1. Introduction

Hemoglobinopathy is a group of inherited disorders characterized by structural variations of the hemoglobin molecule; result from either production of an abnormal haemoglobin chain, such as substitution of one amino acid, or underproduction of a given globin chain ^[1]. It may present as heterozygous or the homozygous form. More than 100 abnormal types of haemoglobin have been identified ^[2]; however, only hemoglobins S, C, and D are commonly seen ^[3].

Sickle cell disease (SCD), first described in the early twentieth century, is an inherited haemoglobinopathy resulting from a mutation mutation occurs in beta-globin gene, on chromosome 11 ^[4]. It is includes disorders affecting the structure, function or production of haemoglobin ^[5]; There is a substitution of glutamate with valine in position 6 of the beta globin resulting in the formation of haemoglobin S ^[6]. The disease is expressed when Haemoglobin S (HbSS) is inherited from both parents, the homozygous patient or HbSS suffers from sickle cell anaemia, while the heterozygous child or Haemoglobin AS (HbAS) is a carrier of a sickle cell trait ^[1]. HbSS is the most common pathological haemoglobin variant worldwide and majority of children born with SCA die before reaching five years of age ^[7]. Other sickle cell haemoglobinopathies like HbAS/C, HbS/C, HbS/D, HbS/ß Thalassaemia pattern has been reported ^[8].

Desaturation of HbSS results in the polymerization of haemoglobin, forming large aggregates called tactoids, which deform the red cells into the typical sickle shape. When compared to HbAS, HbSS begin to sickle at much higher oxygen saturation; hence, sickling with sickle cell trait is rarely a problem without concomitant stasis ^{[1, 5]}. This study aimed to determine the haemoglobin patterns of patients with sickle cell haemoglobinopathies and to measure Hb A, HbA2 and Hb F levels in these patients.

2. Material and Methods

Hospital based descriptive case control study design with well structured interviewer administered research questionnaire developed for this purpose, was used to collect data.

This study was undertaken in a Military Hospital in Khartoum State, one of the biggest hospitals in Sudan.

Blood samples were collected from 70 subjects diagnosed or suspected to have sickle cell disease. Blood samples were also taken from 30 control patients case) were selected from patients attending for follow up and from the co-patients respectively.

Inclusion criteria: All patients who diagnosed or suspecting to have a sickle cell haemoglobinopathies and confirmed by positive sicklling test.

Exclusion criteria: Healthy people who suspected to have sickle cell haemoglobinopathies with negative sickllling test.

Blood sample was obtained by venepuncture of the antecubital vein and 5ml of blood was collected in ethylenediaminetetraacetic acid (EDTA) bottles for determination of haemoglobin genotype using the usual electrophoretic method (electrophoretic equipment model MUPID-EXU, Japan). A small quantity of blood haemolysate from each subject was placed on the cellulose acetate membrane and carefully introduced into the electrophoretic tank containing Tris-EDTA borate buffer at PH 8.9. The electrophoresis was allowed to run for 15 minutes at 160V. Haemolysates from blood samples of known genotypes (Hb A, HbA2 and Hb F,, HbAS, HbSS and HbSC) were run as reference standards. The results were read according to the migration pattern of the haemoglobin variant. The results were treated with utmost confidentiality ^[9]. Sickling test and haemoglobin electrophoresis were carried out using Capillartys2 Flex Piercing (SEBIA).

This study conforms to the ethical principles of medical research developed by the World Medical Association Declaration of Helsinki ^[10]. Ethical clearance was given by the Research Committee in Al Neelain University Faculty of Medicine. Written consents were obtained from each participant before entry into the study.

All data obtained with questionnaire and biochemical analysis were analyzed using the Statistical Package for the Social Sciences (SPSS) version 19. The chi square test was used to test distribution of categorical variables. The differences between test and control groups were assessed with the student's t test. Statistical significance was accepted when P value is ≤ 0.05.

