Open access journal of the Ferrata-Storti Foundation, a non-profit organization
Review Articles

Unresolved laboratory issues of the heterozygous state of β-thalassemia: a literature review

Faculty of Graduate Studies, University of Kelaniya, Dalugama, Sri Lanka; Department of Medical Laboratory Science, Faculty of Allied Health Sciences, University of Peradeniya, Peradeniya
Divisional Hospital, Medawala-Haarispatthuwa
Department of Medicine, Faculty of Medicine, University of Kelaniya, Ragama
Vol. 109 No. 1 (2024): January, 2024 https://doi.org/10.3324/haematol.2022.282667

Abstract

Although considered a mild clinical condition, many laboratory issues of the carrier state of β-thalassemia remain unresolved. Accurate laboratory screening of β-thalassemia traits is crucial for preventing the birth of a β-thalassemia major child. Identification of carriers in the laboratory is affected by factors that influence red cell indices and HbA2 quantification. Silent mutations and co-inheriting genetic and non-genetic factors affect red cell indices which decreases the effectiveness of the conventional approach. Similarly, the type of β mutation, co-inheriting genetic and non-genetic factors, and technical aspects, including the analytical method used and variations in the HbA2 cut-off values, affect the HbA2 results, leading to further confusion. However, the combination of mean corpuscular volume, mean corpuscular hemoglobin, and hemoglobin analysis increases the diagnostic accuracy. Diagnostic problems arising from non-genetic factors can be eliminated by carefully screening the patient’s clinical history. However, issues due to certain genetic factors, such as Krüppel-like factor 1 gene mutations and α triplication still remain unresolved. Each laboratory should determine the population-specific reference ranges and be wary of machine-related variations of HbA2 levels, the prevalence of silent mutations in the community.

Introduction

β-thalassemia is one of the most common hereditary blood disorders.1 It is characterized by the reduction of (β+) or absence of (β0) synthesis of the β globin chains of the hemoglobin (Hb) tetramer. More than 200 different mutations of β globin genes are currently recognized.1 Most of the mutations are single nucleotide substitutions, deletions, or insertions of oligonucleotides leading to frameshifts. More rarely, β-thalassemia results from gene deletions. Clinical severity could be affected by the mutation type.2

Based on clinical and hematologic severity, there are three main forms of β-thalassemia syndromes, i.e., the β-thalassemia carrier (trait) state, thalassemia intermedia (non-transfusion-dependent thalassemia), and thalassemia major (transfusion-dependent thalassemia). The current review will focus on β-thalassemia carrier state (BTT).

It has been estimated that about 1.5% of the global population (80-90 million people) are carriers of β-thalassemia.3 β-thalassemia traits have a 25% risk of having children affected with thalassemia major if their partner is also a carrier. The World Health Organization (WHO) emphasizes the importance of incorporating carrier screening in basic health services in countries with a high incidence of thalassemia.4

Hematology laboratories play a vital role in carrier identification. Screening thalassemia traits using blood analysis has two methodological approaches: 1) primary screening followed by a secondary screening; 2) complete screening. In the former approach, only subjects with reduced mean corpuscular volume (MCV) and/or mean corpuscular hemoglobin (MCH) will be assessed for hemoglobin patterns and HbA2 levels. In contrast, hemoglobin analysis is carried out for all subjects from the outset in the latter.5 The first approach has notable disadvantages over its limited advantages, which include low cost. In the presence of mild/silent mutations, β-promoter mutations, α thalassemia and HbD and/or β variants, β-thalassemia carriers may show normal/marginal red cell indices.6 In addition, also megaloblastic anemia and hereditary persistence of fetal hemoglobin (HPFH) can mask the microcytosis of β-thalassemia carriers.7, 8 Other than these clinical conditions, technical problems such as sample storage can affect MCV results. Thus the first approach will misdiagnose a proportion of |3-thalassemia carriers. The second approach is also not foolproof. The conventional method, high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE) are two techniques used for quantifying HbA2. When the same sample is analyzed using both techniques, machine-related variations in HbA2 have been reported.9,10 Factors such as δ-hemoglobinopathies, δβ°-thalassemia, α-thalassemia, triple-α, hemoglobin variants, iron deficiency anemia (IDA), hyperthyroidism, megaloblastic anemia, HPFH, antiretroviral drugs, and Krüppel-like factor 1 (KLF1) gene mutations can affect the HbA2 levels.8,11,12 Labeling a person as BTT based on HbA2 level without considering the above factors may not be reliable. An HbA2 cut-off of 3.5% is generally used for the diagnosis of BTT,13 though different reference ranges for HbA2 are used in some countries.14 HbA2 values between the upper limit of the normal range and the cut-off value are considered borderline. The diagnosis of borderline samples is usually made only by doing confirmatory genetic tests such as polymerase chain reaction and |3-gene sequencing.

