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October 2009 Clinical Laboratory News: Hemoglobinopathies and Thalassemias

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October 2009: Volume 35, Number 10

Hemoglobinopathies and Thalassemias
The ABCs of Lab Evaluation
By Shirley L. Welch, PhD

Hemoglobin (Hb), the oxygen-carrying protein found in red blood cells, is made up of a non-protein heme group and four globular protein subunits, called globins. Defects in these protein subunits cause a variety of disorders known as hemoglobinopathies and thalassemias that produce various degrees of anemia, morbidity, and even mortality.

The underlying causes of the two types of inherited disorders, however, are quite distinct. Individuals with hemoglobinopathies express a structural variant of one or more of the globin chains, while individuals with thalassemias produce reduced amounts of one of the globin chains. So far, more than 700 globin chain variants have been identified, as well as more than 170 gene mutations and deletions that reduce synthesis of alpha (α), beta (ß) or delta (δ) globin chains.

Today, laboratory testing for hemoglobinopathies and thalassemias is growing in importance. Reasons for testing include: work-up of microcytosis, anemia or erythrocytosis; prenatal testing; and follow-up of abnormal newborn screening results. Lab evaluation of the disorders requires robust methods and a good understanding of the genetic factors involved. This article provides a brief overview of the genetics of hemoglobinopathies and thalassemias and describes the analytical methods used by labs to evaluate them.

Hemoglobin Primer

Hb is composed of four globin chains: two α-globin chains and two other chains. Normally individuals have two copies each of the β- and δ-globin genes on chromosome 11 and four copies of the α-globin genes on chromosome 16. Table 1 summarizes some common genetic mutations of the globin genes and their characteristics.

After birth, the composition of Hb in red blood cells changes. In the fetus, HbF is the major Hb, which is composed of two α- and two γ (gamma)-chains. Production of this Hb type falls to adult levels within 1 to 2 years after birth. In normal adults, HbA, which contains two α-chains and two ß-chains, makes up 95 to 98% of the Hb. Normal adults also have HbA2, which accounts for 2 to 3.8% of Hb and contains two α- and two δ-chains. HbA2 usually is not detected in newborns and reaches adult levels by 1 year of age. HbF is generally undetectable in most adults, although it can be as much as 2% of the total Hb.

Table 1
Types of Mutations Found in Globin Chains

Mutation Type



Point mutation within a coding sequence

Missense mutation

HbS, HbE, HbC, other structural variants

Point mutation within a noncoding sequence

Structurally abnormal mRNA

Mutation of a regulator region

Some β-thalassemias

Conversion of a stop codon to a coding sequence

Elongated globin chain

Hb Constant Spring

Deletion of one or more genes


Most α-thalassemias

Some β-thalassemias

Gene fusion (δ-β)


Hb Lepore


Most hemoglobinopathies, or structural variants of Hb, are caused by point mutations in the globin genes. These mutations may cause changes in the structure of the Hb molecule, its ability to transport O2, its rate of synthesis, and/or its stability. Table 2 lists some of the more common Hb variants.

Other more rare variants have also been observed. For example, a variant called Hb Lepore is formed by fusion of the β- and δ-globin genes. Loss of a stop codon on one of the globin genes also produces a variant known as Hb Constant Spring.

Table 2
Specific Genetic Variations in Globin Genes

Beta Chain Variants


Location of Mutation

Amino Acid Change


β 6

Glu -> Val


β 26

Glu -> Lys


β 6

Glu -> Lys

D-Los Angeles

β 121

Glu -> Gln


β 22

Glu -> Gln

Alpha Chain Variants


α 68

Asn -> Lys

Sickle Cell Disease: HbS

The first Hb variant described by scientists was named sickle cell Hb (HbS). The prevalence of this variant is highest in West and Central Africa, Northeast Saudi Arabia and Kuwait, and East Central India. In heterozygous individuals, HbS provides some protection against the malaria parasite Plasmodium falciparu. But homozygous individuals have shortened life expectancies resulting from the complications of sickle cell disease, with studies reporting an average life expectancy of 42 and 48 years for males and females, respectively.

Today, sickle cell disease is recognized as a severe chronic condition. The substitution of valine in position 6 on the β-globin chain causes a decrease in the solubility of deoxyhemoglobin. This altered Hb molecule forms rigid S polymers that produce the classic sickle-shaped red blood cells for which the disease is named. Once the cell takes on this shape, the process is irreversible, and the individual eventually experiences complications of the disease.

