Methods for hemoglobin analysis and hemoglobinopathy testing

INTRODUCTION — Hemoglobin (Hb), the abundant oxygen-carrying protein found within red blood cells (RBCs), is a tetramer composed of two alpha-like and two beta-like globin chains, each globin chain having an associated heme group. The hemoglobinopathies are a complex group of inherited blood disorders in which one or more genetic abnormalities results in a change in the amount, structure, or function of one or more of the globin chains. In contrast, abnormalities in heme synthesis result in sideroblastic anemias and porphyrias.

The most common hemoglobinopathies include the thalassemias (alpha- and beta-) and sickle cell disease (SCD), but hundreds of abnormalities in the globin genes have been described.

The diagnosis of hemoglobinopathies requires clear understanding of the genetics and structure of the globin chains and Hb. Timely and accurate diagnosis of hemoglobinopathies is important to inform optimal management in affected individuals and to offer genetic counseling and reproductive options in carriers.

This topic reviews methods for hemoglobinopathy testing and their appropriate use. Additional information about the diagnosis of specific disorders and an overview of prenatal hemoglobinopathy testing are presented separately:

●Sickle cell trait and SCD – (See "Diagnosis of sickle cell disorders".)

●Alpha and beta thalassemias – (See "Diagnosis of thalassemia (adults and children)".)

●Hemoglobin C, D, E, and other less common variants – (See "Hemoglobin variants including Hb C, Hb D, and Hb E".)

●Unstable hemoglobin variants – (See "Unstable hemoglobin variants".)

●High oxygen affinity and low oxygen affinity variants – (See "Hemoglobin variants that alter hemoglobin-oxygen affinity".)

●Methemoglobinemia – (See "Methemoglobinemia".)

●Prenatal screening and testing – (See "Hemoglobinopathy: Screening and counseling in the reproductive setting and fetal diagnosis".)

HEMOGLOBIN BIOLOGY

Normal hemoglobins — Human Hbs are tetrameric proteins composed of two pairs of globin chains, each of which contains one alpha-like (α-like) chain and one beta-like (β-like) chain. Each globin chain is associated with an iron-containing heme moiety (figure 1). Throughout life, the synthesis of the alpha-like and the beta-like (also called non-alpha-like) chains is balanced so that their ratio is relatively constant and there is no excess of either type [1]. (See "Structure and function of normal hemoglobins".)

The specific alpha- and beta-like chains that are incorporated into Hb are highly regulated during development (figure 2):

●Embryonic Hbs are expressed as early as four to six weeks of embryogenesis and disappear around the eighth week of gestation as they are replaced by fetal Hb [2,3]. Embryonic Hbs include:

•Hb Gower-1, composed of two ζ globins (zeta globins) and two ε globins (epsilon globins; ζ₂ε₂)

•Hb Gower-2, composed of two alpha globins and two epsilon globins (α₂ε₂)

•Hb Portland, composed of two zeta globins and two gamma globins (ζ₂γ₂)

●Fetal Hb (Hb F) is produced from approximately eight weeks of gestation through birth and constitutes approximately 80 percent of Hb in the full-term neonate. It declines during the first few months of life and, in the normal state, constitutes <1 percent of total Hb by early childhood. Hb F is composed of two alpha globins and two gamma globins (α₂γ₂).

●Adult Hb (Hb A) is the predominant Hb in children by six months of age and onward; it constitutes 96 to 97 percent of total Hb in individuals without a hemoglobinopathy. It is composed of two alpha globins and two beta globins (α₂β₂).

●Hb A₂ is a minor adult Hb that normally accounts for approximately 2.5 to 3.5 percent of total Hb from six months of age onward. It is composed of two alpha globins and two delta globins (α₂δ₂).

The regulation of the globin genes and the developmental switching from one type of Hb to another is discussed in detail separately. (See "Structure and function of normal hemoglobins".)

Types of abnormalities — Hemoglobinopathies include a heterogeneous group of variants caused by genetic alterations in one or more of the different globin genes and their regulatory regions. Many of the clinically important hemoglobinopathies are caused by missense mutations in the coding sequence of a globin gene (single nucleotide substitutions that alter the amino acid sequence of the resulting protein). Copy number variation (eg, deletion, duplication, insertion) is also a common genetic cause of Hb disorders, and complex rearrangements and globin gene fusions can also occur. Mutations in the non-coding (regulatory) regions of the globin genes or their locus control regions that impair the transcription, splicing, and post-transcriptional stability of the messenger RNA are common causes of thalassemia. Over a thousand specific mutations have been described; a listing is available on the Globin Gene Server. Some of the specific defects are discussed in more detail separately. (See "Hemoglobin variants including Hb C, Hb D, and Hb E".)

Hemoglobinopathies can be broadly classified into structural Hb variants (qualitative defects), in which the genetic defect gives rise to a structural and often a functional change in Hb; and thalassemias (quantitative defects), in which the genetic defect leads to a quantitative change in the amount of globin chain produced (figure 3). The disease mechanisms and clinical expression of the hemoglobinopathies also vary. In general, most clinically significant Hb structural variants cause hemolysis in peripheral blood. In contrast, beta thalassemias are predominantly characterized by ineffective erythropoiesis and destruction of erythroid precursors in the bone marrow. Some conditions, like Hb H disease (a form of alpha thalassemia), cause both hemolysis and ineffective erythropoiesis. Anemia may be severe, such as in transfusion-dependent alpha or beta thalassemia (TDT; formerly called thalassemia major). Sickle cell disease (SCD) also causes vaso-occlusive phenomena. In addition to these clinical differences, there are also differences in the approach to diagnostic testing for suspected hemoglobinopathies, which are discussed below. (See 'Available testing methods' below and 'Initial evaluation' below.)

●Hb variants – Hb structural variants are qualitative defects that cause a change in the structure (primary, secondary, tertiary, and/or quaternary) of the Hb molecule. The majority of Hb variants do not cause disease and are most commonly discovered either incidentally or through newborn screening. A subset of Hb variants can cause severe disease when inherited in the homozygous or compound heterozygous state in combination with another structural variant or a thalassemia mutation. Clinical consequences include anemia of several causes (hemolysis, ineffective erythropoiesis, decreased Hb oxygen affinity) or polycythemia due to increased Hb oxygen affinity. Common examples of hemoglobin variants associated with hemolysis include sickle Hb (Hb S) and Hb C. Hb variants can usually be detected by protein-based assay methods; however, DNA-based methods may be required for variants with ambiguous or unusual results from protein-based testing. (See 'Protein chemistry methods' below and 'Molecular genetic (DNA-based) methods' below.)

The major functional consequences of Hb structural variants can be classified as follows:

•Change in physical properties (solubility) – Common beta globin mutations can alter the solubility of the Hb molecule: Hb S polymerizes when deoxygenated and Hb C crystalizes. (See "Pathophysiology of sickle cell disease", section on 'Hb S polymerization and fiber formation'.)

•Reduced protein stability (instability) – Unstable Hb variants are mutations that cause the Hb molecule to precipitate, spontaneously or upon oxidative stress, resulting in hemolytic anemia. Precipitated, denatured Hb can attach to the inner layer of the plasma membrane of the red blood cell (RBC) and form Heinz bodies. (See "Unstable hemoglobin variants" and 'Heinz body prep and other tests for unstable hemoglobins' below.)

•Change in oxygen affinity – High or low oxygen affinity Hb variants are more likely than normal to adopt the relaxed (R, oxy) state or the tense (T, deoxy) state, respectively. High oxygen affinity variants (R state) may cause relative erythrocytosis (eg, Hb Chesapeake, Hb Montefiore). Some low oxygen affinity variants can cause anemia (although tissue oxygen delivery is normal) and even cyanosis due to desaturation. Examples include Hb Kansas and Hb Beth Israel. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity".)

•Oxidation of heme iron – Mutations of the heme binding site, particularly those affecting the conserved proximal or distal histidine residues, can produce M-hemoglobins, in which the iron atom in heme is oxidized from the ferrous (Fe²⁺) state to the ferric (Fe³⁺) state, with resultant methemoglobinemia.

