The β-thalassemia is a hereditary blood disorders, characterized by reduced or absent synthesis of the hemoglobin beta chain that cause microcytic hypochromic anemia. An early diagnosis, economical test, awareness programs and prenatal screening will be a milestone for the eradication of this genetic disorder and to reduce burden of the health sector of a country subsequently the economics. Initially, the diagnosis of β-thalassemia depends on the hematological tests with red cell indices that disclosed the microcytic hypochromic anemia.Hemoglobin analysis shows the abnormal peripheral blood smear with nucleated red blood cells, and reduced amounts of hemoglobin A (HbA). In severe anemia, the hemoglobin analysis by HPLC reveals decreased quantities of HbA and increased the level of hemoglobin F (HbF).The decrease level of MCV and MCH are also associated with β-thalassemia. There are various different molecular techniques such as ARMS PCR, allele-specific PCR, Gap PCR, denaturing gradient gel electrophoresis, reverse dot blotting, DGGE, SSCP, HRM, MLPA, sequencing technology and microarray available to identify the globin chain gene mutations. These molecular techniques can be clustered for detection by mutation types and alteration in gene sequences.
- Microcytic Hypochromic Anemia
- Molecular techniques
There are many tests available for the diagnosis of thalassemia and hemoglobinopathies. However, diagnosis of these conditions that is sufficiently accurate for most of the clinical conditions can usually be established from complete family history (pedigree analysis) and a complete clinical and hematological examination of the patient and their family members.
Generally, doctors everywhere in the world diagnose thalassemia using blood tests which include a complete blood count and special tests for hemoglobin abnormalities. Initially, the primary screening of the thalassemia depends on the complete blood count (CBC), detection of carriers is done by hematological tests with red cell indices and microcytic hypochromic with mild anemia. The high performance liquid chromatography (HPLC) or capillary zone electrophoresis is used for the identification of qualitative and quantitative assessment of hemoglobin (Hb) components. HbA2 test is the most significant identification test for β thalassemia minor, but it can be vary by the presence of defective δ-thalassemia. In earlier days various molecular techniques e.g. amplification refractory mutation specific-polymerase chain reaction ((ARMS-PCR), GAP PCR have been used for the detection of mutations in β- and α-thalassemia, which may help in the prenatal diagnosis of thalassemia hemoglobinopathy in a limited time. Recently the evolution of Next- Generation Sequencing (NGS) has taken an important place for the diagnosis of thalassemia as a confirmatory diagnostic test. The NGS has been introduced for the characterization of both α- and β-thalassemia genes. It gives an accurate diagnosis of thalassemia, although NGS predicts much higher carrier frequencies. The molecular analysis is fundamental to foresee the severe blood transfusion-dependent thalassemia cases to the mild or no blood transfusion. The prenatal diagnosis based on DNA by amniocentesis and chorionic villus sampling (CVS) is equivalent required to detect the genetic abnormalities of the foetus as per expertise. Now a day’s NIPT (Noninvasive prenatal testing) is a technique to identify the genetic abnormalities of the foetus. This testing examines the small remains of foetus DNA that are circulating in a pregnant mother’s blood. An appropriate lab conclusion is urgent for describing the various types of thalassemia hemoglobinopathy with a significant association for prevention and treatment.
This chapter will explain all of these tests, the information of which will be useful for those who are working and interested in the diagnosis of thalassemia hemoglobinopathy.
2. Diagnostic strategies for thalassemia
In many diagnostic labs, the diagnostic strategies have been established for the diagnosis of thalassemia from the simple PCR to NGS for the detection of common, less common and rare thalassemia mutations . Although there are lots of PCR technologies available in the laboratory but most of the diagnostics labs are using the simple and robust technique on allele- specific oligonucleotide hybridization or allele-specific priming, e.g. reverse dot-blotting or ARMS-PCR for the identification of the beta-thalassemia carrier . This approach helps in identifying the common and less common mutations in 90% of cases. The rare mutations will be identifying by secondary screening. The mutation which remains unidentified after these two screenings will be then characterized by DNA sequencing . DNA sequencing is a technique used to identify the specific arrangement of nucleotide bases (A, C, G, and T) in a DNA. The DNA carries the information a cell needs to collect protein and RNA molecule. DNA grouping data is imperative to researchers examining the elements of qualities. DNA sequence information is mandatory to researchers examining the functions of genes .
However worldwide laboratories, it has been noticed that a result of the migration of the natives with different ethnicity has led to increasing the variety of hemoglobinopathy and thalassemia mutations that need to be identified. Because of migration the genetic makeup of the population has mixed, these Hb variations are presently seen everywhere. Thus, the compound heterozygous conditions (Hb D–β thalassemia, Hb E–β thalassemia and Hb Sβ thalassemia) are seen in various places . Molecular indicative research centers in such nations should have the specialized skill, equipment, and diagnostic approach to distinguish an enormous variety of mutations rapidly for prenatal diagnosis, and these labs use DNA sequencing as the principle evaluating strategy for the diagnosis of β-thalassemia point mutations.