The current review aims to discuss these unresolved problems in the BTT state with reference to the laboratory aspects.

Methods

Search strategy

We searched databases of MEDLINE via Pub Med, Google Scholar and Taylor & Francis for research studies published in English over the past 20 years (October 2002 to September 2022) using the following keywords in different combinations: Beta-thalassemia trait, Beta-thalassemia minor, Heterozygous state of beta-thalassemia, Screening carriers, Diagnostic problems, Laboratory issues, HbA2, Borderline, Cutoff, Techniques, Iron deficiency.

Inclusion criteria

Prospective, descriptive, and retrospective studies, including laboratory issues of the |3-thalassemia carrier state in different countries of the world (India, China, Italy, Thailand, Iran, Saudi Arabia, Turkey, Egypt, Malaysia, the US, the UK, Bangladesh, Pakistan, Canada, the Netherlands, the UAE, Portugal, Iraq, Bahrain, Singapore, and Spain) were included in the present review.

Exclusion criteria

Studies including |3-thalassemia traits that did not describe problems in laboratory diagnosis were excluded. In addition, case reports, abstracts, reviews, unpublished studies, and duplicates of previously included studies were also excluded from the present review.

Data extraction

Two researchers (ST and UPJ) independently reviewed all abstracts of journal articles gathered by the web search to identify articles that required full-text review. All selected articles were discussed with a third independent reviewer (AP). Data on study design, objectives, methodology, and results of the selected articles were extracted and methodically reviewed.

Results

Through the investigation strategy, we identified 810 citations, from which 68 articles were selected for qualitative synthesis (Figure 1). The study designs excluding case studies, abstracts, and reviews, included prospective studies, retrospective studies, and short communications. Out of the 68 articles selected, 14 (20.6%) originated from India, ten (14.7%) from Thailand, nine (13.2%) from Italy, eight (11.8%) from China, six (8.8%) from Iran, two (2.9%) each from Malaysia, Saudi Arabia, Pakistan, the Netherlands, and the UK, and one (1.5%) each from Sri Lanka, Bangladesh, Bahrain, Singapore, Egypt, the UAE, Portugal, Iraq, Canada, the US, Turkey, and Spain.

Of these 68 studies, seven (10.3%) analyzed the effectiveness of the conventional approaches of β-thalassemia carrier screening. There were 60 studies (77.9%) focused on factors affecting red cell indices (37 studies) and/or HbA2 value (43 studies). Only one study (1.5%) assessed the conventional cut-off values used in BTT diagnosis.

Effectiveness of the conventional approaches

Out of seven studies that analyzed the effectiveness of conventional hematologic-analysis-based approaches of β-thalassemia carrier screening, 57.1% (n=4) compared the effectiveness of MCV and MCH values in diagnosing BTT based on the first approach.15-18 Sensitivity, specificity, positive predictive values, negative predictive values, and false negatives in different combinations have been used for comparisons. Of four studies, three concluded that MCH is more appropriate than MCV,15,16,18 while one concluded that MCV is more appropriate than MCH.17 Out of seven studies, 14.3% (n=1) showed that using Hb analysis as the first test is unreliable in detecting β-thalassemia traits.19 About 28.6% (n=2) compared the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy among the conventional two approaches for thalassemia carrier detection.20,21 According to both studies, the combination of MCV, MCH and Hb analysis resulted in high sensitivity, specificity, and diagnostic accuracy.

Factors affecting mean corpuscular volume and mean corpuscular hemoglobin among β-thalassemia carriers

Of 37 studies regarding factors affecting RBC indices, 67.6% (n=25) identified silent or mild β-thalassemia carriers with normal or marginal MCV and/or MCH. Of 25 studies, 6 (24%) originated from Italy,6,22-26 5 (20%) from India,14,27-30 4 (16%) from China,31-34 2 (8%) each from Thailand,35,36 Iran37,38 and Pakistan,39,40 one (4%) each from Turkey,41 Bahrain,42 Malaysia,43 and Spain.44 Silent or mild β-thalassemia mutations reported from different countries are listed in Table 1.