●Thalassemias – Thalassemias are quantitative defects that lead to reduced levels of one type of globin chain, creating an imbalance in the ratio of alpha-like chains to beta-like chains. As noted above, this ratio is normally tightly regulated to prevent excess globin chains of one type from accumulating. The excess chains that fail to incorporate into Hb form nonfunctional aggregates that precipitate within the RBC. This can lead to premature RBC destruction in the bone marrow (especially in beta thalassemia) and/or in the peripheral blood (especially in alpha thalassemia). (See "Molecular genetics of the thalassemia syndromes" and "Pathophysiology of thalassemia".)

•Beta thalassemias – Beta thalassemias are characterized by reduced levels of beta globin chains. Beta thalassemias are typically caused by point mutations that disrupt regulatory elements of beta globin gene expression. Beta thalassemia mutations are classified as beta⁺ (β⁺) alleles if some beta globin is produced, or beta⁰ (β⁰) if beta globin production is absent from that gene. (See "Diagnosis of thalassemia (adults and children)", section on 'Overview of subtypes and disease severity'.)

•Alpha thalassemias – Alpha thalassemias are characterized by reduced levels of alpha globin chains. The majority of alpha thalassemias are caused by large deletions of one or both of the alpha globin genes (HBA1 and HBA2), which are located in tandem on chromosome 16 (figure 1). With a normal total of four alpha globin genes, the alpha thalassemias are broadly classified based upon numbers of genes deleted or mutated. One- and two-gene deletions are the most common and are manifested as either the silent carrier state (αα/–α) or alpha thalassemia trait (-α/-α, αα/--). Given their high frequency, it is not uncommon for gene deletions of HBA1 or HBA2 to be co-inherited with beta globin gene mutations. Large deletions that result in loss of both HBA1 and HBA2 on the same chromosome are prevalent in individuals with Southeast Asian ancestry, and when these large deletions are co-inherited with a single alpha gene deletion from the other parent, the result is Hb H disease (three total gene deletions or pathogenic variants; -α/--). Hemoglobin H is a tetramer composed of four beta globin chains. Homozygous inheritance of large deletions involving both alpha globin genes (ie, loss of all four alpha genes) results in alpha thalassemia major or "hydrops fetalis." (See "Pathophysiology of thalassemia".)

The thalassemias are genetically complex and heterogeneous, so the above classification is simplistic. Some other causes of thalassemia include variants that result in altered Hb structure. An example is Hb Lepore, a fusion Hb in which the 5’ δ-globin sequences are fused to 3’ β-globin sequences with deletion of the intergenic DNA, which places the δβ-fusion gene under the transcriptional control of the inefficient δ-globin promoter, causing low expression of the fusion globin. Examples of mutations that create an amino acid change and an aberrant splice site include Hb E, Hb Knossos, and Hb Malay; such mutations produce both qualitative and quantitative abnormalities. Other types of mutations cause "hyper-unstable" globins, in which the nascent globin chains are highly unstable and undergo rapid proteolytic degradation, resulting in a secondary quantitative (thalassemic) disorder [1-4]. (See "Hemoglobin variants including Hb C, Hb D, and Hb E".)

Pathogenic variants or deletions in gamma, delta, and other globin genes may also give rise to a spectrum of disorders ranging from clinically benign hereditary persistence of fetal Hb (HPFH) to symptomatic delta-beta (δβ) thalassemia. (See "Diagnosis of thalassemia (adults and children)" and "Fetal hemoglobin (Hb F) in health and disease".)

Initial screening for thalassemia can be performed using protein-based methods, which may show alterations in the ratio of normal Hbs (eg, increased Hb F or Hb A₂); however, confirmation of the diagnosis of alpha and beta thalassemia requires DNA analysis. (See 'Molecular genetic (DNA-based) methods' below.)

AVAILABLE TESTING METHODS

Overview of methods — Methods for analyzing Hbs continue to advance, and a number of techniques suitable for hemoglobinopathy detection are routinely available [5]. These methods can be broadly divided into protein-based (protein chemistry) and DNA-based (molecular genetic) methods. An overview of the advantages and disadvantages of these approaches is presented in the table (table 1).

Historically, gel-based electrophoresis methods were used to evaluate many common hemoglobinopathies. Electrophoresis can resolve many normal and abnormal Hbs, such as Hb A, Hb A₂, Hb F, Hb S, and Hb C. Gel-based electrophoresis is routinely used in many laboratories; however, high-pressure liquid chromatography (HPLC; also called high performance liquid chromatography), capillary zone electrophoresis (CZE), and isoelectric focusing (IEF) have increasingly become the first-line methods of choice. HPLC and CZE are most commonly employed in high-volume laboratories due to their ability to process large numbers of samples in relatively small amounts of time.

Information from the family history and parent testing including complete blood count (CBC), reticulocyte count, and peripheral blood morphology combined with one or more protein-based methods, is often sufficient for diagnosis of common Hb variants, but not always. DNA-based methods are especially important for diagnosing alpha thalassemias and beta thalassemias. Indeed, they have high clinical utility for the diagnosis of all types of hemoglobinopathies (thalassemias and structural variants).

Protein chemistry methods

Overview of protein chemistry methods — Protein-based methods remain the method of choice for initial qualitative and quantitative Hb analyses (ratios of different Hbs, demonstration of variant Hbs, and absolute quantification of the amounts of Hb A₂ and Hb F). Several protein chemistry techniques are routinely used in clinical laboratories, including HPLC, CZE, and IEF, and alkaline and acid gel electrophoresis. Most laboratories will use one of these methods as a first step to identify an Hb variant, followed by a second and sometimes third protein-based technique to confirm an abnormal result on initial testing (table 1).

Results of all protein-based methods are affected by the presence of transfused blood, requiring >3 months from the last transfusion to determine the patient's endogenous Hb phenotype.

The figures show examples of typical findings:

●HPLC (figure 4 and figure 5 and figure 6)

●CZE (figure 7)

●IEF (figure 8 and figure 9)

●Gel electrophoresis (figure 4 and figure 10)

When results are reported, especially in the context of newborn screening for hemoglobinopathies, the detected Hbs are usually listed in the order of abundance (from the highest to the lowest amount). As examples:

●"AS" indicates that the amount of Hb A is greater than the amount of Hb S, consistent with sickle cell trait.

●"SA" indicates the amount of Hb S is greater than the amount of Hb A, consistent with sickle-beta⁺ thalassemia.

The relative abundance (percentage) of each Hb should also be reported.

Advantages of HPLC and CZE include excellent resolution of Hb fractions (both normal and variant Hbs), high reproducibility, capacity for automation, requirement for a very small blood sample, and rapid turnaround time. These advantages have led to the widespread adoption of these techniques. HPLC and CZE can be used as complementary methods to resolve ambiguous results, and Hbs that are not detectable by one method may be resolved by the other. IEF and gel electrophoresis are less quantitative and thus less useful for accurately quantifying Hbs present in low concentrations. However, they function well for identifying several common hemoglobinopathies.

HPLC — Cation-exchange HPLC separates many normal and variant Hbs, including Hbs A, A₂, F, S, C, O-Arab, D-Punjab, and G-Philadelphia (figure 4 and figure 5 and figure 6). HPLC permits presumptive identification of many more Hb variants than can be distinguished by gel-based electrophoresis (but not CZE). HPLC can quantify normal and abnormal variants and can incorporate internal controls for the most common variants, and results can be compared with an available reference library of unusual variants (retention times for any given Hb can differ across HPLC platforms, so one must refer to each manufacturer's specific documentation for Hb identification).

In HPLC, Hbs are adsorbed to a negatively charged resin column (stationary phase) and then eluted from the column by a positively charged solution that is added in increasing concentration and competes for binding to the negatively charged resin. Hbs are thus differentially eluted at a rate related to their affinity for the column and detected by a photometer. The resulting elution patterns (Hb peaks) are graphically represented in defined "windows" based on their relative time to appearance in the eluate (retention time). Retention times for different Hb fractions differ based on the HPLC equipment and software that is used; one should consult the manufacturer's specific reference for each instrument to identify Hb fractions based on retention times. The size of the Hb fractions can be quantified by computing the area under the curve for each peak.

