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Table of Contents
ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 1  |  Page : 26-31

Haemoglobin F and A2 profiles among sickle cell anaemia patients in Lagos State University Teaching Hospital (LASUTH), Nigeria


1 Department of Hematology and Blood Transfusion, Lagos State University, College of Medicine, Ikeja, Lagos, Nigeria
2 Department of Haematology and Blood Transfusion, Gbagada General Hospital, Lagos, Nigeria
3 Department of Haematology and Blood Transfusion, College of Medicine, University of Lagos, Lagos, Nigeria
4 Department of Community Health and Primary Health Care, College of Medicine, University of Lagos, Idiaraba, Lagos, Nigeria
5 Department of Haematology and Blood Transfusion, Lagos State University Teaching Hospital, Ikeja, Lagos, Nigeria

Date of Web Publication11-Jun-2018

Correspondence Address:
Dr. Akinsegun Akinbami
Department of Hematology and Blood Transfusion, Lagos State University, College of Medicine, PMB 21266, Ikeja, Lagos, Lagos State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/atp.atp_45_17

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  Abstract 

Background: The choice of high-performance liquid chromatography (HPLC) to measure HbF and HbA2in sickle cell disease patients is regarded as a method of choice by many researchers. This study was aimed at using HPLC in determining the mean and gender-specific reference values of HbF and HbA2in sickle cell anemia (SCA) population and bringing to fore all associated implications. Materials and Methods: This was a cross-sectional, retrospective, descriptive study involving SCA patients. All case notes containing HPLC hemoglobin quantification reports were reviewed to extract the percentages of HbA2, HbF, and HbS of patients. The demographic data of individual patients were also obtained from the records. Data were analyzed with IBM SPSS Statistics for Windows, Version 20.0 Armonk, New York, USA. Results: A total of 100 participants' records were reviewed consisting of 40 (40%) males and 60 (60%) females. The overall mean age (±standard deviation [SD]) of participants was 25.89 years ±9.34. The overall mean HbF and HbA2were 6.94% ±5.05 and 3.75% ±0.74, respectively. Thirty percent had HbF <3%, whereas 34% of them had elevated HbA2level >4%. The mean (±SD) HbF and HbA2for both males and females were 6.97% ±5.45 and 3.68% ±0.58, 6.92% ±4.87, and 3.80% ±0.83, respectively. Conclusions: Thirty percent of the study participants had HbF <3%, whereas 34% of them had elevated HbA2level >4% and could indeed be carrying beta thalassemia trait with the sickle cell gene.

Keywords: Hemoglobin profiles, HbA2, HbF, high-performance liquid chromatography, sickle cell anemia


How to cite this article:
Akinbami A, Uche E, Dosunmu A, Osikomaiya B, Adediran A, Sarah JO, Esther O, Badiru M, Bamiro R. Haemoglobin F and A2 profiles among sickle cell anaemia patients in Lagos State University Teaching Hospital (LASUTH), Nigeria. Ann Trop Pathol 2018;9:26-31

How to cite this URL:
Akinbami A, Uche E, Dosunmu A, Osikomaiya B, Adediran A, Sarah JO, Esther O, Badiru M, Bamiro R. Haemoglobin F and A2 profiles among sickle cell anaemia patients in Lagos State University Teaching Hospital (LASUTH), Nigeria. Ann Trop Pathol [serial online] 2018 [cited 2019 Dec 7];9:26-31. Available from: http://www.atpjournal.org/text.asp?2018/9/1/26/234157


  Introduction Top


Sickle cell anemia (SCA) is common in Nigeria with prevalence values ranging from 2% to 3% of the 140 million populations.[1]

SCA is characterized by an abnormal hemoglobin structure consequent on replacement of adenine with thymine on the β-globin gene resulting in valine replacing glutamic acid on the β chain of the hemoglobin structure.[2] Co-inheritance of α-thalassemia gene with SCA causes a reduction in mean corpuscular hemoglobin concentration which inhibits hemoglobin S polymerization, thus causing an increase in hemoglobin levels which may increase blood viscosity in SCA.[3] Conversely, co-inheritance of β thalassemia gene with SCD impacts negatively on disease severity.