Figure 1.Flow chart of article selection for the systematic review.

Out of 37 studies, 13.5% (n=5) analyzed the effect of the coinheritance of α- and β-thalassemia on MCV and MCH.34,35,45-47 SEA type was the most common α-thalassemia type that co-existed with β-thalassemia in three studies from Thailand, China and Singapore while 3.7 kb deletion was the most common type in the other two studies from Thailand and Iran. Of five studies, four (80%) reported a significant increase in MCV and MCH values among heterozygotes for α- and β-thalassemia compared to the simple β-thalassemia carriers.34,35,46,47 The same four studies revealed that this increase in MCV and MCH values is higher when one or two α-globin gene deletions co-existed with β-thalassemia than in other α-gene arrangements. However, in all four studies, mean values of MCV and MCH in heterozygotes for α- and β-thalassemia were less than 80 fL and 27 pg. Out of 37 studies, 2.7% (n=1) showed the effect of fetal Hb in HPFH on MCV and MCH of BTT.8 The study showed a significant increase in MCH and MCV values, accounting for 29% and 30% of their respective variances. This effect was highest in individuals who have inherited two copies of the HPFH quantitative trait loci (QTL), least in those without HPFH, and intermediate in subjects with only one copy of the QTL. However, mean values of MCV and MCH were less than 80 fL and 27 pg even in individuals who have inherited two copies of the HPFH QTL.

In the present review, we observed some non-genetic factors affecting MCV and MCH. Out of 37 studies, 2.7% (n=1) focused on normalized red cell parameters of BTT in patients with megaloblastic anemia.7 Treatment with vitamin B12 and folic acid reduced the mean MCV of patients from 85.28±12.49 to 74.45±9.74. According to 13.5% of studies (n=5), antiretroviral therapy (ART) causes an increase in MCV and MCH among HIV-infected patients.48-52 Pornprasert et al.50 revealed that at 6 and 12 months after the ART therapy, the mean MCV of β-thalassemia patients shifted from microcytic level (<80 fL) to normocytic level (80-100 fL). Mean MCH increased to normal levels (27-31 pg).

Factors affecting HbA2

Of 43 studies focusing on factors affecting HbA2 levels, 34.9% (n=15) reported the type of β-thalassemia mutation itself as one of the factors associated with reduced HbA2 level among β-thalassemia carriers. Four studies (26.7%) from Thailand,36,53-55 three (20%) from Italy,6,11,56 two (13.3%) from China,33,34 two (13.3%) from India,14,30 and one (6.7%) each from Iran,38 Malaysia,57 Saudi Arabia,58 and Portugal59 reported different types of β-thalassemia mutations resulting in borderline or normal HbA2 levels in β-thalassemia carriers.

Out of 43 studies, 39.5% (n=17) investigated the effect of α-thalassemia on HbA2 level. Of 17 studies, two (11.8%) compared pure α-thalassemia traits and normal individuals, and both reported reduced HbA2 levels in α-thalassemia traits.60,61 One study reported that the reduction in HbA2 expression depends on the number of α-globin gene defects.60 Co-inheritance of β-thalassemia with Hb Constant Spring was associated with lower HbA2 expression when compared to other types of α-thalassemia.60 Out of 17 studies, 47.1% (n=8) compared HbA2 levels between |3-thalassemia traits and |3-thalassemia traits co-inherited with α-thalassemia.34-36,45-47,55,60 Although four studies (50%) observed a reduction in HbA2 level when a-thalassemia co-exists,34,35,47,55 only two studies (25%) reported a possible effect of this on BTT diagnosis.47,55 Of these two, one showed that HbA2<4.0% can occur when HbH disease (a-thalassemia intermedia) co-exists with the BTT.55 Surprisingly, out of 17 studies, 47.1% (n=8) reported a marginal increase in HbA2 levels among a-thalassemia traits without β-thalassemia heterozygosis.6,11,33,38,54,55,57,58 None of the studies could explain the underlying mechanism.

Table 1.Silent/mild β-thalassemia mutations causing normal or marginal mean corpuscular volume.