The government of the country should make various screening and diagnosis mandatory for the eradication of thalassemia. They are indicated as under mentioned . The country Greece has followed this rule now Greece is a thalassemia free country.
2.1 Premarriage screening
Premarriage screening should be implemented to detect β thalassemia carriers and hemoglobinopathies such as sickle cell trait. In developing country, it is not frequently acceptable because of social stigma and reasons in the general public. The premarriage screening should be possible in universities and colleges, schools, or public places such as theater, shopping mall etc. Earlier it was performed in where the predominance of thalassemia is high. But nowadays due to the mixing up of the gene pool this screening is recommended in all colleges where students are of marriageable age. Our lab has performed this screening in the Tharu tribal area of the Kheri Lakhimpur, Uttar Pradesh, India. We have collected more than 700 samples from the college and carrier screening of sickle cell disease has been performed. It is a belt of HBS .
2.2 Antenatal screening
In antenatal screening irrespective of gestational age of all pregnant women should be screened for carrier status of thalassemia and hemoglobinopathies. The spouse of the affected female should be test for mutation such as α-, β thalassemia and hemoglobinopathies (HbS trait, Hb E trait, Hb D trait etc.). The Prenatal diagnosis have to be encouraged if the foetus is in danger for the having thalassemia mutations e.g. α-, β thalassemia and hemoglobinopathies (HbS trait, Hb E trait, Hb D trait etc.). If the couple found positive for thalassemia during antenatal screening they can decide for prenatal diagnosis and subsequent pregnancy [8, 9, 10, 11].
2.3 Preconception screening
While troublesome circumstances are in the developing country like India, this ought to be done but mostly females frequently do not enroll in antenatal centres before 12 weeks of gestation. A similar methodology with respect to antenatal screening ought to be followed. The preconception screening is important for all couples coming to IVF in infertility clinics. If the female is a thalassemia carrier then her husband or sperm donor should be screened and vice versa [8, 9, 10, 11].
2.4 Neonatal screening
Infant screening is mainly suggested in haemoglobinopathies such as sickle cell diseases, prevalent in tribal and urban populations. If possible, neonatal screening has to be implemented universally where infants are at high-risk for homozygous β-thalassemia and all instances of HbS–β thalassemia. This methodology will miss a couple of instances of sickle β-thalassemia when the mother is a β-thalassemia carrier and the father is a carrier of HbS. All babies with significant hemoglobinopathy should be re-tested using molecular technology to confirm the diagnosis within three months after birth [8, 9, 10, 11].
2.5 Preoperative/pre-anesthesia screening
Preoperative/pre-anesthesia screening of patients is preventive measure where the prevalence of HbS is high. As the presence of sickle hemoglobin might interfere during preoperative and postoperative procedures [8, 9, 10, 11].
3. Screening methods for thalassemia
3.1 Hematological screening
The primary screening of thalassemia is based on (MCV, MCH) values, Hb A2 levels, and F levels by automated blood analyzer, Hb electrophoresis and HPLC, respectively. The secondary screening encompasses further hematological studies to detect suspected variant such as sickle solubility, iron levels and heat instability tests. The
The variations from the typical hematological phenotypes of β-thalassemia carrier include:
Decrease level of MCV and MCH with marginal or normal levels of Hb A2 when person must consider α -thalassemia, heterozygosity for mild β-thalassemia mutations, iron deficiency, heterozygosity for eγδβ-thalassemia.
Normal/Borderline MCV and MCH levels with higher Hb A2 when person must deliberate co-inheritance of alpha and beta-thalassemia.
Normal Hb A2 with Normal or decreased red cell indices but raised Hb F level when person should consider heterozygous δβ -thalassemia or HPFH.
The deranged hematological and biochemical value after primary and secondary screening is confirmed by molecular analysis.
3.2 Biochemical screening
As per the International Committee for Standardization in Hematology (ICSH) in 1978, has recommended three kinds of lab tests for the diagnosis of thalassemia and hemoglobinopathies . In that rule, the screening research facility ought to have the option to perform the alkaline electrophoresis. The reference lab approved by ICSH needed to perform challenging tests like globin electrophoresis and citrate agar electrophoresis. It is necessary to have manual strides all through hemoglobin investigation from reagent availability, electrophoresis, and information examination, and accordingly, the experience of the research centre proficient was a key to fruitful recognizable proof. Late improvement of lab strategies and expanded information on hemoglobinopathy and thalassemia has determined the distribution of refreshed rules . The British Committee for Standards in Hematology suggests possible recognizable proof of hemoglobins on at least two procedures and gets conclusive ID as that dependent on DNA examination, protein sequencing or mass spectrometry.