The World Health Organization recognized SCA as a global public health problem in 2006.[1] It also included SCA in 2010 Global Burden of Diseases, Injuries and Risk factors.[4],[5] The sickle cell trait (HbAS), individuals who are carriers of the disease, have a normal hemoglobin and a sickled hemoglobin, they are usually asymptomatic and the prevalence ranges from 10% to 40% in sub-Saharan Africa.[6]

SCA is the symptomatic presentation of the inherited mutation which can present depending on inheritance as severe which is SCA in which the inheritance is HbSS or HbSβ thalassemia, HbSS is seen in 2%–3% of Nigerians.[7],[8]

Hemoglobin structure

The functional properties of hemoglobin are determined by their characteristics folds of amino acid chains of the globin proteins including seven stretches of the polypeptide α helix in the α chains and eight β helix in the β chains.[9] The reversible binding of O2, CO, and NO to the four ferrous iron atoms of the heme is responsible for transportation of these gases by hemoglobin.[10]

Hemoglobin phenotypes

Normal adult hemoglobin structure of α2β2 is known as hemoglobin A, it accounts for 97% of total hemoglobin. Other hemoglobin types present in adult are hemoglobin A2 which is α2δ2. It accounts for 2% and hemoglobin F α2γ2 which accounts for 1% of adult hemoglobin.[11]

Hb F is the best-suited hemoglobin in-utero because it has a slightly higher oxygen affinity than hemoglobin A and a much higher oxygen affinity than hemoglobin S being able to bind 2,3 bi-phosphoglycerate less strongly. In the HbF γ chain at birth, γG is more abundant, while γA is the predominant form in adulthood.[12] The α gene remains fully active at birth, following delivery, the γ is downregulated while more of the β and less of δ genes are up-regulated. Combinations of α gene with β,δ and γ genes give rise to the formation of 97% normal adult hemoglobin A (α2β2), 2% hemoglobin A22δ2) and 1% of hemoglobin F2γ2) by the end of the first year of life. In individuals whose γ gene downregulation is not effective following delivery, it results in higher percentage of HbF (α2γ2) known as hereditary persistent of fetal hemoglobin HPFH.[13] When glucose is covalently glycosylated to the β-chain amino-terminal residue it forms hemoglobin A1C.[14]

Fetal hemoglobin and its benefits in sickle cell anemia

Xu et al. in their study reported amelioration of the symptoms associated with SCA in individuals with accentuated expression of γ globin genes resulting in a high level of HbF which compensates for the defective β-globin products of SCA.[15] High HbF levels are inherited as quantitative traits dependent on many gene loci outside the β-globin cluster [16] including 2q16, 6q23, 8q, and Xp22.2.[17] As early as 1948, Janet Watson noted that symptoms associated with SCA were not fully manifested until a hemoglobin switch from fetal to adult takes place.[18] Several laboratory studies have confirmed levels of HbF needed to ameliorate symptoms associated with SCA and high level of HbF is reported to be inversely related to degree of severity in SCA.[19]

High HbF retards polymerization of sickled cells in the deoxygenated state by reducing HbS concentration thus inducing a lower rate of vaso-occlusive crises, leg ulcers, avascular necrosis of the neck of femur, acute chest syndrome, and ultimately a reduced disease severity. However, elevated HbF does not impact on priapism, stroke, systemic blood pressure, and sickle vasculopathy.[20]

The percentage of F cells in females is reported to be higher than in males because it is partially controlled by the Xp 22.2 locus on the X chromosome. A total of 70% of the variation in HbF levels in SCA patients may be associated with variation in percentages of F cells.[21]

Olaniyi et al.[22] in 2010 determined the HbF ranges in Nigerian SCA patients, they categorized the level into low HbF <2% moderate 2.1%–10% and high 10.1%–16%.[22]