Of the studies that investigated factors affecting HbA2, 13.9% (n=6) from four different countries (Italy,6,11,56 India,62 Thailand,60 and Portugal59) identified that co-inheritance of δ-hemoglobinopathy decreases the HbA2 level in |3-thalassemia carriers resulting in misdiagnosis. About 11.6% (n=5) evaluated the effect of a-gene triplication on the HbA2 level. Out of these five studies, four reported the presence of an aaa condition in healthy individuals with borderline increased HbA2 levels but without a |3-globin mutation.6,11,33,34 Only one study stated that a-gene triplication does not affect hematologic parameters.63 Similarly, out of 43 studies, only 2.3% (n=1) found that co-inherited HPFH in BTT reduced HbA2 levels.8 The effect increased with the number of copies of the HPFH allele.

Similarly, 18.6% (n=8) eligible studies commented on the effect of KLF1 gene mutations. KLF1 or Erythroid KruppelLike Factor is a zinc-finger transcription factor that has different functions in erythropoiesis including modifying chromatin architecture, regulating |3-like globin gene switching, and activating or repressing gene transcription.64 Of eight studies on the effect of KLF1 gene mutations, seven (87.5%) observed a high prevalence of KLF1 gene defects among individuals with borderline increased HbA2 levels without an HBB gene mutation.12,26,33,34,54,65,66 They concluded a possible effect of KLF1 gene mutations on increased HbA2 levels among normal individuals. Only one study (12.5%), which was a cross-sectional analysis in Saudi Arabia, reported that KLF1 gene mutations are not only associated with borderline high HbA2 results but also normal (HbA2<3%) and high (HbA2>4.3%) results.58 Out of seven studies which determined the effect of KLF1 gene mutations on HbA2 level, the studies of Perseu et al.,12 Hariharan et al.,65 and Liu et al.66 revealed 0.8%, 1.1%, and 0.5% increases, respectively, in HbA2 level among individuals with KLF1 gene mutations when compared to the group without KLF1 gene mutations. Interestingly, Liu et al.66 had shown a high prevalence of KLF1 genes in β-thalassemia endemic regions in China and an amelioration of the severity of β-thalassemia by KLF1 gene changes.66 We were able to determine the most common KLF1 mutations in different countries; G176Afsx179 was the most common KLF1 mutation in China and Thailand, while p ser. 270 and -148 (G<A) were the most common in Italy and India, respectively. In the present study, we analyzed the effect of non-genetic factors on HbA2 level. Out of 43 studies, 20.9% (n=9) investigated the association between iron deficiency anemia (IDA) and HbA2 levels. Of these nine studies, four (44.4%) determined the effect of IDA on HbA2 results of normal (non-thalassemic) individuals.56,60,67,68 All concluded that IDA causes a significant reduction in HbA2 levels in normal individuals. Eight studies (88.9%) compared the results of HbA2 among β-thalassemia traits with and without IDA. Out of these eight studies, five (62.5%) showed significantly higher HbA2 values for β-thalassemia traits without IDA than in those with IDA;30,61,67,69,70 in the other three, no difference was observed.56,68,71 Of five studies that observed higher values, three (60%) suggested possible effects on β-thalassemia diagnosis,30,61,67 while others showed no effect due to the steadying of all HbA2 values above the cutoff.69,70 Among those studies that suggested possible effects, one proposed a dose effect of severity of iron deficiency on HbA2 level of β-thalassemia traits.61

