Strictly speaking, there is no thalassemia hemoglobin. Thalassemia comprises a complex group of genetic abnormalities in globin chain synthesis. There are three major clinical categories: thalassemia major, associated with severe and often life-threatening clinical manifestations; thalassemia minor, with mild or minimal clinical manifestations; and a combination of the thalassemia gene with a gene for another abnormal hemoglobin.

The genetic situation is much more complicated than arbitrary subdivision into the major clinical syndromes would imply.

The globin portion of normal hemoglobin (Hb A1) is composed of two pairs of polypeptide (amino acid) chains, one pair (two chains) called alpha (a) and the other two chains called beta (b). All normal hemoglobins have two alpha chains, but certain hemoglobins have either one or both beta chains that have a polypeptide amino acid sequence different from usual beta chains. Thus, Hb A2 has two delta (d) chains, and Hb F has two gamma (g) chains in addition to the two alphas. All three of these hemoglobins (A1, A2, and F) are normally present in adult RBCs, but A2 and F normally are present only in trace amounts. One polypeptide chain from each pair of alpha and beta chains is inherited from each parent, so that one alpha chain and one beta chain are derived from the mother and the other alpha and beta chain from the father. The thalassemia gene may involve either the alpha or the beta chain. In the great majority of cases, the beta chain is affected; genetically speaking, it would be more correct to call such a condition a beta thalassemia. If the condition is heterozygous, only one of the two beta chains is affected; this usually leaves only one beta chain (instead of two) available for Hb A1 synthesis and yields a relative increase in Hb A2. This produces the clinical picture of thalassemia minor. In a homozygous beta thalassemia, both of the beta chains are affected; this apparently results in marked suppression of normal Hb A1 synthesis and leads to a compensatory increase in gamma chains; the combination of increased gamma chains with the nonaffected alpha chains produces marked elevation of Hb F. This gives the clinical syndrome of thalassemia major. It is also possible for the thalassemia genetic abnormality to affect the alpha chains. The genetics of alpha thalassemia are more complicated than those of beta thalassemia, because there are two alpha-globin gene loci on each of the two globin-controlling body chromosomes, whereas for beta-globin control there is one locus on each of the two chromosomes. In alpha thalassemia trait (“silent carrier”), one gene locus on one chromosome only is deleted or abnormal (one of the four loci). In alpha thalassemia minor, two of the four loci are affected. This may be produced either by deletion or abnormality in both loci on one of the two chromosomes (a genotype called alpha-thalassemia-1, more common in Asians), or by deletion of one of the two loci on each of the two chromosomes (a genotype called alpha-thalassemia-2, more common in Africans and those of Mediterranean descent). Hemoglobin A1 production is mildly curtailed, but no Hb A2 or F increase occurs because they also need alpha chains. Hemoglobin H disease results from deletion or inactivation of three of the four loci. All four globin chains of Hb H are beta chains. Hemoglobin H disease occurs mostly in Asians but occasionally is found in persons from the Mediterranean area. The most serious condition is that resulting from deletion or inactivation of all four alpha-globin gene loci; if this occurs, the hemoglobin produced is called Bart’s hemoglobin, and all four globin chains are the gamma type. In most cases functional hemoglobin production is curtailed enough to be lethal in utero or neonatal life. Another abnormal hemoglobin that should be mentioned is Hb-Lepore, which is called a fusion hemoglobin, and which consists of two normal alpha chains and two nonalpha fusion chains, each containing the amino terminal portion of a delta chain joined to the carboxy terminal portion of a beta chain.

Thalassemia major (Cooley’s anemia). This globin variant is the homozygous form of beta thalassemia and consists of two alpha chains and two gamma chains. The condition generally does not become evident until substantial changeover to adult hemoglobin at about 2-3 months of age and clinically is manifest by severe hemolytic anemia and a considerable number of normoblasts in the peripheral blood. The nucleated RBCs are most often about one third or one half the number of WBCs but may even exceed them. There are frequent Howell-Jolly bodies and considerable numbers of polychromatophilic RBCs. The mature RBCs are usually very hypochromic, with considerable anisocytosis and poikilocytosis, and there are moderate numbers of target cells. The mean corpuscular volume (MCV) is microcytic. WBC counts are often mildly increased, and there may be mild granulocytic immaturity, sometimes even with myelocytes present. Platelets are normal. Skull x-ray films show abnormal patterns similar to those in sickle cell anemia but even more pronounced. Death most often occurs in childhood or adolescence.