HPLC is a preferred method to identify beta thalassemia because it accurately quantifies Hb A₂ and Hb F. HPLC is also suitable for population screening for hemoglobinopathies because it can be greatly automated. An example is prenatal screening for hemoglobinopathies (as performed in the United Kingdom) or primary newborn screening, especially in populations with high frequencies of beta thalassemia or alpha thalassemia. HPLC is suitable for beta thalassemia carrier detection because it can easily quantify Hb A₂ and Hb F. HPLC is also effective for settings in which quantification is important, such as assessing Hb S and Hb F percentages in individuals receiving transfusion or hydroxyurea therapy for sickle cell disease. (See 'Population screening (eg, routine newborn screen)' below.)

Disadvantages of HPLC include overlap (poor resolution; very similar or identical retention times) of some Hbs. As an example, Hb E, Hb Korle-Bu, and Hb Lepore may all overlap with Hb A₂, so accurate quantification of each fraction cannot be performed. Hb A₂ may also be falsely elevated in the presence of Hb S (due to post-translational Hb adducts). Separation of Hbs varies somewhat between instruments, reagents, and programs used for HPLC. The presence of glycosylated or acetylated Hbs and artifacts may yield complex patterns, and interpretation should be performed by experienced laboratory personnel.

Rather than separation of intact Hb tetramers, cation-exchange HPLC (and the electrophoretic techniques discussed in the next section) resolve or separate different Hbs as dimers (alpha:beta and alpha:gamma dimers). To individually resolve alpha, beta, gamma, and other globin monomers, reverse-phase HPLC can be used. This is referred to as globin chain separation. Globin chain separation may be useful in the investigation of certain non-deletional forms of HPFH (to determine the relative abundance of the Gγ globin and Aγ globin chains). (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Hereditary persistence of fetal hemoglobin (HPFH)'.)

Capillary zone electrophoresis — Capillary zone electrophoresis (CZE) can also be used as a first-line testing for hemoglobinopathies, for primary newborn screening, or as a complementary method to detect and quantify abnormal Hbs and to resolve ambiguous results from another protein-based method. CZE can be performed on red blood cell (RBC) hemolysates from dried blood spots, cord blood, or whole blood samples, making it potentially useful for newborn screening. CZE is comparable to HPLC in its resolution, ability to quantify low-abundance Hbs, ability to be automated, and speed (multiple samples can be run together with an eight-minute run time). CZE lacks interference from most posttranslationally modified (eg, glycated) globin chains. CZE is also a preferred method to identify beta thalassemia because it accurately quantifies Hb A₂ and Hb F. CZE also cleanly separates Hb E and Hb A₂, allowing quantification of both, unlike HPLC.

CZE is a type of electrophoresis that uses a liquid buffer rather than a gel to separate and quantify normal and abnormal Hbs. Hb variants are separated by electro-osmotic flow using negatively charged silica capillaries and a high voltage current. The resulting tracing is divided into 15 zones in which normal and variant Hbs are displayed as peaks, which can be quantified (figure 7).

Disadvantages of CZE include cost, need for technical expertise, and incomplete separation of Hb S from Hb D.

Examples of complementary methods include using CZE to resolve a falsely elevated Hb A₂ detected in the presence of Hb S by HPLC, or using HPLC to resolve Hbs that fall in the same window as Hb A by CZE.

Isoelectric focusing — Isoelectric focusing (IEF) is a common method to qualitatively identify abnormal Hbs because it has great precision and accuracy (greater than standard gel electrophoresis). IEF detects Hb S, C, E, A₂, Hb D-Punjab (Hb D-LA), and Hb G-Philadelphia. IEF can also detect fast-migrating Hbs characteristic of alpha thalassemia such as Hb H (a tetramer of beta globin chains, β₄) and Hb Barts (a tetramer of gamma globin chains, γ₄), making it particularly useful for newborn screening. (See 'Population screening (eg, routine newborn screen)' below.)

IEF is a robust methodology with relatively simple technology that requires limited laboratory expertise, making it a preferred diagnostic method, particularly for newborn screening, in limited resource settings compared with HPLC or CZE.

IEF is an electrophoretic technique for separating Hbs based on their net charge within a pH gradient on a gel medium (eg, polyacrylamide, thin-layer agarose). Hbs migrate through a pH gradient to a point at which their net charge is zero (ie, isoelectric point [pI]) (figure 8 and figure 9). The Hb migration pattern of IEF is similar to alkaline gel electrophoresis, but with resolution of Hb C from Hb E and Hb O, and Hb S from Hb D and Hb G. In addition, Hb A and Hb F are clearly resolved.

Disadvantages of IEF are that it cannot be used to distinguish Hb E from Hb C-Harlem or Hb O-Arab, and it cannot distinguish Hb G-Philadelphia from Hb Lepore. Scanning densitometry of the gel can be used to obtain a semiquantitative measurement of the size of the bands, but it is not precise enough for accurate quantification of low-abundance Hbs such as Hb A₂. Thus, IEF is not as useful as HPLC or CZE for routine, initial testing in adults.

Gel-based electrophoresis — Despite technical advances with newer methodologies, many smaller laboratories continue to use gel electrophoresis as the initial method to diagnose hemoglobin disorders; it can separate several normal Hbs including Hb A, Hb A₂, and Hb F; and a number of variant Hbs including Hb S and Hb C. Its simplicity and low cost make Hb gel electrophoresis a valuable complementary method for detection of common Hb variants. However, for first-line screening for hemoglobinopathies, gel electrophoresis has been replaced in most laboratories by more sensitive, reliable, and automatable methods such as CZE, HPLC, and IEF.

Electrophoresis is the classical method used to separate Hb proteins based on their net charge. An electric field is applied to a gel medium at a specific pH. The gel can be run at alkaline pH (cellulose acetate) or acidic pH (citrate agar). At alkaline pH, Hbs with a greater net negative charge will migrate more rapidly towards the positively charged anode of an electrical field (figure 10). Scanning densitometry of the gel can be used to obtain a semiquantitative measurement of the size of the bands.

There are some disadvantages of gel electrophoresis:

●At alkaline pH, it is not possible to distinguish Hbs that migrate to the same position, such as Hb A₂, Hb C, Hb E, and Hb O; or separating Hb D, Hb G, and Hb S. Separation of some co-migrating Hbs can be accomplished at acidic pH (eg, separation of Hb C and Hb O from Hb E and Hb A₂; separation of Hb S from Hb D and Hb G). However, it is not possible to distinguish Hb E from Hb A₂ or to separate Hb D from Hb G.

●It is also not possible to quantify Hb variants present in low concentrations or to quantify the low abundance adult Hb A₂ accurately.

●It is difficult to detect fast-moving Hb variants seen in alpha thalassemia (Hb H and Hb Barts).

●It is not ideal for high-throughput analysis because it requires manual processing.

Mass spectrometry — Mass spectrometry (MS) is also being investigated or used for the diagnosis or newborn screening of hemoglobinopathies. There are a number of MS techniques, all of which can be used to infer the peptide sequences (normal and variant) of globin chains of interest. This allows even the identification of "silent" variants that cannot be resolved or discriminated by HPLC and CZE. The proportion (relative abundance) of different globin chains can also be assessed to infer the presence of some thalassemic mutations. Considerable expertise and sometimes custom-built equipment are required.

Molecular genetic (DNA-based) methods

Overview of DNA-based methods

Use of genetic testing — Genetic testing is not a single entity. There are different genetic testing methods, each with specific strengths and limitations. For all the genetic techniques, it is necessary for the clinician to understand the methods that are used to properly interpret the results. For hemoglobinopathies, sequencing alone may be inadequate, and copy number variation analysis is often necessary. For example, sequence analysis done by itself will not detect large deletions (eg, hemizygosity for the Hb S mutation, as in Hb S/gene-deletion-HPFH, will appear by sequence analysis to be homozygosity for the Hb S mutation, although these two conditions are vastly different, and their distinction has important implications for genetic counseling and other aspects of management). Moreover, analysis of both alpha and beta globin loci (both sequence analysis and copy number variation analysis of both loci) is often necessary to explain or predict the phenotype. It may not be apparent a priori which patients "require" genetic testing (and/or which type of genetic testing), arguing for comprehensive genetic testing for all patients with suspected hemoglobinopathies.