However, individuals with one copy of HbS, called HbS trait, usually are asymptomatic and have normal red blood cell indices and morphology. Typical laboratory findings for these patients are 35 to 45% HbS. In simple cases of HbS trait, the percentage of HbS is always greater than the percentage of HbA. For individuals with HbS levels <30%, labs should consider concurrent iron deficiency or α-thalassemia trait.

Individuals with two copies of HbS develop sickle cell disease. Symptoms include hemolytic anemia, with Hb levels of 6 to 10 g/dL. In addition, peripheral-blood smears show sickle cells, target cells, and Howell-Jolly bodies. Their electrophoretic patterns and HPLC results typically show 90 to 95% HbS, no HbA, and often slightly elevated HbF in the 5 to 10% range. It is important to know a patient’s transfusion history in cases of sickle cell disease, as small amounts of HbA post-transfusion can produce misleading test results. In neonates, blood counts are usually normal, but as HbS levels increase and HbF levels decrease, hematologic abnormalities appear.

Individuals can also have HbS in combination with other variants and/or mutations. For example, a person may co-inherit a β−chain variant such as HbC, an α-chain variant such as HbG-Philadelphia, or have concurrent β-thalassemia and/or α-thalassemia. (See HbC section below.) A diagnosis of concurrent β+-thalassemia is likely when test results show HbS >50% and HbA <50% with increased HbA2 and HbF. If HbA is not present and HbA2 and HbF are elevated, concurrent β0 thalassemia should be considered. Patients with S/β0-thalassemia have a clinical course similar to sickle cell disease, but patients with S/β+-thalassemia generally have milder disease due to the presence of some HbA.

A Southeast Asia Variant: HbE

HbE is the most common variant found in individuals from Southeast Asia, with the highest prevalence occurring in people from Thailand, Laos, and Cambodia. Up to 50% of the population residing where the boundaries of the three countries meet are affected. In these individuals, the βE-chain is synthesized at a reduced rate compared with βA-chain, leading to an imbalance in globin chains and thalassemic red cell indices.

Individuals with HbE trait do not exhibit disease symptoms and generally have around 30% HbE. Their complete blood counts (CBCs) often show a slight microcytosis and erythrocytosis, with some target cells on the peripheral blood smear, and HbF is usually normal at <2%.

Homozygous HbE individuals have no major health problems, but they exhibit microcytosis and erythrocytosis. The mean cell volume (MCV) of red blood cells is generally lower than that seen in HbE trait individuals. Their HbA2 levels are normal, but the molecule is often not measurable as it co-migrates with HbE in many analytical systems. HbF may be slightly elevated.

Both α- and β-thalassemia are also common in many regions of Southeast Asia. Consequently, many individuals co-inherit HbE and either α- or β-thalassemia. Individuals who inherit HbE and α-thalassemia have lower percentages of HbE compared to those with HbE trait. Furthermore, the amount of HbE decreases with the number of α-globin genes that are deleted. CBC findings show microcytosis with no anemia; however, if anemia is present concurrently with a decreased amount of HbE, iron deficiency must be ruled out.

Other co-inheritance patterns are also observed for HbE. One example is HbE trait with HbH disease. Individuals with HbH disease carry deletions in three of the α-globin genes. In combination with HbE trait, this inheritance pattern results in moderately severe thalassemic blood findings, very similar to HbH disease. HbE levels in these individuals ranges from 10 to 15%.

HbE/β0-thalassemia is another combination that causes serious health problems including anemia, microcytosis, icterus, and splenomegaly, similar to β-thalassemia major. In these individuals, HbE levels range from 40 to 80% of the total Hb with the remainder being HbF.

A West African Variant: HbC

HbC originated in West Africa and occurs in about 3% of African Americans. Individuals with one copy of HbC may show mild microcytosis, with occasional target cells seen on the peripheral smear and 35 to 40% HbC. However, there are no other hematologic abnormalities.

Homozygosity for HbC results in a mild hemolytic anemia with microcytosis. Laboratory findings show HbC with normal amounts of HbA2 and HbF levels that are normal or only very minimally elevated. HbC is less soluble than HbA and sometimes forms crystals within red blood cells, which may be observed in blood smears along with target cells.

HbC can also be inherited along with other β-chain variants such as HbS. HbSC disease is a sickling disorder, with a milder presentation than sickle cell disease. While the exact mechanism of this sickling is unclear, these patients have mild to moderate anemia, with less frequent and less disabling vaso-occlusive crisis. Hb analysis of these individuals shows 50% HbS, with slightly less HbC.

A Common α-Chain Variant: HbG-Philadelphia

The most common α-chain variant seen in the U.S. is HbG-Philadelphia, with a 1 in 5,000 prevalence in African Americans. This variant has no hematologic or clinical effect. As an α-chain variant, it can be co-inherited with β-chain variants, such as HbC and HbS. Interpretation of laboratory findings for these individuals can be difficult, as the combination of Hbs results in multiple bands by electrophoretic analysis and multiple peaks on HPLC.