3. Results

As illustrated in Table 1, 36 (51.4%) of patients were female and the remaining 34 (48.6%) were male, while in control group 10 (33.3%) were female and 20 (66.7%) were male. Moreover, Figure 1 demonstrated the age distribution among the study participants. The frequent of each sickle cell haemoglobinopathies were shown in Figure 2 as follow: 37 of patients (52.9%) showed AS pattern, 1 patient (1.4%) showed AS/C Pattern, 8 patients (11.4%) showed S/β-Thalassaemia pattern, 1 patient (1.4%) showed SC pattern, 3 patients (4.3%) showed S/D pattern and 20 patients (28.6%) showed SS pattern.

Table 1. The demographic characteristic of patients and control group according gender

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View next table

Figure 1. Age distribution in study group

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View next figure

Figure 2. The frequency of variants sicklling haemoglobinopathies among patients group

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View previous figure

Table 2. The mean level of Haemoglobin A, HbA2, HF and HbS level in AS patients compared with control group

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View previous table

View next table

AS patient demonstrated a significant higher mean level of Hb F (2.14%) ± 2.7, and significant lower level of the mean of Hb A (55.4%) ±15.2 in comparison with control group, while Hb S was only show in patients group. while Hb A2 show no significant different of mean level when compared with control group as show in (Table 2).

In addition, AS/C patient showed no significant different in Hb A2 and Hb F level when compared to control group, while a significant lower level of Hb A (47.8%) ±0.31was observed when compared with control group. Hb S as well as Hb C were only showed in AS patient group with mean level of (42.8%) ± 1.1 and (6.4%) ± 0.1 respectively as show in (Table 3). As demonstrated in (Table 4) Hb A2 in S/C patient show no significant different in mean level when compared with control group (2.1%) ± 2.5, Hb F shows significant higher mean level (4.4%) ± 2.54, but there is no level of Hb A, Hb S or Hb C (0.00%) ±0.0.

Table 3. The mean level of Haemoglobin A, HbA2, Hb F, Hb S and Hb C in AS/C patients compared with control group

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View previous table

View next table

Table 4. The mean level of Haemoglobin A, Hb A2, Hb F, Hb S and Hb C level in S/C patients compared with control group

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View previous table

View next table

In patients with S/βThalassaemia Hb A2 show statistically significant higher mean levels when compared with control group (6.2%) ± 2.3, Hb F also showed significant higher mean level (4.4%) ± 2.54, Hb A level showed lower level than control group (42.58%) ± 28.3. While Hb S was only showed in S/β Thalassaemia patients with mean level of (45.33%) ± 23.9 as show in (Table 5).

Table 5. The mean level of Haemoglobin A, Hb A2, Hb F and Hb S level of S/βThalassaemia patients group compared with normal control group

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View previous table

View next table

While (Table 6) for S/D patients Hb A2 have no significant different of mean level when compared with mean level in control group (2.5%) ± 0.44, Hb F showed slightly significant increase in mean level (1.9%) ± 2.3, but there is no level of Hb A (0.00%) ±0.0 SD. While Hb S as well as Hb D were only showed in S/D patient group with mean level (56.33%) ± 8.31 and (43.5%) ± 0.03 respectively.

Table 6. The mean level of Haemoglobin A, Hb A2, Hb F, Hb S and Hb D in S/D patients compared with control group

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View previous table

View next table

In SS patient group Hb A2 show slightly increased mean level when compared with group ( 3.6% ) ± 0.66, Hb F shows statistically significant increase in the mean level (12.9%) ± 9.5, but there is no level of Hb A (0.00%) ±0.0 SD. While Hb S was only showed in SS patients group with mean level of (84.1%) ± 8.96 as show in (Table 7).

Table 7. The mean level of Haemoglobin A, Hb A2, Hb F and Hb S in SS patients compared with control group

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View previous table

4. Discussion

In this study, Capillary Hb electrophoresis results obtained from normal healthy control group revealed that the means of HbA, HbA2 and HbF were consistent with study in New York USA which defined the mean of HbA A (α2β2) of the normal healthy adults was 95% with small amounts (<3.5%) of Hb A2 (α2δ2) and Hb F (α2γ2) present ^[11].