Technical considerations — Rather than the globin protein's amino acid composition, DNA testing (also referred to as genotyping or molecular genetic testing) determines the nucleotide sequence of one or more globin genes as well as the presence of copy number variations (eg, deletions, duplications) and complex rearrangements. These tests analyze the DNA in white blood cells (WBCs), which carry all of the individual's germline DNA sequence, rather than red blood cells (RBCs), which are anucleate. There are a number of available methods, and it is possible to analyze one gene or multiple genes at a time, depending on the method and equipment used (table 1).

DNA-based methods are rarely used as a screening test for a hemoglobinopathy, although the role of first-line DNA testing is evolving. Incorporating DNA testing into routine use as a second-tier testing method is becoming the standard of care. Molecular methods are especially useful for confirming clinically significant disease in patients with suspected thalassemia, and in the setting of prenatal and newborn screening. Potential advantages of DNA testing in newborns includes the ability to identify defects in globin chains before these chains are expressed (eg, identification of beta globin defects during early infancy, when the predominant beta-like chain has not yet shifted from gamma globin to beta globin) (figure 2). By contrast, diagnosis using protein-based methods may require waiting until the infant is three months of age or older and producing predominantly adult Hbs. Earlier identification of thalassemia in these settings offers the possibility of early diagnosis, genetic counseling, and early institution of appropriate treatment. DNA-based analysis has shown clinical utility as a component of newborn screening for sickle cell disease (SCD) [6,7].

Other advantages of DNA-based testing include rapid turnaround time (hours) for some methods, high reliability, potential for automation, continually decreasing costs, and ongoing protocol improvements. In addition, introduction of next-generation sequencing (NGS) and array comparative genomic hybridization (array CGH) techniques may allow broader screening for multiple hemoglobinopathies simultaneously. (See 'Array CGH' below and 'NGS' below.)

There are several settings in which molecular techniques are especially useful:

●In newborns with certain initial newborn screening results:

•Hb F only, to confirm and distinguish β⁰-thalassemia (transfusion-dependent thalassemia [TDT]) from homozygous hereditary persistence of fetal Hb (HPFH)

•Hb FE, to distinguish Hb E/β⁰-thalassemia from Hb EE

•Hb Barts (tetramer of gamma globin chains) present at >25 percent of total Hb, to determine the specific genotype of alpha thalassemia (Hb H disease, Hb H-Constant Spring, and others).

●If prenatal carrier screening tests of one of the parents suggests alpha or beta thalassemia, including one or more of the following:

•RBC indices showing microcytosis or hypochromia (eg, mean corpuscular volume [MCV] <80 fL or mean corpuscular hemoglobin [MCH] <27 pg)

•Elevated Hb A₂ (>3.5 percent) or elevated Hb F (>5 percent)

●If a patient is being evaluated for a hemoglobinopathy and protein-based methods reveal an unknown variant or ambiguous result, DNA-based methods can be used to identify the specific genetic defect. (See 'Patient with unknown variant' below.)

●If a patient is being evaluated for a possible thalassemia diagnosis, including heterozygous, homozygous, compound heterozygous with another thalassemia mutation or an Hb variant, and a definitive diagnosis is desired. (See 'Patient with suspected thalassemia' below.)

The choice of DNA testing method depends on the type of variant suspected. Traditional (Sanger) sequencing and allele-specific polymerase chain reaction (PCR) are used to detect point mutations and to evaluate breakpoint mutations when a deletion has been identified. Gap-PCR, multiplex ligation-dependent probe amplification (MLPA), and array comparative genomic hybridization (aCGH) are used to detect copy number variations such as deletions, duplications, and fusion genes.

All of the DNA-based methods can be performed on DNA from peripheral blood leukocytes. For prenatal diagnosis, amniotic fluid cells or chorionic villus samples can be used. The DNA is typically amplified by PCR (see "Polymerase chain reaction (PCR)") and then genotyped for a definitive diagnosis. Use of these methods on cell-free fetal DNA in the maternal circulation for hemoglobinopathy diagnosis remains investigational. (See "Prenatal screening for common aneuploidies using cell-free DNA".)

There are some general limitations to DNA-based testing, which depend on the specific testing method used. As examples, certain sequencing and PCR methods may fail to identify large gene deletions due to absent (deleted) primer annealing sites. This can be circumvented by performing initial testing at the protein level (CZE, HPLC, or IEF) or by using DNA-based methods specifically designed to identify large deletions (see 'Gap-PCR and MLPA' below and 'Array CGH' below). Primer annealing sites may also be polymorphic, which may lead to failure of amplification. If this is suspected, alternative primer pairs can be used. Another disadvantage of DNA-based methods is the cost of equipment and reagents, which remain significantly higher than for most of the protein-based techniques.

Traditional DNA sequencing and allele-specific PCR — DNA sequencing determines the nucleotide sequence of a gene or genes. Traditional (Sanger) sequencing is the gold standard for DNA sequencing. It is often used when a specific type of globin gene defect (eg, alpha or beta thalassemia variant) is suspected based on preliminary testing or family history, in which case it can be targeted to one or more of the affected globin genes, as shown in the figure (figure 11). However, the phenotype of thalassemias in particular and hemoglobinopathies in general is determined by the interaction of both the alpha globin and beta globin gene clusters, so a correct, definitive diagnosis often requires testing of both loci.

Allele-specific assays can be used to target the presence or absence of a specific allele. Allele-specific PCR employs a set of manufactured oligonucleotide primers that hybridize to and specifically amplify sequences of DNA containing the target alleles of interest (eg, Hb A, Hb S, Hb C, Hb D, Hb E and Hb O-Arab). Analysis of the PCR products generated can be done by electrophoresis based on the length of the fragments generated. Alternatively, restriction enzymes can be used to cut DNA at specified sequences that are present only in certain variants, followed by electrophoresis for the restriction enzyme products; this is referred to as restriction fragment length polymorphism (RFLP) analysis.

Sanger sequencing can also be used to scan for a spectrum of point mutations causing the most common Hb variants and beta thalassemia genotypes using an automated 96-well format in which overlapping PCR fragments and universal tagged primers are employed. Such screening techniques only detect the subset of Hb mutations for which they are designed, so negative or normal results should be interpreted with caution.

Sanger sequencing can be used to determine the full coding sequence (and noncoding/regulatory sequences of interest) of any of the globin genes. Therefore, this method can be used to identify definitively any hemoglobinopathy caused by a sequence variant (point mutation) or a small insertion/deletion. It is important to consider simultaneous copy number variation (deletion/duplication) analysis because of the high frequency of large deletions, duplications, and other rearrangements as a cause of hemoglobinopathies.

Gap-PCR and MLPA — Gap-polymerase chain reaction (gap-PCR) techniques are used to test for large deletions in the alpha globin or beta globin genes that cannot be detected by DNA sequencing (because one or both primer-annealing sites are deleted). This method is particularly useful for detection of common alpha thalassemia deletions.

Gap-PCR is a variation of PCR, but unlike PCR used for gene sequencing, gap-PCR is used to identify deletion breakpoints. This is done with PCR primers that flank a known deletion breakpoint rather than a normal gene sequence. Thus, the primers will only generate a product if the patient's DNA contains the unique sequence characteristic of that gene deletion. The most common example is alpha thalassemia, for which primers specific for the most common deletions can be combined; an example is shown in the figure (figure 12). Gap-PCR is also routinely used to detect deletions in the delta and beta globin genes that result in a delta/beta hybrid variant known as Hb Lepore (see 'Types of abnormalities' above), as well as globin gene deletions causing HPFH and alpha globin gene duplications. Products of gap-PCR are analyzed by gel electrophoresis and compared with known positive control samples; the presence of the abnormal band on the gel is indicative that the individual carries the particular deletion.

Gap-PCR is not as useful for evaluating unusual or uncharacterized thalassemia deletions, because it will only detect deletions for which the breakpoints are known.

Gap-PCR will not detect alpha gene triplication (and higher-order copy number gains), which can have a major impact on clinical phenotype (beta thalassemia heterozygotes with alpha triplication may have clinically significant anemia).