Thalassemias are caused either by mutations that reduce the rate of synthesis of a globin chain or by deletion of one or more of the globin genes. α-Thalassemias usually are caused by deletions of one or more of the four α-globin genes. These deletions decrease the synthesis of the protein, thereby creating an overabundance of γ-chains in a fetus or β-chains after HbF disappears. These γ- or β- chains can aggregate and form HbBart’s or HbH, respectively.

For patients whose Hb electrophoresis or HPLC analyses and iron studies are normal and the MCV is low, α-thalassemia trait should be considered. However, DNA studies are required for a definitive diagnosis. Table 3 summarizes the effects of α-gene deletions. 

Table 3
Common α-Thalassemias


Genes Deleted


Clinical Findings



Silent Carrier




Homozygous α2-Thalassemia




α-thalassemia 1 trait

+/- anemia



HbH Disease

Chronic hemolytic anemia



Hydrops fetalis



At least 150 mutations are known to cause β-thalassemia. The condition occurs mainly in people from the Mediterranean region, the Middle East, India, and Southeast Asia. These mutations have been divided into two categories: β0-thalassemias, which involve complete absence of β-chain production; and β+-thalassemias, which result in reduced synthesis of the β-chain. The severity of the disorder varies widely depending on the amount of β-globin produced.

Laboratory findings for individuals with β-thalassemia trait include microcytosis, hypochromia, no or mild anemia, and normal or slightly increased RBCs. HbA2 levels are elevated in these individuals, and HbF may be normal or increased. Inheritance of two β-thalassemia genes causes more severe disease ranging from β-thalassemia intermedia to Cooley’s anemia or β-thalassemia major.


δβ-thalassemia is caused by large deletions of both the δ- and β-globin genes. These individuals have persistent increased levels of HbF, although the increased production of γ-chains is not enough to completely compensate for the decrease in β-chain production. This situation leads to imbalance of the two globin chains and the classic thalassemic RBC indices. In individuals with δβ-thalassemia trait, HbF levels are increased to 5 to 15%, and HbA2 is normal or decreased. There have been rare cases of homozygous δβ-thalassemia. These individuals have 100% HbF for their entire lives.

Hereditary Persistence of HbF

Hereditary persistence of fetal Hb (HPFH) is a group of disorders in which HbF levels remain persistently elevated, ranging from 5 to 35% of the total Hb. Several different mutations can be found in different ethnic groups. Individuals with HPFH may have normal or slightly decreased MCV but no anemia. The condition can also be co-inherited with other β-chain variants, such as HbS. The high HbF levels in these individuals appears to moderate the severity of the sickling disorder.

Laboratory Methods

Labs can choose from a variety of methods for indentifying abnormal Hbs and quantifying abnormal Hbs, HbA, HbA2, and HbF. Electrophoresis on cellulose acetate or agarose has been used for decades, and according to 2007-2009 CAP survey data, it still is the most commonly used technique. However, the method is loosing popularity compared with high pressure liquid chromatography (HPLC) and capillary electrophoresis (CE) methods, as these types of instruments become more automated and user-friendly. Many labs use a combination of two or more techniques to improve identification of hemoglobin variants.

Electrophoresis: A Major Tool

Historically, labs have used alkaline electrophoresis (pH 8.2 to 8.6) on agarose or cellulose acetate as the initial screening method for hemoglobinopathies. Separation is based predominantly on the charge of the Hb molecule. At this pH, hemoglobin is negatively charged and moves toward the anode. The method allows preliminary identification of HbA and HbF; however HbS, HbD, HbG and HbLepore co-migrate, as do HbA2, HbE, HbC, and HbO-Arab. Because so many Hbs co-migrate with the common Hb variants, such as HbS, HbC, and HbE, labs frequently use a second technique to definitively identify the variant.

When labs detect variant Hb by alkaline electrophoresis, they often confirm its identity by acid electrophoresis (pH 6.0 to 6.2). Under these conditions, HbS separates from HbD, HbG, and HbLepore, and HbC separates from HbE, HbA2, and HbO-Arab.

Many excellent articles and books have been published that provide comparisons of the mobilities of Hb variants; therefore, it will not be described here.