Out of 43 studies, 4.6% (n=2) reported the effect of megaloblastic anemia in elevated HbA2 levels among normal individuals without β-thalassemia.7,6 8 One of the above studies reported a change of mean HbA2 of the study group from 4.56% to 3.81% with vitamin B12 and folic acid treatments, whereas 93.75% of subjects diagnosed as BTT (HbA2>3.5%) before treatment became normal (HbA2<3.5%) after the treatment.7 Among 43 studies that discussed factors affecting HbA2 levels, 2.3% (n=1) indicated the effect of thyroid hormones on the production of HbA2.72 According to the results, HbA2 was significantly higher in hyperthyroid (mean: 2.77%) patients than in the controls (2.39%). However, this increase in HbA2 did not pass the cut-off level of HbA2, meaning there was less chance of misdiagnosing BTT. Five studies analyzed the effect of antiretroviral therapy (ART) on HbA2 results.48-52 All of them showed an increase in HbA2 levels among HIV-infected patients treated with ART leading to a misdiagnosis of BTT. Of five studies, three (60%) confirmed that zidovudine increased HbA2 levels.48,51,52 Bhagat et al.48 evaluated the effect of three different drugs, and concluded that both zidovudine and stavudine increase HbA2 levels, while tenofovir had no effect. In the present review, we observed technical issues due to the variations in analytical methods used to determine HbA2 level in β-thalassemia screening. Out of 43 studies, 6.9% (n=3) compared HbA2 results by different analytical systems in the presence of Hb variants.9,10,73 All three studies determined falsely increased HbA2 in HPLC reports of patients with HbE and HbS variants. However, the CE method clearly separated HbE and HbA2. One study reported the variant Hb Lepore, which co-elutes with HbA2 in the HPLC method causing problems in diagnosis.73 Similarly, when the HPLC technique is used, falsely reduced HbA2 had been observed in the presence of HbD Punjab in two studies.9,73 According to two studies, HbA2 peak was included in HbC peak when the CE method was used.9,10 Interestingly, all three studies reported variations in HbA2 values between HPLC and CE methods when comparing HbA2 measurements in samples within the normal range. Two studies reported higher HbA2 values by the Bio-Rad variant II HPLC method (mean: 2.95% and 2.67%, respectively) than the Sebia CE method (mean: 2.49% and 2.51%, respectively).9,73 On the contrary, one study reported HbA2 levels by the Sebia CE method (mean: 2.8%) was higher than that of the Primus HPLC method (mean: 2.3%).10

Problems with HbA2 cut-off values

In the present review, we analyzed the variations in cutoff values of HbA2 used in β-thalassemia screening. Out of 68 studies, 54.4% (n=37) articles (excluding studies based on borderline HbA2 values) clearly mentioned the HbA2 cut-off value used in diagnosing β-thalassemia carriers. Of 37 articles, the majority (48.6%; n=18) used 3.5% as the cut-off value, while 32.4% (n=12) of studies used 4.0% as the cut-off. Eighteen studies that used 3.5% as the cut-off include five from China,18,21,32,34,70 five from Iran,15,16,37,47,67 two from Thailand,46,53 one each from India,51 Bangladesh,20 Saudi Arabia,19 Portugal,59 the UAE,61 and the UK.52 Similarly, 12 studies that used 4.0% as the cut-off include four from India,7,17,27,48 four from Thailand,35,49,51,55 and one each from Malaysia,43 Bahrain,42 Italy,24 and the Netherlands.73 A 3.9% cut-off was used for three studies from India (n=2)68,71 and Italy (n=1).12 Two studies from Italy26 and Turkey41 used 3.8% as the cut-off while 3.4%, 3.6%, and 3.7% were used as the cut-off values by three separate studies from Italy,56 Thailand,36 and India,28 respectively. Abdel-Messih et al.74 determined the effectiveness of the two most common cut-off values used in BTT screening. The results revealed the sensitivity (100.0% and 97.4%), specificity (70.0% and 72.7%), positive predictive value (75.0% and 92.6%), negative predictive value (100.0% and 88.8%), and accuracy (70.0% and 92.0%) in identifying β-thalassemia carriers at 3.5% and 4.0% cut-off values, respectively.

There were 16 (23.5%) studies which analyzed borderline HbA2 results in BTT diagnosis; nine different borderline ranges were identified among these studies (Table 2).

Discussion

The diagnosis of β-thalassemia using red cell indices as the primary screening or using HPLC or CE as the first test each seem to have their own deficiencies. In the first approach, MCV and MCH are crucial hematologic parameters to rule out possible β-thalassemia traits. Several studies reported that MCH is more appropriate than MCV.15,16,18 Most of these studies generate sensitivity figures based on their own cut-off values for MCV and MCH. MCH is considered to be more stable in ambient temperature while MCV increases with time after being stored for several hours before testing;75 this stability of MCH may be the reason for its higher sensitivity. Some clinicians use only MCV to exclude β-thalassemia in the primary screening.36 In such cases, falsely high MCV values due to stored samples may give misleading results. Therefore, it is important to consider not one but both MCV and MCH results in screening for β-thalassemia. Reduction in any parameter should be considered as having diagnostic importance.