Diagnosis. Diagnosis is suggested by a severe anemia with very hypochromic RBCs, moderate numbers of target cells, many nucleated RBCs, and a family history of Mediterranean origin. The sickle preparation is negative. Definitive diagnosis depends on the fact that in thalassemia major, Hb F is elevated (10%-90% of the total hemoglobin, usually >50%). Hemoglobin F has approximately the same migration rate as Hb A1 on paper electrophoresis but may be separated by other electrophoretic techniques. In addition, Hb F is much more resistant to denaturation by alkali than is Hb A1. This fact is utilized in the alkali denaturation test. The hemoglobin solution is added to a certain concentration of sodium hydroxide (NaOH), and after filtration the amount of hemoglobin in the filtrate (the undenatured hemoglobin) is measured and compared with the original total quantity of hemoglobin. One report cautions that if the RBCs are not washed sufficiently before the hemoglobin solution is prepared, reagents of some manufacturers may produce a false apparent increase in Hb F.

The gene producing the Mediterranean type of homozygous beta thalassemia does not synthesize any beta chains; sometimes referred to as b°. When homozygous beta thalassemia occurs in African Americans, the gene is apparently slightly different because small amounts of beta chains may be produced as well as the predominant gamma chains. This gene is referred to as b+, and the degree of anemia tends to be less severe than in the Mediterranean b° type.

Thalassemia minor. This clinical subgroup of the thalassemias is most frequently the heterozygous form of beta thalassemia (beta thalassemia trait). Besides a relatively high incidence (1%-10%) in Americans of Mediterranean extraction, there is an estimated frequency of 1% (0.5%-2%) in African Americans. About 75% of patients have anemia, which is usually mild; fewer than 10% of patients with anemia have hemoglobin levels less than 10 gm/100 ml. Patients with beta thalassemia trait have microcytic MCV values in a great majority of cases (87%-100%) whether or not anemia is present. The mean corpuscular hemoglobin (MCH) value is also decreased below reference range in almost all cases. Peripheral blood smears typically contain hypochromic and somewhat microcytic RBCs, usually with some target cells and frequently with some RBCs containing basophilic stippling. Nucleated RBCs are not present. The reticulocyte count is frequently elevated (50% of patients in one study had reticulocytosis >3%).

The main laboratory abnormality in beta thalassemia trait is an increased amount of Hb A2 (A2 is not elevated in alpha thalassemia trait). As noted previously, A2 is a variant of adult Hb A and is normally present in quantities up to 2.5% or 4%, depending on the method used. In beta thalassemia trait, A2 is elevated to some degree with a maximum of approximately 10%. (If >10% is reported using cellulose acetate electrophoresis, this suggests another Hb migrating in the A2 area, such as Hb C.) Hemoglobin F is usually normal but can be present in quantities up to 5%. Hemoglobin A2 cannot be identified on paper electrophoresis, and demonstration or quantitation necessitates cellulose acetate or polyacrylamide gel electrophoresis or resin column methods. DNA probe tests are available in some university centers for diagnosis of beta thalassemia.

Thalassemia minor due to alpha thalassemia trait is probably more common in the United States than previously recognized. There is a relatively high incidence in persons of Southeast Asian origin and in African Americans. (Limited studies have detected 6%-30% affected persons from African Americans.) There is also a significant incidence in persons of Mediterranean extraction. The majority of affected persons do not exhibit any clinical symptoms of any anemia, and of the minority that do, symptoms of anemia are most often relatively mild. In one limited study of African Americans in Los Angeles, the majority of affected persons had decreased MCV but only about 10% were anemic. The average MCH value was about 2% less than the average value for normal persons, and the average MCH concentration (MCHC) was about the same as that of normal persons. Hemoglobin H disease can be detected by appropriate hemoglobin electrophoretic techniques. Hemoglobin H disease is mostly restricted to Asians and is manifest by a chronic hemolytic anemia of moderate degree (which may, however, be mild rather than moderate). There is also an acquired form of Hb H disease, reported in some patients with myelodysplastic or myeloproliferative syndromes.

Currently there is no easy laboratory method to diagnose genetic alpha-globin abnormality. However, in the newborn’s cord blood, Bart’s hemoglobin is generally elevated (by electrophoresis) in rough proportion to the severity of the alpha thalassemia syndrome. Bart’s hemoglobin thus constitutes about 25% (range 20%-40%) in Hb H disease, about 5% (range 2%-10%) in alpha thalassemia trait, and about 1%-2% in the silent carrier state. After about 4 months (range 4-6 months) of age, Bart’s hemoglobin has mostly disappeared. Thereafter, globin chain synthesis studies or DNA probe techniques are the current methods used, but these techniques are available only in research laboratories. Hemoglobin H inclusions in RBCs can be seen using standard reticulocyte count methods, but their sensitivity is disputed (especially in alpha thalassemia trait), possibly due in part to differences in methodology.