In early long stretches of finding cellulose acetic acid derivation electrophoresis is an agent custom electrophoresis strategy. It gives distinguishing proof of Hb A, F, S/G/D, C/E, and H and different variations .
HPLC is useful for the finding of β-thalassemia carrier since that HbA2 can be correctly quantitated . Similar other HPLC methods, a wary control of insightful conditions like segment temperature, stream rate, and backup conditions are important.
3.3 Advance molecular techniques for the characterization of thalassemia
There are various different molecular techniques available to identify the globin chain gene mutations. These molecular techniques can be clustered for detection by mutation types such as structural variations (Gene deletion, duplication, or triplication) and alteration in gene sequences (Insertion, substitution, or short insertion/deletions) [18, 19]. Over 90% of α-thalassemia patients are caused by gene deletion. Approximate 10% of the α-thalassemia cases are due to the alteration in the gene sequence such as single nucleotide insertion, deletion or substitution . The α-globin gene group encode identical protein which consists of exceptionally homologous genes as well as 2 HBA genes. The gene deletions in α-thalassemia are mainly cause due to imbalanced crossing over between these homologous regions during meiosis . The most well-known deletion of 3.7 kb and 4.2 kb has been reported . More than 90% of β-thalassemia cases are caused by the alteration in the gene sequence as compared to α-thalassemia. Approximately, 280 gene sequence alterations are related with β-thalassemia in which some mutations are caused by the deletion of gene including the HBB gene .
Gap polymerase chain reaction (PCR) technique is mainly used for the detection of deletion. The southern blotting may be used for unknown deletions with the help of using probes. The MLPA (multiplex ligation-dependent probe amplification) technique can identify both known and unknown deletions. It is commonly used in diagnostics labs for its highly sensitive and is easy to use. Common mutation or alteration in gene sequences can be detected by using techniques such as amplification refractory mutation specific (ARMS) PCR, allele-specific PCR, denaturing gradient gel electrophoresis, reverse dot blotting, DGGE, SSCP, HRM (High resolution melting), sequencing technology and microarray in a cost-effective manner.
Recently, ARMS PCR technology has been improved for the detection of both normal and altered alleles with internal positive control are detected in a single tube assay , referred to as tetra primer ARMS-PCR. Two pairs of primers are used in tetra, ARMS-PCR in which one pair of primers for flanking regions and other pair primers are complementary to different strands. These primers amplify the two different bases that are located in a single position of the globin gene. The different alleles (mutant and wild type) can be detected on an agarose gel based on their sizes (Figure 3). This ARMS PCR technique has been useful in the diagnosis of β-thalassemia mutations. Multiplex ARMS PCR can be screened for more than one mutation a single reaction by multiplexing the ARMS primers attached with a common primer .
The target genomic DNA containing the mutation is amplified by PCR with the help of specific primers. The PCR products are digested by a specific restriction enzyme. The digested PCR products are separated on the agarose gel. The digested PCR products are separated according to their molecular weight (size). Based on restriction site (presence or absence) is determine the size or pattern of PCR products. This RE analysis is simple, relatively economical, and powerful prompting unequivocal outcomes; the RE-PCR-based technique is a precious molecular diagnostic tool. Be that as it may, it is restricted in its application as just an extent of the alpha-thalassemia, β-thalassemia mutation, and hemoglobin variations, naturally generate restriction sites [26, 39].
The multiplex PCR technique that amplify up to 60 probes by using one pair of primer. The PCR amplicon with novel genomic target and length are fluorescently labeled and identified by capillary electrophoresis. The number of genomic sequence of interest is determined by comparing the peak pattern with reference sample .
Since, the varied molecular basis of α-thalassemia and β- thalassemia mutation are uncommon and difficult to detect. In addition to well-established methods, MLPA is known as an effective, simple and unambiguous technique for the identification and classification of deletions and duplications in thalassemia [48, 49, 50].