Multiplex ligation-dependent probe amplification (MLPA) is increasingly used to detect large deletions and duplications within the globin genes, similar to the indications for gap-PCR. However, unlike gap-PCR, which can only identify deletions for which breakpoints are known, MLPA can also identify deletions with unknown breakpoints, especially large deletions in the alpha or beta globin genes. MLPA is a variation of PCR in which a pair of ligated probes rather than the target gene sequences are amplified by PCR. A pair of probes will only be amplified when they hybridize close enough together on the target gene in the patient's DNA to be ligated by DNA ligase, and they will only hybridize close together on the target gene when the full gene sequence is present. Thus, failure to generate a PCR product indicates deletion of a region of DNA in that part of the gene. Further, the relative amounts of the PCR products can be used to determine whether the deletion is homozygous (absence of the product) or heterozygous (50 percent reduction in the amount of the product). Copy number gains can be identified similarly.

While MLPA is a useful tool to screen for deletions, it does not define the exact deletion breakpoints; if necessary, determination by traditional sequencing or NGS is required. Identification of the exact deletion breakpoints may be helpful in predicting phenotype and determining if the patient has a deletion that arose de novo and/or was not previously described.

Array CGH — Array-based comparative genomic hybridization (aCGH; also known as fine tiling array) is another molecular approach to confirming thalassemia defects comprehensively across the alpha and beta globin genes. Besides point mutations and small deletions, aCGH also detects copy number variations (CNVs), inversions, insertions, deletions, and other complex rearrangements, most of which are not detected by standard DNA sequencing.

aCGH is primarily used to characterize a deletion that has been detected by MLPA, such as when MLPA identifies that a deletion is present in an alpha or beta globin gene but does not allow determination of the exact breakpoint. In this setting, aCGH may be less time-consuming and cumbersome than gap-PCR to characterize deletions and duplications caused by recombination (eg, delta-beta thalassemias).

In aCGH, the patient's genomic DNA is hybridized to a microarray containing tens to hundreds of thousand overlapping oligonucleotide probes, each approximately 60 to 80 nucleotides in length. Custom, high resolution tiling arrays are available for thalassemia diagnosis in which the collection of probes represents the majority of the DNA sequence in the complete alpha and beta globin gene clusters as well as the neighboring regions. Thus, aCGH can identify any areas of either gene, including coding regions and regulatory regions (such as introns) that are present in normal levels, reduced levels, excess levels, or completely absent.

The major disadvantages of aCGH include cost, need for trained personnel, and longer turnaround time.

NGS — Next-generation sequencing (NGS), also referred to as massively parallel or deep sequencing, refers to sequencing technologies that simultaneously determine the sequence of multiple short DNA fragments ("reads"), allowing rapid and less costly sequencing of multiple genes (or the whole exome or even the entire genome). NGS sequencing is not routinely used for hemoglobinopathy testing at this time, but is being actively explored. (See "Next-generation DNA sequencing (NGS): Principles and clinical applications".)

An investigational NGS platform covering the entire spectrum of known beta thalassemia mutations as well as the common Hb variants including Hb S, Hb C, and Hb D, has been developed to test 100 patient samples in a single run. Potential advantages of using NGS for hemoglobinopathy testing include the ability to perform simultaneous sequencing of all of the globin genes in a single test, along with other genes that modify hemoglobin expression such as BCL11A, MYB, KLF1, and GATA1. NGS can be performed on a smaller size sample than Sanger sequencing and has the potential for automation and reduced labor. However, the accuracy of NGS depends on the platform used and the depth of coverage, and results from NGS require confirmation by Sanger sequencing.

Point-of-care assays — Point-of-care (POC) assays (also called POC tests [POCTs]) are tests that can be used directly at the place and time of patient evaluation in areas without access to a specialized clinical laboratory. These tests may be especially useful in resource-limited settings with a high prevalence of hemoglobinopathies or when a rapid intervention is required.

There are several advantages to using POCTs for the diagnosis of SCD and other hemoglobinopathies in limited-resource settings.

●The ability to inform the parent/caregiver of an abnormal result at the time of testing and eliminate the need to subsequently track the patient to allow linkage to care.

●POCTs require limited laboratory expertise and often are disposable, not requiring continued replenishment of reagents.

●Most POCTs are designed to be inexpensive, allowing for widespread use even in the most resource-limited settings.

While several POCTs can provide accurate diagnosis, important barriers to effective widespread use remain, and there is greater need to improve the global diagnosis of SCD and other clinically important hemoglobin disorders.

There are several innovative POC assays (table 2) that have been validated and are being used across the world, primarily to diagnose sickle cell disease (SCD) [8]. Examples include the following:

●Gazelle Hb Variant – Gazelle Hb Variant is a POC device that uses a miniaturized version of the cellulose acetate electrophoresis method described above. (See 'Gel-based electrophoresis' above.)

The device can detect and distinguish Hbs by their traveling distance from the sample application point, which is based on their charge, in a process referred to as "microchip electrophoresis" [8]. A small amount of blood, most commonly obtained as a finger (or heel) stick sample, is processed on a piece of cellulose paper in alkaline buffer housed within a micro-engineered plastic chip, wherein cellulose acetate electrophoresis is performed using a battery-powered electric field to separate Hbs (figure 13). Images of the hemoglobin electrophoresis results are captured by a portable reader for analysis in real-time (within 8 minutes), including quantification of relative percentages of each hemoglobin present in the sample. Quantitative percentages of hemoglobin fractions are displayed on an electronic tablet embedded in the machine.

Several field studies have demonstrated the accuracy and utility of this device in a variety of clinical settings. Compared with HPLC, the "Gazelle Hb Variant" test correctly identified patient samples as Hb AA, Hb SA, Hb SS, and Hb SC with 96.6 to 100 percent accuracy. Field-testing for SCD screening has been ongoing in India and sub-Saharan Africa. The HemeChip test also performs comparably to laboratory electrophoretic methods [9-11].

The Gazelle Hb Variant method has the same limitations as cellulose acetate electrophoresis, most importantly the inability to overlap in the separation of Hb C, Hb E, and Hb O and Hb D, Hb G, and Hb S. When Hb S and Hb C are the most important Hbs to identify, such as in most of sub-Saharan Africa, these limitations are not as important, but this method has less utility in the diagnosis of thalassemias or in areas where Hb E, Hb D, and Hb O are more common. (See 'Gel-based electrophoresis' above.)

●HemoTypeSC – "HemoTypeSC" (figure 14) is a POC immunoassay for SCD that uses a fingerprick blood sample to prepare a hemolysate that is added to a vial containing a test strip containing immobilized monoclonal antibodies against Hb A, Hb S, and Hb C [12]. The assay uses a competitive lateral flow immunoassay (LFIA) format in which labeled reagent Hbs compete with the patient's Hbs for binding to the immobilized monoclonal antibodies. Thus, the presence of an Hb variant in the patient sample is indicated by the absence of a red-colored band at the test line.

When evaluated on whole blood samples from 100 individuals, this test correctly identified all of the hemoglobin phenotypes including homozygous and heterozygous forms of Hb A, Hb S, and Hb C [12]. In a subsequent study involving 587 participants, sensitivity and specificity were >99 percent [13]. The test is easy to perform because it does not require extensive training or a separate dilution step, but interpretation may be nonintuitive for some personnel because the absence of a band indicates the presence of a variant. Results are available within 10 minutes. Field testing continues to show very high sensitivity and specificity [14,15]. The test is designed to diagnose forms of SCD (Hb SS and Hb SC disease); it is not designed to diagnose thalassemia or other abnormal hemoglobin patterns.

●Sickle SCAN – "Sickle SCAN" (figure 15) is a POC lateral flow immunoassay (LFIA) that requires a fingerprick blood sample mixed in a tube prefilled with buffer to form hemolysate. Five drops of the hemolysate are added to a test cartridge containing a test strip to which monoclonal antibodies against human Hb S, Hb C, and Hb A are fixed. As the hemolysate diffuses through the absorbent test strip, patient Hbs first bind to a labeled monoclonal antibody against the alpha globin chain present in all Hbs. These labeled antigen-antibody complexes selectively bind to the test lines containing the antibodies for visual detection of each type of normal and variant Hb present. Results are available in 5 minutes, and the device can be used on dried blood spots or blood samples stored at 4°C for up to one month.