Hemoglobin A2 and implications of its high level in sickle cell anemia

When compared with HbSS individuals, HbA2 has been reported to be elevated in Hb S/β0-thalassemia.[23] An advantage of quantification of HbA2 level is to differentiate between the two types of sickle cell anemia, i.e. Hb SS and Hb S/β0-thalassemia.[24],[25]

High-performance liquid chromatography (HPLC) is regarded as the method of choice by many researchers for the measurement of HbA2 in patients with SCA.[26],[27],[28] However, this was faulted by Suh et al. in 1996.[29]

They proposed that HbA2 estimation in SCA may be falsely estimated by HPLC because HbA2 values obtained by HPLC increased significantly in samples containing HbS resulting in wrongly labeling HbSS as Hb S/β0-thalassemia.[29] Based on 1996 Suh et al. hypothesis, HPLC analyzed HbA2 value of up 5.9% in HbSS individuals was considered as normal value by Shokrani et al. in 2000.[30]

Posttranslational modifications in some HbS make them have same retention time as HbA2. This was postulated as the reason for falsely elevated HPLC measured HbA2 in patients with SCA by Head et al. in 2004.[24] The presence of other β chain variants such as in HbE, HbD, and Hb Lepore has also impacted on the falsely elevated levels of HbA2 measured by HPLC.[31],[32]

However, Giambona et al.[33] defined borderline β-thalassemia as HbA2 of a range of between 3.1%-3.9%, in an Italian population with a high prevalence of β-thalassemia, in which a total of 23,485 patients were retrospectively studied between 2000 and 2006.


  Materials and Methods Top


Study population

Patients attending adult SCA Clinics of Lagos State University Teaching Hospital, Ikeja, were the study population. The clinic has an estimated total of 500 patients with SCA.

Inclusion criterion

HPLC Hemoglobin quantification results of homozygous HbSS patients found in the case notes.

Exclusion criteria

  1. HPLC Hemoglobin quantification results of HbSC or other double heterozygous phenotype such as HbSD, and Hb CD, etc
  2. All HbSA patients
  3. All patients already on hydroxyurea
  4. HIV-positive HbSS patients
  5. HbSS participants with elevated mean corpuscular volume >100.


Study design

This was a descriptive, cross-sectional, and retrospective study involving SCD patients. The study was conducted in November 2017. Patients' sociodemographic data and the HPLC results were retrieved from their folders. Proportions of HbA2, HbF, and HbS of the patients were generated. HbF values were categorized as done by Olaniyi et al. and HbA2 by Giambona et al.

Sampling technique

A nonprobability convenience sampling was used that is applicable to both quantitative and qualitative studies, in which members of a target population that meet certain criteria, such as accessibility, geographical proximity, availability at a given time, or willingness to participate are included for the purpose of the study. Percentages of HbA2, HbF, and HbS from the case notes were abstracted.

Data analysis

The data were entered and analyzed using IBM SPSS Statistics for Windows, Version 20.0 Armonk, New York, USA. The mean ± standard deviation (SD), median, SDs were generated as necessary for continuous data. Tests of statistical significance between variables such as Chi-square analysis and Fischer's exact for discrete data were used. Level of significance was set at P ≤ 0.05.

Ethical consideration

Ethical approval was obtained from Health and Ethics Research Committee. The reference number is LREC.06/10/916, approved on October 10, 2017.


  Results Top


One hundred (100) out of a total of 129 participants who met the inclusion and exclusions criteria were used. It consisted of 40 (40%) males and 60 (60%) females. The overall mean ± SD age of participants was 25.89 ± 9.34 years. The overall mean ± SD HbF was 6.94%±5.05. [Table 1] shows overall and gender-specific parameters of age, HbF, HbA2, and HbS.
Table 1: Overall and gender-specific mean age, hemoglobin F, hemoglobin A2 and hemoglobin S

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The overall mean ± SD HbA2 was 3.75%±0.74. [Table 2] shows overall and gender-specific HbF ranges. Analyzing HbA2 in all participants, [Table 3] highlights overall and gender-specific Hb A2 ranges.
Table 2: Overall and gender-specific hemoglobin F ranges