This is a significant result for the haemoglobinopathies as most cases include carrier testing, subsequently arrangement follows much of the time require checking by eye. There are two diverse sequencing sciences accessible dependent on the Sanger technique [54, 55, 56], dye primer and dye eliminator; they differ from each other in the way wherein the fluorescent level is incorporated during linear cycling. Despite the fact that the dye eliminator is more straightforward to set-up the signal from each nucleotide is less dependable making the dye primer chemistry more suitable for heterozygote detection and it is friendlier with sequence analysis programming. The dye eliminator would have more application in X- linked diseases, for instance, G6PD transformations in which influenced males are hemizygous and will show up as a homozygous change. Taking everything together, the way toward getting a succession from whole blood and the examination may require 4–5 days, with certification using another PCR based test before the change is accounted for. The only drawbacks of using sequencing as a routine investigation technique are the cost and time taking examination compared to PCR. Sequencing is a multistage procedure requiring PCR intensification, cycle sequencing and precipitation before the sequence can be distinguished. After this the sequence ought to be researched and checked and any movements noted. Notwithstanding the grouping examination programming is available it is not 100% compelling at identifying heterozygotes.
Microarray analysis of gene expression has formed into a great tool for the characterization of various pathophysiological processes. The fundamental idea is that RNA isolated from tissue is hybridized to probes for specific genes that are fixed in a grid in small microscopic spots. The microarray is a quick, simple to perform, and precise strategy for concurrent identification of α and β-thalassemias. But, this technique needs should be improved and approved in a bigger number of specimens with hemoglobinopathies before further routine laboratory use [62, 63, 64].
3.4 Prenatal screening
The prenatal diagnosis based on DNA by amniocentesis and CVS sampling is required to detect the genetic abnormalities of the foetus. Now a day’s NIPT is a technique to identify the genetic abnormalities of the foetus. This testing examines the small remains of foetus DNA that are circulating in a pregnant mother’s blood. An appropriate lab conclusion is urgent for describing the various types of thalassemia with a significant association for prevention and treatment. Because of population migration and mixing of the gene pool of different populations in many immigration countries as well as regions the hemoglobiopathies and thalassemia are more prevalent over their [65, 66, 67, 68].
The importance of prenatal diagnosis comes in the diagnostic field as it helps and early diagnoses the growing foetus in the mother wombs for thalassemia and hemoglobinopathies. It plays impartment role in the eradication of these genetic disorders such as β-thalassemia major, sickle cell disease and hemoglobin Bart’s nonimmunehydropsfetalis [69, 70]. The prenatal diagnosis includes the investigation of fetal material from chorionic villi, amniotic liquid, string blood, and fetal DNA in maternal dissemination. In spite of the way that examination of fetal hemoglobin types is successfully performed by means of robotized HPLC, it is assessable through assessment of fetal blood got by cordocentesis and the technique is inclined to mistake because of mixing of sample by maternal tissue .
Advances in molecular testing have worked with the assurance of complex thalassemias and hemoglobinopathies saw in ethnically varying population. Comprehensive screening programs highlighted recognizing carriers and offering prenatal diagnosis in pregnancies for thalassemia have been incorporated in Canada and European countries [72, 73]. Different procedures have been implimented to distinguish thalassemia like genotyping assay, genotyping measur next-generation sequencing and mass spectrometry [74, 75, 76]. The methods are as yet testing; consequently, more investigations are expected to create and approve them and eventually lead to proficient, exact and concrete non-invasive prenatal diagnosis of thalassemia and hemoglobinopathies .
4. Cost comparison for testing
|Low cost techniques||High cost techniques|
|MCV and MCH||Mass spectrometry|
|Molecular Techniques||ASO, RDB,||MLPA|
|ARMS PCR||Direct sequence|
|DGGE and SSCP|
5. Point of care (POC) testing for thalassemia
As there is a saying that prevention is better than cure. The thalassemia and hemoglobinopathies are genetic disorder and due to the migration of population in the different regions and endogamy the new combinations of thalassemia hemoglobinopaty are arising fast. For the eradication and further treatment and management of thalassemia disease an effective diagnostic test at the point of care (POC) is the need of hour.
As thalassemia has been spread worldwide an early diagnosis, economical test, awareness programmes and prenatal screening will be a milestone for the eradication of this genetic disorder and to reduce burden of the health sector of a country subsequently the economics.
The initial hematological, biochemical screening to the advance molecular testing under one roof will not only help to diagnose the thalassemia patients but shall be also helpful in the treatment and management of the disease (Figure 1). The objective of POC for testing thalassemia will be achieved if these quick testing methods like NESTROFT. This has to be in the approachable distance to the patients.
The government should include the thalassemia screening as a mandatory tool for screening. The best example Italy and Cyprus there with the initiation of prenatal diagnosis and screening of the carrier now this is a thalassemia free country.
Likewise in India, the Uttar Pradesh state government has initiated the task by giving free of cost of screening of thalassemia carrier, blood transfusion and iron chelators to several medical colleges. The Indian government has provided the HPLC machines to provide the screening of the carrier of thalassemia hemoglobinopathies. Basically the eradication, treatment and management of thalassemia are joint efforts of government, stake holder, policy makers, pediatrician, pathologist, transfusion medicine and geneticist.