One of the main disadvantages of using red cell indices for screening includes the possibility of missing silent β-thalassemia carriers, as red cell indices may be normal in these individuals. Silent β-thalassemia mutations exist not only in codons, but also in the promoter region and/or the cap region of the HBB gene.24,28,41,76 Screening for thalassemia based on red cell indices will also result in missing individuals with HbD trait and individuals with triple α genes.77 Although phenotypically not important, and globally rare, the co-inheritance of the above conditions with |3-thalassemia mutations leads to severe phenotype in the offspring.

In the present review, we analyzed the multitude of factors that may affect HbA2 levels, and thus the diagnosis of β-thalassemia (Table 2). The problems may arise in two circumstances: decreased HbA2 levels in β-thalassemia traits, or deceptively elevated HbA2 levels in non-carriers. The type of |3-thalassemia mutation is the main reason for reduced or borderline HbA2 levels among carriers.30 Silent or mild |3-thalassemia mutations show reduced/borderline HbA2 levels along with normal or slightly reduced RBC indices. Most of the time, homozygotes for classic and silent |3-thalassemia mutations have thalassemia intermedia phenotype.76,78 Unfortunately, silent carriers will be missed until the birth of a child with |3-thalassemia intermedia. Co-inheritance of α-thalassemia with |3-thalassemia results in decreased HbA2 levels due to the unavailability of a free α-chain pool to partner with δ-globin chains.2 This reduction seems insufficient to cause problems in BTT diagnosis36,60 unless mild or silent |3-thalassemia also coexists. Satthakar et al.55 showed HbA2 less than 4.0% only in HbH and HbH-CS diseases; since both these conditions are apparent in HPLC patterns, |3-thalassemia traits will not be missed. Surprisingly, borderline increases in HbA2 levels have also been identified among individuals with α-thalassemia but without β-thalassemia mutations.33,55,58 The exact reason for that has still not been discovered, while some researchers consider regulation factors or mutations in regulatory genes to be causes.33,38 Co-inheritance of δ-hemoglobinopathy60 and HPFH8 cause reduced HbA2 levels in |3-thalassemia carriers. The synthesis of insufficient amounts of δ-globin monomers results in lower HbA2 levels in δ-hemoglobinopathy. On the other hand, co-inheritance of heterocellular HPFH leads to a primary increase in γ-chain synthesis resulting in high HbF levels.8 However, the reason for decreased HbA2 level is not clear.

The effect of α-triplication and KLF1 gene mutations on increased HbA2 levels is contradictory. α-triplications are considered uncommon because de novo crossover events generating triplications are rare.63 The KLF1 gene has a regulatory effect on erythropoiesis.79 Perseu et al.12 first identified a delay in the transcriptional switch from the HBD to the HBB gene by KLF1 mutations. The majority of the studies regarding α-triplication and KLF1 gene mutations in the current review reported possible effects on increased HbA2 levels. Most of these studies have specifically selected cohorts with borderline HbA2 results; because of the marginally increased HbA2 levels and absence of β-thalassemia mutations, possible effects have been hypothesized. Among eligible studies, there were a few of a cross-sectional nature, with some studies including members of the general population, but they revealed no association between increased HbA2 level and α-triplication/KLF1 mutations.58,63 Therefore, larger scale studies that include the general population are needed in order to gather more comprehensive and informative data. IDA is the major cause of anemia in the world.80 Co-existence of IDA may decrease the HbA2 level in |3-thalassemia traits. Intracellular lack of iron reduces α-globin chain synthesis more than non-α-globin chains. In |3-thalassemia, β-globin chain synthesis is also limited. Therefore, |3 chains compete more effectively for α-globin chains than δ-globin chains, resulting in reduced levels of HbA2.81 It is widely believed that IDA cannot reduce HbA2 levels of |3-thalassemia traits below the cut-off values. If this is the case, the effect of IDA on |3-thalassemia diagnosis would be minimal, except in cases of severe IDA or silent/mild |3-thalassemia mutations.

Table 2.Factors affecting HbA2 results of high-performance liquid chromatography and capillary electrophoresis methods.