Considerable researches were done to determine the different variants of sickle cell haemoglobinopathies ^{[1, 12, 13, 14]}. The current work revealed that (52.9%) of patients were AS pattern which a very high rate when compared to previous reports ^[9]. Nnaji and other in Southeastern Nigeria reported that the majority (72.6%) of the respondents had HbAA, only 26.4% were HbAS, while 0.94% were HbSS did not find any other variants of haemoglobinopathies ^[9]. Moreover, similar finding exposed in Turkey by Altay et al ^[15]. Several reports agreed that other variants of haemoglobinopathies were very rare when compared to the Mediterranean region ^{[8, 16]}. This dispute may reflect different ethnic mixes in Sudanese population.

The results of the current study are further supported by a study in USA, which revealed in simple cases of HbS trait, the percentage of HbS is always greater than the percentage of HbA, also individuals with two copies of HbS develop sickle cell disease their capillary electrophoretic patterns results typically show 90 to 95% HbS, no HbA, and often slightly elevated HbF in the 5 to 10% range ^[17]. Moreover, the study also divulged that the mean of haemoglobin A pattern in patient group were statistically significant lower than means of control group (P value < 0.05) which also consistence with previous mentioned study at US ^[17].

The study reflect that patients with sickle cell haemoglobinopathies (with exception S/βThalassaemia) have no significant differences of haemoglobin A2 in comparison with control group, while Hb F and Hb S show significant elevation respectively in comparison with control group (P. value < 0.05), this is consistent with study in Belgium was obtained [Hb A2 CZE (%) = 1.233 ; Hb FCZE (%) = 1.118 Hb], and new reference values had to be determined (Hb A2 2.7–3.8%; Hb F <1.2%). The quantification of Hb A2 was not influenced by Hb S ^[18].

It is apparent from the current study; that hemoglobinopathies and sickle cell anaemia are important health problems in Sudan; that emphasis the important of premarital screening programmes and their value in such country where these diseases are endemic ^{[14, 19, 20]}. The use of such programmes must critically evaluated before implementation, including recent experiences in Saudi Arabia, followed by discussion of the outcomes of such programmes. The success of these programmes is highly depends on adequate religious support, government policy, education and counseling ^{[21, 22, 23]}.

5. Conclusion

The study concluded that the distribution rate of sicklle cell haemoglobinopathies was among all age groups, affected both sexes equally. Haemoglobin A2 have no significant in pateints with sickle cell haemoglobinopathies in comparison with control group, while Hb F and Hb S show significant elevation respectively in comparison with control group.

Competing Interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Acknowledgment

We have collaborated with many colleagues for whom we have great regard.