•Testing of this assay on blood samples from 137 heterozygous and homozygous individuals accurately detected Hb A, Hb S, and Hb C with 99 percent sensitivity and specificity [16]. Further validation on samples collected at the point of care from 71 individuals with known hemoglobin genotypes showed 100 percent sensitivity and near-perfect specificity [16]. In a separate analysis of 139 newborns and adults, the device had high sensitivity (98 to 100 percent) and specificity (93 to 99 percent) for detecting Hb A and Hb S, and for distinguishing sickle cell trait from SCD and unaffected controls [17]. The test does not detect Hb A at levels below 40 percent, and individuals with sickle-beta⁺ thalassemia cannot be distinguished from those with Hb SS or sickle-beta⁰ thalassemia genotypes. There is potential for interference from cross-reacting Hb variants that share epitopes with Hb A.

•The accuracy, usefulness, and cost-effectiveness of real-world use of this assay have been demonstrated in resource-limited settings including Nigeria and Haiti, with sensitivities of 90 to 100 percent and specificities of 97 to 98 percent, using HPLC as the gold standard [18,19]. The study in Nigeria included a survey of 197 health care workers and 221 patients, in which over 97 percent indicated a willingness to recommend Sickle SCAN as a screening test for SCD [18]. Importantly, the study from Haiti documented the feasibility and accuracy of Sickle SCAN as a tool for newborn screening [19]. This study noted that the ability to notify parents/caregivers immediately about the results rather than having to seek out and notify them at a later date resulted in earlier initiation of interventions such as penicillin prophylaxis, pneumonia vaccination, and counseling about the disorder. Subsequent analyses have shown very high sensitivity and specificity in other populations [19-21].

These POC tests are not appropriate for diagnosing thalassemias or for identifying abnormal hemoglobin variants other than Hb C and Hb S.

Other disease-specific tests — Other tests may be available in some cases, such as iron studies (ie, serum, ferritin, and transferrin saturation) to assess for iron deficiency, or erythrocyte protoporphyrins, including metal-free erythrocyte protoporphyrin (also called free erythrocyte protoporphyrin [FEP]) and zinc protoporphyrin, to evaluate for lead toxicity and iron deficiency. (See "Iron deficiency in infants and children <12 years: Screening, prevention, clinical manifestations, and diagnosis" and "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults".)

Hemoximetry, Heinz body prep, Hb H staining, and the sickle cell prep may provide additional useful information but generally are not sufficient to make a definitive diagnosis. However, the information guides further testing and may be useful, especially in resource-limited settings, screening programs, research, or when it can be obtained more easily or rapidly than the testing described above.

Hemoximetry — This assay measures the oxygen affinity of Hb (ie, it determines the oxygen-Hb dissociation curve). The results of this assay are reported as the partial pressure of oxygen at which Hb is 50 percent saturated with oxygen (the p50). A low p50 indicates increased oxygen affinity. A high p50 indicates decreased oxygen affinity. This test can aid in the diagnosis of inherited conditions that affect Hb oxygen affinity (eg, hemoglobinopathies and other intrinsic RBC abnormalities), which may present with anemia, polycythemia, or cyanosis. This test can also help to narrow the differential diagnosis of acquired forms of anemia, polycythemia, or cyanosis, some causes of which might be associated with abnormal oxygen affinity (eg, carboxyhemoglobinemia or methemoglobinemia). (See "Methemoglobinemia".)

Heinz body prep and other tests for unstable hemoglobins — Heinz bodies are Hb precipitates inside the RBC that form when Hb denatures and heme dissociates from the heme pocket and binds elsewhere in the partially denatured globin chains (hemichrome formation). These denatured aggregates typically adhere to the internal surface of the RBC membrane.

The Heinz body prep involves incubating RBCs with a supravital stain such as methylene blue or crystal violet. This may be helpful for cases of suspected unstable Hb variants. However, the test is nonspecific and is abnormal in many other disorders besides unstable hemoglobins [22]. Any cause of oxidant hemolysis, whether an enzymopathy (eg, G6PD deficiency), oxidizing agents (eg, dapsone), or unstable Hbs can result in the formation of Heinz bodies. Further, the absence of Heinz bodies does not exclude the diagnosis, and the interpretation is highly operator-dependent.

Thus, the Heinz body prep is only done in a laboratory with appropriate expertise and evaluated in the context of other clinical and laboratory information. (See "Unstable hemoglobin variants", section on 'Blood smear and Heinz body preparation'.)

Heat or isopropanol denaturation techniques are more specific screening tests for unstable hemoglobins than the Heinz body prep [22]. Increasingly, genetic testing is used instead of (or in addition to) Hb denaturation tests. Details and resources for obtaining this testing are presented separately. (See "Unstable hemoglobin variants", section on 'Specialized testing'.)

Hb H staining — Hb H is an insoluble tetramer of beta globin chains that precipitates inside the RBC when there are insufficient alpha globin chains with which to pair. Hb H is produced in individuals with alpha thalassemia. Hb H can be visualized by staining with new methylene blue (a chemical dye related to methylene blue) or brilliant cresyl blue as a type of RBC inclusion. Hb H staining is nonspecific, as other protein or nucleic acid precipitates (including Hb Barts, a tetramer of gamma globin chains) are also stained. The presence of typical inclusions provides supportive evidence for a presumptive diagnosis of Hb H disease. The absence of Hb H inclusions does not eliminate the possibility of alpha thalassemia trait. Hb H staining may also be useful for identification of the Alpha Thalassemia X-linked Intellectual Disability (ATR-X) syndrome, caused by pathogenic variants in the ATRX gene.

Sickle cell prep — The sickle cell prep is a method for detecting Hb S-containing RBCs that involves treating a blood sample with a reducing agent such as sodium metabisulfite or dithionite; these agents reduce the oxygen tension in the sample and promote sickling, which is then examined visually. It does not distinguish sickle cell trait from disease. This method is not recommended for clinical use and is generally of historical interest. There are a few exceptions:

●The sickle cell solubility test can be useful to detect sickling variants that are caused by a second mutation in addition to the Hb S mutation (on the same allele) but show a different pattern from that of Hb S on electrophoresis and HPLC. Examples include Hb S-Antilles and Hb S-Oman. (See "Sickle cell trait", section on 'Symptoms of sickle cell disease' and "Overview of compound sickle cell syndromes", section on 'Sickle cell syndromes that appear to be sickle cell trait on hemoglobin analysis'.)

●This method is used in many blood banks to screen RBC units for the presence of Hb S when Hb S-negative blood is needed for transfusion.

INITIAL EVALUATION

Indications for hemoglobin analysis — Hb testing may be used for general population screening as well as diagnostic evaluation for a suspected clinical condition:

●In many (mostly high-resource) regions of the world, hemoglobinopathy testing is a component of routine prenatal testing or routine newborn screening. Additional testing is used to confirm preliminary positive results. (See 'Population screening (eg, routine newborn screen)' below and "Hemoglobinopathy: Screening and counseling in the reproductive setting and fetal diagnosis" and "Overview of newborn screening".)

●For a patient with a suspected hemoglobinopathy diagnosis, additional testing is used to confirm and characterize the defect(s). (See 'Patient with suspected sickle cell disorder' below and 'Patient with suspected thalassemia' below.)

●Other individuals may have a known variant or thalassemia in combination with an unknown or uncharacterized variant (eg, a patient may be heterozygous for a sickle Hb mutation plus another variant or mutation that cannot be further characterized by initial testing methods) [3]. (See 'Patient with unknown variant' below.)

In contrast with the above scenarios, hemoglobinopathy testing generally should not be performed as part of the initial evaluation of individuals with new-onset anemia or those with unexplained anemia for whom other common diagnoses such as iron deficiency have not yet been addressed. (See "Approach to the child with anemia" and "Diagnostic approach to anemia in adults".)

Overview of approach — The appropriate testing strategy depends on the indication for testing (population screening versus clinical diagnostic evaluation), the type of hemoglobinopathy being investigated, the associated clinical findings, and the available resources.

Other important information to be incorporated into the diagnostic evaluation includes results of the complete blood count (CBC), reticulocyte count, and red blood cell (RBC) morphology, as well as geographic ancestry and the results of testing of first-degree relatives (eg, parents, siblings). (See 'Clues from the CBC' below.)

A number of important considerations are worth keeping in mind:

●Results of hemoglobinopathy testing should always be interpreted in the context of the clinical picture. Additional information, including transfusion history, geographic ancestry, and relevant hematologic studies are extremely helpful in guiding the approach to laboratory diagnostic testing. (See 'Clues from the CBC' below.)