HPLC: Growing in Popularity

Recently, use of cation exchange HPLC has grown in labs as a hemoglobinopathy screening method. The method is now automated and allows preliminary identification of a large number of Hb variants. The precision of these systems is such that quantitation of most variants, as well as HbF and HbA2, is possible from a single sample injection onto the column. But interpreting HPLC chromatograms can also be complicated (Figure 1). Major Hb components occasionally co-migrate, and minor Hb components such as glycosylated and acetylated forms sometimes do not separate from major components. Some serum components like bilirubin can also produce a peak in the same area as HbH. If possible, labs should confirm variants by another method.

Figure 1
Examples of Hb HPLC Chromatograms


Automated cation exchange HPLC provides preliminary identification of a large number of Hb variants. Confirmation, however, may require a second method.

Capillary Electrophoresis: The Newest Method

CE is a relatively new technique for separating Hbs that more labs are using. The charged Hb molecules separate by their electrophoretic mobility in an alkaline buffer. With automated versions of this method now available, it is more suitable for clinical labs. Furthermore, the sensitivity is good, and accurate quantitation of HbA2 in the presence of HbE or HbS can be achieved. Again, confirmation of Hb variants by another method can be helpful.

Isoelectric Focusing: Suitable for Newborn Screening

In IEF, Hb chains separate based on their isoelectric point. The method is more sensitive than routine electrophoresis, and analysis requires smaller sample volumes. Dried blood spots are also amenable to analysis. These characteristics make the method particularly advantageous for neonatal screening labs. However, minor components, such as glycosylated Hbs, also separate, making interpretation somewhat more difficult.

Quantitation of HbA2 and HbF

Diagnosis of β-thalassemia requires quantitation of HbA2. In general, labs use anion-exchange column chromatography, HPLC or CE for this analysis. Both HPLC and CE provide acceptable quantitation of HbA2 and HbF in the presence of many Hb variants; however, anion exchange chromatography only measures HbA2 quantitatively. Therefore, labs should use another method in conjunction with anion exchange chromatography to identify Hb variants.

Labs can also measure HbF by alkali denaturation if the levels are >50%. Labs should confirm by a second method levels of HbF >10% detected by HPLC.

Sickle Solubility Test

Another test used by labs specifically for screening or confirming the presence of HbS is the sickling solubility test. Several commercial kits are available that can detect HbS down to a level of 20%. Solubility testing as a screen is not indicated in infants under 6 months of age, as there is a high potential for false-negative results.

DNA Studies

Although most clinical labs do not routinely perform DNA analysis for Hb studies, this testing is important for genetic counseling purposes in confirming diagnosis of α-thalassemia trait. PCR primers are now available for the most common α-globin gene deletions. DNA studies are also useful in confirming HbD-Punjab.

Interpretation of Results

Laboratory evaluation of hemoglobinopathies and thalassemias can be quite complex, and other information is required to adequately interpret the results. Age, CBC indices, and iron studies are critical. For example, HbA2 can be reduced in iron-deficient individuals; therefore, any iron deficiency must be resolved before thalassemia can be ruled out as a cause of microcytosis. Iron deficiency will also reduce the amount of β-globin chain variants such as HbS and HbE present, confounding the diagnosis. Reticulocyte counts can also be useful in some instances, and family studies can help in particularly complex cases.


Of the many genetic Hb variants and thal-assemias identified, only a few have substantial clinical significance. In general, the severity of clinical manifestations relates to the amount of globin chain produced and the stability of residual chains present in excess. Laboratory investigation to identify hemoglobinopathies and thalassemias requires precise methods and an understanding of the genetic variations that contribute to the disorders.

Suggested Readings

  • Bunn HF, Forget BG. Hemoglobin: Molecular, Genetic and Clinical Aspects. WB Saunders Co, Philadelphia.1986.
  • Riou J, Godart C, Hurtrel D, Mathis M, et al. Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants. Clin Chem 1997;43:34–39.
  • Clarke GM, Higgins TN. Laboratory investigation of hemoglobinopathies and thalassemias: review and update. Clin Chem 2000;46:1284–1290.
  • Ou C-N, Rognerud CL. Diagnosis of hemoglobinopathies: electrophoresis vs HPLC. Clin Chem Acta 2001;313:187–194.
  • Bain BJ. Haemoglobinopathy Diagnosis. 2nd Edition. Blackwell Publishing LTD. 2006.
  • Hoyer JD, Korft SH, ed. Color Atlas of Hemoglobin Disorders: A Compendium Based on Proficiency Testing. College of American Pathologists, Northfield, IL. 2003.
  • Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60,000 samples in a clinical diagnostic laboratory. Clin Chem 2004;50:1736–1747.


Shirley L. Welch, PhD, is director of chemistry at Kaiser Permanente NW Regional Laboratory in Portland, Ore. She is an AACC Outstanding Speaker and has served on many AACC committees, including as chair of the 2006 AACC Annual Meeting.