Megaloblastic anemia, ART therapy, and hyperthyroidism are other non-genetic factors which increase HbA2 levels in β-thalassemia traits. Both megaloblastic anemia and long-term ART therapy (in particular, zidovudine and stavudine) affect red cell synthesis. Nuclear maturation of red cell precursors is delayed in megaloblastic anemia, while antiretroviral medications inhibit nucleic acid synthesis.50 So, more Hb synthesis occurs in immature erythroid precursors. Since the synthesis of ö chains is relatively greater in these cells, HbA2 level is increased.81 Besides this, thyroid hormones are considered to specifically increase the expression of ö globin resulting in high HbA2 levels in hyperthyroidism.72 No literature was found which demonstrated an increased HbA2 level above the cut-off in hyperthyroidism. Nevertheless, a careful medical history of the individual is warranted prior to β-thalassemia screening.

Table 3.Nine different borderline ranges for HbA2 in diagnosing β-thalassemia traits by 16 studies included in the present review.

There are machine-related variations in HbA2 results of normal individuals or β-thalassemia traits. A comparison of the Bio-Rad variant II HPLC and Sebia CE methods showed consistently high results have been obtained from the HPLC method,9,73 although there is good correlation between the two. When using a single cut-off value to diagnose β-thalassemia carriers, this will be a major problem, especially in borderline HbA2 results. Therefore, when issuing a diagnosis of BTT based on borderline values, machine-related variation of the HbA2 level should be borne in mind. The main reason for this unresolved problem is the non-availability of standardized HbA2 results between methods. Non-availability of a reference method and international reference HbA2 calibrators makes this problem more complex.9 In addition, in the present review, we identified marked differences in the HbA2 cut-off values (3.0-4.0%) used for diagnosis of BTT. Although it is recommended to define population or country specific cut-off values, sometimes 2 or 3 different cut-offs have been used within a single country. Similarly, different authors have defined borderline ranges arbitrarily. In the present review, the percentages of β-thalassemia heterozygotes in each borderline group ranged from 1.9% to 83.3%, which is very broad (Table 3); the reason may be the differences in inclusion/exclusion criteria of the study group and variations in the analytical methods used to identify β-thalassemia mutations. However, it is noteworthy that all these β-thalassemia traits would be missed if HbA2≥4.0% was used as the cut-off. We believe a cut-off with a higher sensitivity than specificity is suitable for screening to prevent missing β-thalassemia traits. If problems arise in the accurate diagnosis of β-thalassemia heterozygosis due to the borderline results, the parental studies may provide prompt solutions. If not, further genetic or molecular tests are necessary.

Conclusions and recommendations

Many laboratory issues of the heterozygous state of β-thalassemia remain unresolved. During the screening of β-thalassemia, both RBC indices and HbA2 level should be taken into consideration. Population-specific reference ranges and machine-related variations in HbA2 level should be determined within the laboratory. Borderline HbA2 levels should be further investigated for factors affecting HbA2 levels. Careful screening of the patient’s clinical history can identify most non-genetic factors that affect HbA2 screening. IDA and α-thalassemia are unlikely to affect the BTT diagnosis unless in individuals with silent β mutations. Based on the prevalence of silent mutations and other co-inheriting genetic factors, the diagnostic approach of BTT should be modified for each population. For example, screening the partners of patients with β-thalassemia heterozygosity for common silent β mutations, HbE, HbD, etc. is possible. However, effects of some genetic factors, such as KLF1 gene mutations and α-triplication on HbA2 level are still controversial, and further investigations are needed. Moreover, there may be other as yet unidentified factors, such as the environment, lifestyle, regulation factors or mutations in regulatory genes affecting HbA2 level. Further studies should aim to identify these factors and should include large numbers of participants.

Footnotes

  • Received February 15, 2023
  • Accepted March 19, 2023

Correspondence

S. Thilakarathne shyamali5thilakarathne@gmail.com shyamali.thilakarathne@ahs.pdn.ac.lk

Disclosures

No conflicts of interest to disclose.

Contributions

ST and UPJ wrote the first version of the manuscript. AP revised the manuscript critically for important intellectual content. All authors read, revised, and approved the final manuscript.

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Vol. 109 No. 1 (2024): January, 2024 : Review Articles
 
DOI
https://doi.org/10.3324/haematol.2022.282667
Pubmed
37259577
 
Published
2023-06-01
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Ferrata Storti Foundation, Pavia, Italy
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0390-6078
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1592-8721
 
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