●Recent blood transfusion may interfere with the interpretation of hemoglobin analysis using protein-based methods. In this setting, the options are to wait until >3 months have elapsed since the last transfusion, or to use molecular genetic (DNA-based) methods, which are unaffected by recent transfusion. (See 'Molecular genetic (DNA-based) methods' above.)

●An initial abnormal test result suggestive of thalassemia or Hb variant is typically confirmed using an alternative method, especially when the initial testing method is protein-based (eg, high-performance liquid chromatography [HPLC], isoelectric focusing [IEF], capillary zone electrophoresis [CZE]). A common practice is to use HPLC for newborn screening followed by IEF for confirmation, or vice versa (see 'Available testing methods' above). Confirmation by a separate technique is likely to clarify ambiguous results and guide the decision regarding whether further evaluation using molecular testing methods is required for definitive diagnosis, especially in the thalassemias. This practice of using a separate confirmatory test is also important when the variant is uncommon or its clinical significance is unclear.

●Co-inherited alpha thalassemia may alter the proportion of variant Hbs, which may make interpretation more challenging. As a general rule, concomitant alpha thalassemia reduces the levels of abnormal beta globins such as Hb S, Hb C, and increases levels of abnormal alpha globins.

●Concomitant iron deficiency may alter the findings on the blood smear as well as the ratios of normal Hbs. In adults being evaluated for thalassemia, it is usually appropriate to perform iron studies to aid in the interpretation of protein-based hemoglobinopathy testing. An exception may be an individual being evaluated for thalassemia trait who has a relatively normal CBC.

●In all cases and at all stages of testing, quality controls should be in place to reduce the likelihood of errors and to enhance the communication between the laboratory, treating clinicians, and the patient [5].

These issues are discussed in greater detail in a 2015 document from the Centers for Disease Control (CDC) in the US Department of Health and Human Services [5]. General approaches for the more common clinical scenarios are presented in the following sections. (See 'Common clinical scenarios' below.)

Clues from the CBC — The complete blood count (CBC) provides information that should always be considered when evaluating a possible hemoglobinopathy. Relevant features include the degree of anemia (or polycythemia), RBC count, RBC indices, reticulocyte count, and RBC morphology from the peripheral blood smear. Examples of morphologic findings that are characteristic of specific conditions are listed separately. (See "Evaluation of the peripheral blood smear", section on 'Red cell abnormalities'.)

The following findings are suggestive of specific hemoglobinopathies:

●Sickled cells (picture 1) suggest sickle cell disease (SCD), including homozygous Hb SS disease (Hb SS) and Hb S-beta thalassemia. Individuals with sickle cell trait do not have sickled cells on the routine peripheral blood smear.

●Hexagonal Hb crystals (picture 2) suggest homozygous Hb C disease (Hb CC).

●"Canoe" cells (also called "boat cells" or "pita bread cells") (picture 3) and target cells are often present in Hb SC disease.

●Target cells (picture 4) may be seen in increasing abundance in beta thalassemia trait (picture 5), intermedia (picture 6), and major; they are also seen in other hemoglobinopathies such as Hb C, Hb D, and Hb E.

●Teardrop cells (picture 7) suggest beta thalassemia.

●Bite cells (picture 8) and/or spherocytes (picture 9) may be seen with certain unstable Hb variants.

Some of these RBC abnormalities are also seen in other (non-hemoglobinopathy) conditions, such as target cells in liver disease, teardrop cells in bone marrow fibrosis, and spherocytes in immune hemolytic anemias and hereditary spherocytosis.

Abnormalities in white blood cells (WBCs) or platelets should also be considered. In general, hemoglobinopathies do not cause alterations in WBC or platelet counts, and abnormalities in number or distribution of these cells suggests an alternative or additional diagnosis. A notable exception is SCD; in the untreated state, there is often leukocytosis and thrombocytosis, and with hydroxyurea therapy, there is macrocytosis and potential, mild decreases in leukocyte and platelet counts.

Common clinical scenarios

Population screening (eg, routine newborn screen) — In the United States and many other high-resource countries, newborns are routinely screened for hemoglobinopathies from a blood spot specimen obtained by heel puncture, often using HPLC. Hb S is one of the most commonly detected findings from the screening. In some populations, thalassemias are more prevalent. (See "Overview of newborn screening".)

HPLC is particularly useful in the newborn period for diagnosing Hb H disease. Normally, the major Hb in the newborn is Hb F (a tetramer of two alpha and two gamma chains [α2γ2]). In Hb H disease, there is marked deficiency of alpha globin chain production, and the excess gamma globin chains form a tetramer known as Hb Barts. Elevated levels of Hb Barts (eg, >25 percent) within 12 to 48 hours after birth is the hallmark of Hb H disease in the newborn. The threshold for detection is based on validation data from a reference population; this may differ by region depending on which mutations are common in the population [23]. The timing is also important because Hb Barts levels fall rapidly after birth. Newborn screening programs may also detect some cases of alpha thalassemia trait by identifying low levels of Hb Barts.

All abnormal newborn screening results require confirmatory testing on a second sample, regardless of the initial screening test used and the clinical findings. For patterns consistent with SCD and beta thalassemia (Hb S or Hb F-only pattern, respectively), this may involve a second protein-based test. A complementary method is used to ensure that no methodologic or clerical errors were made. Molecular testing is required to make a definitive diagnosis in newborns with suspected alpha thalassemia, suspected coinheritance of two beta thalassemia mutations, or suspected Hb E in combination with a beta thalassemia mutation. Some infants are retested after several months, especially those born prematurely who have very low levels of Hb A or only Hb F (see 'Normal hemoglobins' above) or infants who receive a blood transfusion prior to collection of the initial newborn screening specimen. Alternatively, DNA-based methods may be used at any age. DNA-based testing has been demonstrated to be useful as a component of newborn screening for SCD [6,7]. (See 'Molecular genetic (DNA-based) methods' above.)

Prenatal testing — Prenatal testing for hemoglobinopathies is discussed in detail separately. (See "Hemoglobinopathy: Screening and counseling in the reproductive setting and fetal diagnosis".)

Patient with suspected sickle cell disorder — Patients may be suspected of having a sickle cell disorder based on a known familial mutation, results of prenatal testing, results of newborn screening, or clinical features suggestive of SCD (eg, vaso-occlusive phenomena, hemolytic anemia, sickled cells on the blood smear) (picture 1). Symptoms are not present at birth, because the sickle mutation only affects beta chains, and the switch from gamma globin to beta globin production occurs gradually during early infancy (figure 2). (See "Overview of the clinical manifestations of sickle cell disease".)

A standard evaluation involves HPLC, CZE or IEF followed by additional testing depending on the pattern found (table 3). (See "Diagnosis of sickle cell disorders" and "Overview of compound sickle cell syndromes".)

Patient with suspected thalassemia — Patients may be suspected of having thalassemia based on a known family history, results of prenatal testing, symptoms of anemia, or characteristic findings on the CBC (eg, microcytic anemia high RBC count, low reticulocyte count) and blood smear (varying degrees of microcytosis, target cells, and other bizarre forms depending on disease severity) (picture 6 and picture 5 and picture 10).

The major differential diagnosis in individuals with suspected thalassemia is iron deficiency anemia; both conditions cause microcytic anemia. Iron deficiency can also affect (lower) the Hb A₂ level, potentially making diagnosis of thalassemia more challenging [24-26]. Thus, testing for iron deficiency is often indicated. In some cases, testing for iron deficiency (and, if needed, treatment) precedes testing for thalassemia; this would be the case for an adult with microcytic anemia and possible blood loss. In other cases, testing for iron deficiency is performed concurrently with testing for thalassemia trait. This would be the case for an individual in whom prenatal testing demonstrates microcytic anemia. Various calculations that take into account the RBC indices and RBC count have been used to predict the likelihood of thalassemia trait versus iron deficiency (eg, ratio of mean corpuscular volume [MCV] to RBC count [Mentzer index]), although none of these is diagnostically robust for an individual patient [27-31]. In practice, it may be easier and more cost-effective to test for iron deficiency and thalassemia trait simultaneously. (See "Iron deficiency in infants and children <12 years: Screening, prevention, clinical manifestations, and diagnosis" and "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults".)

