economic status. All patients coming for a complete blood count with differential (CBCD) underwent a free-of-charge sickling test.
Inclusion and exclusion criteria: Eligible for this study were all subjects referred to our laboratory for CBCD testing, without any exclusion based on age, gender, socioeconomic status or MCV findings. The sample included in our analyses comprised all eligible subjects, where no sampling was applied. Moreover, repetitive results for same patients were excluded.
Blood testing method: Blood samples (3 ml) were drawn from patients using EDTA (Ethylenediaminetetraacetic acid) tubes. As a primary screening procedure, the sickling test was carried out for each patient using two methods. In the first method (the new "sickling test method"), one drop of blood was placed on a slide and a cover slide which was sealed using nitrocellulose dissolved in butyl acetate usually found in nail varnish. The slides were incubated at 37°C for 24 hours. As for the second method ("sodium metabisulfite sickling test"), one drop of 2% Sodium metabisulfite was added to a drop of blood between a slide and a cover slide, sealed using the same sealing method and incubated at 37°C for 10 minutes or at room temperature for one hour. For both sets, the slides were inspected under the microscope (Olympus, dry objective 40), and the red blood cells found to be sickling in shape were indicative of a positive sickling test (Figure 1-2). Patients found to be positive were notified of their condition and advised to confirm their results by haemoglobin electrophoresis.
As a confirmatory test, haemoglobin electrophoresis was run on a random sample of 223 subjects who were found to be positive using the sickling test. Another sample of whole blood was drawn from these patients in EDTA tube, and then the red cells were separated from plasma by centrifugation. The Capillarys, an  Data Collection: Data were collected by extracting the information from the electronic and paper files. For the analysis of the prevalence, information available were a date, age, gender, and MCV. On the other hand, for the confirmatory analysis, information collected included age, gender, and the hematologic parameters such as RBC, Hb (haemoglobin), Hct (hematocrit), MCV (mean corpuscular volume), MCH (mean cell hemoglobin), and MCHC (mean corpuscular hemoglobin concentration). Moreover, information about different types of haemoglobin: HbA1 (haemoglobin, alpha 1), HbA2 (hemoglobin, alpha 2), HbF (fetal hemoglobin) and HbS (hemoglobin S) were included.
Ethical Considerations: Patients enrolled in our study provided oral consent for the tests to be performed, as well as to be included in the study. The sickling tests were carried out free of charge. Moreover, institutional review board (IRB) of the American University of Science and Technology (AUST) in Lebanon was obtained. Data collected were kept confidential at the principal investigator's office.
Statistical analyses: Data entered into a Microsoft Excel spreadsheet and then transferred into the Statistical Package for Social Sciences (SPSS) version 21, which was used for data cleaning, management and analyses. Descriptive analyses were carried out by presenting the number and percent for categorical variables, whereas mean and standard deviation were performed for continuous ones. The prevalence of the sickle cell trait was calculated along with the 95% confidence interval (95% CI). The difference in the prevalence of sickle cell trait, as well as the other hematological parameters between males and females, were assessed by using the students t-test. Statistical significance was identified at 0.05 level.  Worth noting is the finding that the prevalence of sickle cell in each year was lower among males compared to that among females. In some years this   Finally, Table 5 presents the differences in age and the different hematological parameters between males and females, for the 223 cases with positive HbS screening. Females were older than males with the average age being 26.9 years (±14.3) and 22.1 years (±17.0) for females and males, respectively, p-value = 0.03. A statistically significant difference between males and females was found in the RBC, Hct, and HbA2, the males having highest levels. The p-values were <0.0001 for each of the parameters.

Discussion.
In this retrospective cohort study, that was carried out in a private laboratory in the Sowthern Suburb of Beirut between 2002 and 2014, we assessed the prevalence of Sickle Cell Trait, and time trends over a 12-year period, and any differences in prevalence between the two genders. Moreover, we described a new technique for screening of sickle cell trait carriers, assessing the correlation of the results with those of the known sickling test, and confirmed the findings with the haemoglobin electrophoresis technique (Capillarys). We found the prevalence of Sickle Cell Trait to be 0.49% (95% CI: 0.46-0.52). No  Table 4. Demographic and hematological parameters for 223 cases with positive HbS screening.  36 and Yemen (2.2%). 37 This difference could be explained in part by the lower rate of consanguineous marriages among the Lebanese population compared to other Arab countries.
The results of the present study showed some gender differences, where the prevalence of Sickle Cell Trait in females (0.62%, 95% CI: 0.57 -0.67) was higher than that of males (0.35%, 95% CI: 0.32 -0.39), consistently over the years. Similarly, significant gender differences have been also reported in adults with Sickle Cell Trait carriers. For instance, In a study carried out by Kamble et al. in India, it was reported that male: female ratio was 1.71:1 in HbAS cases. 38 We did not find any significant differences in the trends of the Sickle Cell Trait over the 12-year period. No studies have been found in the literature reporting on the trends of the Sickle Cell Trait prevalence over time. Nevertheless, our findings indicate a relatively stable prevalence of Sickle Cell Trait over time probably because our studied subjects were born in Lebanon and of Lebanese ancestry. Accordingly, no crucial demographic changes or marital habits (consanguineous) were observed in the past 12 years.
In our study, the overall average MCV was found to be 84.3 fl (±7.6). Similarly, a comparable standard MCV value has been reported among sickle cell trait carriers in France by Tripette et al. in 2009. 39 On the other hand, in a study done in India, Dangi, et al. in 2010 reported an MCV value of 73.4 fl among the sickle cell trait carriers, which was lower than that reported in our study. 40 However, this study was performed in an anemic population, and most of these subjects were affected by sickle anemia with beta thalassaemia trait, 40 and also the iron deficiency cannot be excluded in this setting of patients. We found that the MCV level of the 223 cases of positive sickle cell trait, which were confirmed by the Hb electrophoresis, to be 80.6 (±9.1). MCV levels among the whole sample, as well as the 223 cases, that were tested carrying the Sickle Cell Trait, were within the normal range. Thus, MCV is not useful for screening sickle cell trait, and the microcytosis does not exclude the presence of Sickle Cell Trait as reported by Dangi, [40] and an accurate screening test, like the sickling test, should also be made in patients with microcytosis (microdrepanocytosis according to Silvestroni). 41 Of course, the results should be confirmed by Hb electrophoresis (100% accuracy).
The present study had several limitations. First, it was an observational study without a control group. Another limitation is that the sample was collected from a single center that might not be representative of the whole Lebanese population. As such, the results of the study should be interpreted with these limitations in mind. On the other hand, the study had several strengths, such as the large sample size, the extended period, the prospective data collection, as well as being among the few studies carried out in Lebanon and the region assessing the prevalence of Sickle Cell Trait.

Conclusion.
In this study, we found that the prevalence of Sickle Cell Trait is lower as compared to other Arab countries, higher in females, and with no significant trend over the 12-year period. Moreover, we showed that the MCV, though was not a screening test for Sickle Cell Trait carriers, was useful. In fact, subjects with microcytosis should not be excluded from the selection and could have a contemporaneously thalassemic trait, or sideropenia. On the other hand, the sickle cell test was found to be an accurate, simple and cheap test (It costs approximately two US dollars for both sickling tests) which could be easily added as a requirement for the pre-marital testing to screen for Sickle Cell Trait carriers.