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Table 3: Overall and gender-specific hemoglobin A2 ranges

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The mean age of male participants was 23.77 ± 8.13 years with a range of 14 and 49 years [Table 1]. The mean HbF for males was 6.97 ± 5.45%, a minimum of 1% and maximum of 21.6%. HbF values for males showed majority 24 (60%) had a range of between 2.1% and 10%, followed by 10.1% and 16% and <2%, both groups had 6 of 40 each (15%) and only 4 (10%) had HbF >16.1% [Table 2].

Majority of males participants 25 (62.5%) had HbA2 of 3.1%–3.9%, followed by 10 (25%) with a range of 4%–5.9% and 5 (12.5%) had HbA2<3.1 [Table 3].

The mean HbF for females was 6.92 ± 4.87%. HbF values for females showed, majority 38 (63.3%) had HbF of between 2.1% and 10% followed by 11 (18.3%) with HbF of between 10.1% and 16%, similarly, 9 (15%) had HbF of <2% and only 2 (3.3%) females had HbF >16.1% [Table 2]. The mean HbA2 was 3.80 ± 0.83. Majority 25 (41.7%) are within the range of 3.1%–3.9%, followed by 24 (40%) with a range of 4%–5.9% and 11 (18.3%) had a range of <3.1% [Table 3].

HbF and HbA2P values were 0.56 and 0.44, respectively, when compared with the ages of participants, similarly, HbF and HbA2P values were 0.53 and 0.43, respectively, when compared with the gender of participants.


  Discussions Top


HPLC has been proven to be a rapid, sensitive and accurate method for quantifying various types of normal and abnormal hemoglobins despite its limitations, one of which includes co-elution of HbS byproducts with HbA2 resulting in falsely elevated level of the latter.[34] It is however very suitable for HbF estimation with little or no limitations.

This study analyzed HPLC generated results of HbF and HbA2 in one hundred SCA patients with a view to determining their, overall and gender-specific reference values for HbF and HbA2 in Lagos, South West, Nigeria, determine the percentage of our SCA patients with low, moderate or high values of HbF, the value has been proven to impact on the clinical severity of SCA patients and to determine SCA patients with elevated HbA2 which is a reliable diagnostic marker of beta thalassemia trait.

Epidemiological study has demonstrated that the lower rates of recurrent clinical events such as acute chest syndrome, vaso-occlusive crises, and frequent rate of hospitalizations are associated with HbF levels above 20%.[35] Similarly, patients with values above 10% had reduced incidence of strokes and avascular necrosis of head of femur.[35]

This study reported a mean HbF value of 6.94% ±5.05, which is lower than the previous studies done in, Lagos,[36] but similar to values reported in Ibadan,[37] and Benin.[38]

Despite the wide range noted in this study, we observed that 7% of SCA patients in this study have HbF levels below 1% unexpectedly much lower than 14% reported in HbAA individuals, although, methodology used differs,[39] 20% had HbF ≥10% which confers advantage of reduced incidence of strokes and avascular necrosis of the head of femur.[35] However, majority of the participants in this study (62%) had value between 2% and 10%. Only 2% have HbF >20% which is considered as hereditary persistent of fetal hemoglobin (HPFH) coexisting with HbS.[14] HPFH is caused by deletions of the β-globin gene cluster, which induces a compensatory γ-globin synthesis increase. It is also thought to be due to mutations in the HBG promoter regions or inheritance of HbF modulating quantitative trait loci, like HBSIL-MYB intergenic region (6q23) and BCL11A (2p16).[40],[41]

In a contrary study by Adeyemo et al.,[36] they reported a higher mean value in females than males as compared to this study. The study in Ibadan reported a higher value in males than females despite a reported higher percentage of F cells in females than males and HbF levels being partially controlled by an X-Linked Gene.[21] However, the value obtained in this study is not statistically significant when compared to the study by Adeyemo et al.[36]