A standard evaluation for thalassemia involves initial protein testing, typically with CZE, HPLC or IEF, and subsequent testing determined by the Hb pattern found (table 4 and table 3):

●Suspected alpha thalassemia – Clinical manifestations of alpha thalassemia may develop in utero because alpha chains are a component of Hb F (α₂γ₂). The most dramatic example is alpha thalassemia major, in which all four alpha globin genes are affected; this usually causes hydrops fetalis. (See "Nonimmune hydrops fetalis" and "Alpha thalassemia major: Prenatal and postnatal management".)

If the initial protein-based testing suggests alpha thalassemia (eg, increased Hb Barts at birth, microcytic anemia, presence of Hb H) (table 4), a DNA-based method must be used for confirmation. Most alpha thalassemia variants are deletions, but there are many non-deletional alpha thalassemia variants. Thus, a combination of methods to detect deletions and sequence variants is critical. Gap-polymerase chain reaction (gap-PCR) can detect approximately 80 percent of alpha thalassemia deletions; alternatively, multiplex ligation-dependent amplification (MLPA) can be used instead. (See 'Gap-PCR and MLPA' above.)

DNA sequencing can be used to detect point mutations that account for approximately 15 percent of alpha thalassemias (Hb Constant spring, poly-A mutation). (See 'Traditional DNA sequencing and allele-specific PCR' above.)

For the remaining 5 percent (or as a substitute for gap-PCR entirely), MLPA can be used to detect a deletion, followed by sequencing to define the breakpoints and characterize the deletion mutation, or array-based comparative genomic hybridization (aCGH) can be used to detect the presence of a deletion and characterize the deletion mutation simultaneously. (See 'Gap-PCR and MLPA' above and 'Array CGH' above.)

●Suspected beta thalassemia – Symptoms of beta thalassemia are not present at birth, because predominantly Hb F (α₂γ₂) is produced; beta globin production increases gradually during early infancy (figure 2). (See "Diagnosis of thalassemia (adults and children)".)

If the initial protein-based testing suggests beta thalassemia (eg, increased Hb A₂) (table 3), a second protein-based method or a DNA-based method can be used (see 'Protein chemistry methods' above). Most beta thalassemia variants are point mutations. Thus, when DNA testing is used, beta thalassemia can be detected by targeted PCR-based testing or traditional sequencing (see 'Traditional DNA sequencing and allele-specific PCR' above). If a point mutation is not found, screening may be done using MLPA, with deletion breakpoints defined by allele-specific PCR or traditional sequencing. The use of aCGH to diagnose beta thalassemia is limited to reference laboratories that has appropriately trained personnel.

Patient with unknown variant — Unknown variants generally are identified during an evaluation for common hemoglobinopathies (SCD or thalassemia) [32,33]. Unknown variants may also be suspected in patients who are being evaluated for unstable Hbs, inherited methemoglobinemia, or high or low oxygen affinity disorders, all of which are relatively uncommon. (See "Unstable hemoglobin variants" and "Methemoglobinemia" and "Hemoglobin variants that alter hemoglobin-oxygen affinity".)

REFERRAL TO A SPECIALIZED LABORATORY — The decision to send the patient's blood for analysis by an outside reference laboratory is generally made in one of the following settings:

●A hemoglobin variant is present, but the specific abnormality cannot be characterized using routine testing.

●An abnormal hemoglobin is suspected on clinical or laboratory grounds but cannot be detected by routine methods discussed above. Examples include:

•Heinz body positive hemolytic anemia and possible unstable hemoglobin variant

•Unexplained erythrocytosis and possible high oxygen affinity hemoglobin variant

•Unexplained normocytic, normochromic anemia and possible low oxygen affinity hemoglobin variant

•Cyanosis, methemoglobinemia, and possible hemoglobin M disease

•Microcytosis or microcytic anemia with negative testing for iron deficiency

The choice of reference laboratory may be determined by contractual agreements with one of the national reference laboratories or local preference for certain specialized laboratories. Ideally, a laboratory should be chosen that includes interpretation and oversight by an expert in hemoglobinopathies. Academic laboratories in the United States that specialize in the analysis of abnormal Hbs include:

●Erythrocyte Diagnostic Laboratory at Cincinnati Children's Hospital Medical Center, Cincinnati, OH

●Hemoglobin Diagnostic Reference Laboratory at Boston University, Boston, MA

●Hemoglobinopathy Reference Laboratory at UCSF Benioff Children’s Hospital, Oakland, CA

●Mayo Medical Laboratories at Mayo Clinic, Rochester, MN

●Titus HJ Huisman Hemoglobinopathy Laboratory at Augusta University, Augusta, GA

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Sickle cell disease and thalassemias".)

SUMMARY AND RECOMMENDATIONS

●Globin chains – Human hemoglobins (Hbs) are tetrameric proteins composed of two pairs of globin chains (figure 1); the specific chains are highly regulated during development (figure 2). Hemoglobinopathies include a heterogeneous mixture of conditions caused by genetic alterations that affect one or more of the alpha-like (α-like) globin chains, the beta-like (β-like) globin chains, or some combination of these. They can be broadly classified into structural hemoglobin variants (qualitative defects), and thalassemias (quantitative defects). (See 'Hemoglobin biology' above.)

●Testing methods – Protein-based methods remain the method of choice for initial qualitative and quantitative Hb analyses. Routinely used techniques include high-pressure liquid chromatography (HPLC; also called high performance liquid chromatography), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), and gel electrophoresis. Molecular genetic (DNA-based) methods listed above are not commonly used as a first-line or screening test for a hemoglobinopathy, although this role is evolving and entering standard comprehensive diagnostic practice. Advantages and disadvantages of the various methods are listed in the table (table 1). Several innovative, protein-based point-of-care tests are under development that can be used directly at the place and time of patient evaluation, particularly in limited-resource settings without access to a specialized clinical laboratory (table 2). (See 'Available testing methods' above.)

●Settings in which testing is appropriate – Hb testing may be used for general population screening (eg, prenatal testing, newborn screening) as well as diagnostic evaluation for a suspected clinical condition. The appropriate testing strategy depends on the indication for testing (population screening versus clinical diagnostic evaluation), the type of hemoglobinopathy being investigated, the associated clinical findings, and the available resources. The diagnostic evaluation also incorporates review of the peripheral blood smear, results of the complete blood count (CBC), reticulocyte count, and red blood cell (RBC) morphology, as well as racial/ethnic background and the results of testing of first-degree relatives. (See 'Indications for hemoglobin analysis' above and "Hemoglobinopathy: Screening and counseling in the reproductive setting and fetal diagnosis" and 'Overview of approach' above and 'Clues from the CBC' above.)

●Approach to choosing a test – Common approaches to newborn screening and the laboratory evaluation of suspected sickle cell disease (SCD), suspected thalassemia, and unknown hemoglobin variants are described above. Typical Hb patterns in these conditions are listed in the table (table 3). (See 'Common clinical scenarios' above.)

●Advanced testing – Analysis by a reference laboratory is generally indicated if an abnormality cannot be characterized using routine testing, or if an abnormal hemoglobin is suspected on clinical grounds but cannot be detected by routine methods (eg, Heinz body hemolytic anemia, unexplained erythrocytosis, unexplained cyanosis, or unexplained anemia for which other testing is unrevealing), or when the phenotype of the disease differs from the results of first-line clinical testing. Contact information for reference laboratories in the United States is listed above. (See 'Referral to a specialized laboratory' above.)

●Diagnosis of specific hemoglobin disorders – Additional information about the clinical manifestations and diagnosis of specific hemoglobinopathies is presented separately. (See "Diagnosis of sickle cell disorders" and "Diagnosis of thalassemia (adults and children)" and "Hemoglobin variants including Hb C, Hb D, and Hb E" and "Unstable hemoglobin variants" and "Hemoglobin variants that alter hemoglobin-oxygen affinity" and "Methemoglobinemia".)

ACKNOWLEDGMENTS

The UpToDate editorial staff acknowledges Ferdane Kutlar, MD, Abdullah Kutlar, MD, and Carolyn Hoppe, MD, who contributed to earlier versions of this topic review.

The UpToDate editorial staff also acknowledges the extensive contributions of William C Mentzer, MD, to earlier versions of this and many other topic reviews.