References

[1]	Wilson M, Forsyth P, Whiteside J: Haemoglobinopathy and sickle cell disease. Continuing Education in Anaesthesia, Critical Care & Pain 2009, 10.
	In article
[2]	Ohls RK CR: Development of Haematopoeitic system. In: Nelson Textbook of Paediatrics. Edited by Jenson HB e, 17th edn; 2004.
	In article
[3]	Haima GK: Haemoglobinopathy. In: The American Heritage Medical Dictionary. vol. 8th: Houghton Mifflin Company; 2009.
	In article
[4]	Herrick J: Peculiar elongated and sickle-shaped red blood corpuscles in a case of severe anemia. Arch Intern Med 1910, 6: 517-521.
	In article		CrossRef
[5]	Firth PG HC: Sickle cell disease and anesthesia. Anesthesiology 2004, 101: 766-785.
	In article		CrossRef
[6]	Serjeant GR, Serjeant BE: Sickle Cell Disease, 3rd edn: Oxford: Oxford University Press.; 2001.
	In article
[7]	Weatherall D, Akinyanju O, Fucharoen S, Olivieri N, Musgrove P: Disease Control Priorities in Developing Countries. In. Edited by Jamison DT ea: Oxford University Press; 2006: 663-680.
	In article
[8]	Taiwo IA, Oloyede OA, Dosumu AO: Frequency of sickle cell genotype among the Yorubas in Lagos: Implications for the level of awareness and genetic counseling for sickle cell disease in Nigeria. J Community Genet 2011, 2: 13-18.
	In article		CrossRef
[9]	Nnaji GA, Ezeagwuna DA, Nnaji I, Osakwe JO, Nwigwe AC, Onwurah OW: Prevalence and pattern of sickle cell disease in premarital couples in Southeastern Nigeria. Niger J Clin Pract 2013, 16(3): 309-314.
	In article		CrossRef
[10]	World Medical Association Medical Ethics Committee,Updating the WMA Declaration of Helsinki. Wld Med J 1999, 45: 11-13.
	In article
[11]	Hedlund B: Haemoglobinopathies and thalassaemias. In: Haemoglobins of human embryos, fetuses, and neonates. Edited by Fairbanks V. New York; 1980.
	In article
[12]	Tarer V, Etienne-Julan M, Diara JP, Belloy MS, Mukizi-Mukaza M, Elion J, Romana M: Sickle cell anemia in Guadeloupean children: pattern and prevalence of acute clinical events. Eur J Haematol 2006, 76(3): 193-199.
	In article		CrossRef
[13]	Al-Allawi NA, Al-Dousky AA: Frequency of haemoglobinopathies at premarital health screening in Dohuk, Iraq: implications for a regional prevention programme. East Mediterr Health J 2010, 16(4): 381-385.
	In article
[14]	Bayoumi RA, Abu Zeid YA, Abdul Sadig A, Awad Elkarim O: Sickle cell disease in Sudan. Trans R Soc Trop Med Hyg 1988, 82(1): 164-168.
	In article		CrossRef
[15]	Altay C, Yilgor E, Beksac S, Gurgey A: Premarital screening of hemoglobinopathies: a pilot study in Turkey. Hum Hered 1996, 46(2): 112-114.
	In article		CrossRef
[16]	RK Ohls, Christensen R: Development of Haematopoeitic system. In: Nelson Textbook of Paediatrics. Edited by RE Behram, RM Kliegman, Jenson H, 17th edn: Philadelphia: Saunders; 2004: 1601-1604.
	In article
[17]	Shirley L: Haemoglobinopathies and Thalassaemias. In: The ABCs of Lab Evaluation. vol. 35; 2009.
	In article
[18]	Cotton F, Lin C, Fontaine B, Gulbis B, Janssens J, Vertongen F: Evaluation of a capillary electrophoresis method for routine determination of hemoglobins A2 and F. Clin Chem 1999, 45(2): 237-243.
	In article
[19]	Mohamed AO: Sickle cell disease in the Sudan. Clinical and biochemical aspects. Minireview based on a doctoral thesis. Ups J Med Sci 1992, 97(3): 201-228.
	In article
[20]	Molineaux L, Fleming AF, Cornille-Brogger R, Kagan I, Storey J: Abnormal haemoglobins in the Sudan savanna of Nigeria. III. Malaria, immunoglobulins and antimalarial antibodies in sickle cell disease. Ann Trop Med Parasitol 1979, 73(4): 301-310.
	In article
[21]	Memish ZA, Owaidah TM, Saeedi MY: Marked regional variations in the prevalence of sickle cell disease and beta-thalassaemia in Saudi Arabia: findings from the premarital screening and genetic counseling program. J Epidemiol Glob Health 2011, 1(1): 61-68.
	In article		CrossRef
[22]	Alhamdan NA, Almazrou YY, Alswaidi FM, Choudhry AJ: Premarital screening for thalassaemia and sickle cell disease in Saudi Arabia. Genet Med 2007, 9(6): 372-377.
	In article		CrossRef
[23]	Al-Mendalawi MD: RE: Six-year outcome of the national premarital screening and genetic counseling program for sickle cell disease and beta-thalassaemia in Saudi Arabia. Ann Saudi Med, 31(6): 661.
	In article