Another point worthy of note is 7%, and 23% of our patients had very low HbF <1% and low level of HbF <3%, respectively. These groups might benefit from hydroxycarbamide, which is known to elevate HbF level by conversion of the hydroxycarbamide to nitric oxide (NO) in vivo. NO stimulates intracellular soluble guanylate cyclase which in turn, elevates cGMP and causes an increase of HbF level through cGMP-dependent protein kinase G.[42]

SCA patients with variants in BCL11A have been reported to have a higher HbF response to hydroxycarbamide [43] elevated HbF improves or reduces severity of crises in the patients.[44] However, Green et al. in 2016 reported a blunted response of HbF to hydroxycarbamide use overtime leaving SCA patients to worsening disease complications and increased hospitalizations.[45]

Determining HbA2 levels in SCA in order to identify SCA patients with beta thalassemia trait may not have physiological significance in SCA, its knowledge is desirable in genetic counseling for couples at risk of having affected child with β-thalassemia [46] Apart from using elevated HbA2 to diagnose beta thalassemia trait, which is considered the best approach for beta thalassemia trait diagnosis, various methods could help in making in the diagnosis of beta thalassemia trait, these include microcytic (M)/hypochromic (H) ratio estimation,[47] red blood cell flags,[48] red cell distribution width.[49] Adeyemo et al. in 2014 proposed that elevated HbA2 in SCA patients above 4%[36] is a valuable screening tool in diagnosing beta-thalassemia trait, while borderline range of beta thalassemia trait is considered to be 3.1%–3.9% of HbA2.[24],[33],[50]

However, apart from SCA, other factors that could cause elevation of HbA2 above 3.4% are thyrotoxicosis, HIV infection with zidovudine therapy and some cases of megaloblastic anemia. Furthermore, alpha thalassemia, severe iron deficiency, anemia of chronic diseases, sideroblastic anemia, lead poisoning, and acute myeloid leukemia could cause a reduction on HbA2 lower than 2.2%.[51],[52]

The mean HbA2 obtained in this study was very similar to value obtained from SCA patients in Brazil in which the authors quantified HbA2 with HPLC.[53]

Another study by Craver et al.[54] reported a lower value of HbA2 as compared to this study. This could be due to the fact that isoelectric focusing was used by Craver for the quantification of HbA2 underscoring substantial co-elution of HbS with A2 byproducts in HPLC resulting in falsely elevated level. However, the three values in SCA are much higher than HbAA reference range (2.4% ±0.9) reported by Craver et al.[54]

Thirty-four percent of our study participants had HbA2 > 4% hence could be considered to be SCA with beta thalassemia trait and wrongly labeled as HbSS. This is higher than the proportion of participants with SCA with beta-thalassemia trait reported by Adeyemo et al. in their study probably because unlike this study they considered red cell indices as a screening tool apart from level of HbA2 > 4%.[36] Our reported value is also higher than value reported by Inusa et al. in Northern Nigeria in 2015 because they used a higher HbA2 cutoff value of 6% and the general population unlike SCA cohort used in this study.[55]

Thalassemia is considered a Mediterranean disease, the presence of beta thalassemia trait among our sickle cell population in Lagos, Nigeria which is a non-Mediterranean country might be higher than excepted going by this study, a polymerase chain reaction (PCR)-based method to determine the exact percentage of the beta thalassemia in our population is the gold standard and long overdue.


  Conclusions Top


Thirty percent of the study participants had HbF <3% while 34% of them had elevated HbA2 level >4% and could indeed be carrying beta thalassemia trait with the sickle cell gene.

Limitations

A limitation of the study is reliability on the HPLC measured HbA2 percentages despite its widely reported deficiencies. Another important limitation of this study is the use of hospital-based data to determine reference range instead of data generated from a population-based study, lack of use of controls and appropriate matching, done in a community survey. A more sensitive test like PCR-based study will be appropriate to confirm a diagnosis of thalassemia in the absence of a population-based study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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