Thalassemia minor vs. chronic iron deficiency. Thalassemia minor must sometimes be differentiated from iron deficiency anemia because of the hypochromic-microcytic status of the RBCs. Certain guidelines have been suggested to increase suspicion for thalassemia minor or to presumptively rule it out in patients with microcytic anemia. The RBC distribution width (RDW; Chapter 2) is usually normal in uncomplicated thalassemia minor and elevated in chronic iron deficiency anemia. Uncomplicated thalassemia minor typically has an RBC count greater than 5 million/mm3 in spite of decreased MCV. Uncomplicated thalassemia minor very uncommonly has a hemoglobin level less than 9.0 gm/100 ml (90 g/L) and usually has a MCHC of 30% or greater (adult reference range, 33%-37% or 330-370 g/L), whereas 50% of patients with chronic iron deficiency anemia have a hemoglobin level less than 9.0 gm (90 g/L), and 50% or more have an MCHC less than 30% (300 g/L). There are also several formulas to segregate thalassemia trait from chronic iron deficiency, of which the best known is the discriminant function of England and Frazer. Unfortunately, there is enough overlap to severely limit the usefulness of these formulas for any individual patient. Serum iron is usually decreased in uncomplicated chronic iron deficiency anemia or anemia of chronic disease and is usually normal in uncomplicated beta thalassemia trait. An elevated total iron-binding capacity (TIBC) suggests iron deficiency, and a decreased TIBC suggests chronic disease. Serum ferritin is decreased in uncomplicated chronic iron deficiency and is normal in uncomplicated beta thalassemia trait. Bone marrow iron is absent in iron deficiency and normal or increased in thalassemia trait. The word “uncomplicated” is stressed because patients with beta thalassemia trait may have concurrent chronic iron deficiency and because anemia of chronic disease may be concurrent with either condition. The anemia of chronic disease may itself be microcytic and hypochromic in about 10% of cases.

Definitive diagnosis of beta thalassemia trait involves measurement of Hb A2, which is increased in uncomplicated beta thalassemia trait but not in chronic iron deficiency or chronic disease. However, chronic iron deficiency decreases Hb A2 levels so that iron deficiency coexistent with beta thalassemia trait could lead to falsely normal Hb A2 results. In one study, 15% of children with beta thalassemia minor initially displayed normal A2 levels that became elevated after 2 months of therapy with oral iron. Conditions other than beta thalassemia that raise Hb A2 levels include folate or B12 deficiency, increase in Hb F level, and the presence of certain abnormal hemoglobins that migrate with A2, the particular variety of interfering hemoglobin being dependent on the A2 assay method used.

Sickle thalassemia. This combination produces a condition analogous to SC disease, clinically similar in many respects to SS anemia but considerably milder. Sickle cell test results are positive. There frequently are considerable numbers of target cells. Approximately 60%-80% of the hemoglobin is Hb S. In the S-thalassemia b° (S-Thal-b°) type, most of the remaining hemoglobin is Hb F, so that the pattern resembles SS anemia. However, S-Thal-b° is clinically milder, and the peripheral smear may display RBCs that are more hypochromic than one would expect with SS disease. Also, Hb A2 is increased. The S-thalassemia b+ (S-Thal-b+) pattern might be confused with the SA pattern of sickle trait. However, in sickle trait Hb A predominates rather than Hb S, so that an electrophoretic SA pattern in which more than 50% of the total Hb is Hb S suggests S-thalassemia.

Screening for thalassemia. Several reports indicate that a decreased MCV detected by automated hematology cell counters is a useful screening method for thalassemia as well as for chronic iron deficiency anemia. One report suggests an 85% chance of thalassemia when there is a combination of MCV of less than 75 femtoliters (fL) with a RBC count of more than 5 million/mm3 (5 Ч 106/L). A hemoglobin level less than 9.0 gm/100 ml (90 g/L) plus an MCHC less than 30% suggests chronic iron deficiency rather than thalassemia. As noted previously, cord blood has been used for detection of the alpha thalassemias. It is now becoming possible to screen for homozygous hemoglobinopathies and severe forms of thalassemia in utero employing DNA analysis of a chorionic villus biopsy at 8-10 weeks or amniotic fluid cells from amniocentesis at 16-18 weeks.