Anemia Associated with Hemoglobin E

At a Glance

A family history of anemia in the absence of iron deficiency should prompt consideration of a hemoglobinopathy, and a number of these appear in the differential diagnosis. Hemoglobin E is an inherited mutation in the ß-globin gene. Persons of SE Asian decent frequently carry this mutation, which is the 4th most common variant worldwide. Homozygosity is 4% in Cambodians, and the carrier rate is as high as 50% in some areas.

Hemoglobin E is a normally-functioning oxygen carrier that is under-produced and, thus, behaves as a β+-thalassemia, but is thought to confer protection against malaria. The peripheral smear of persons with hemoglobin E (trait - 1 mutated gene vs. disease - 2 mutated genes) is characterized by microcytosis, hypochromia, and target cells. There is no hemolysis or significant anemia, and most patients are asymptomatic. Carriers of the mutation are usually only revealed through family studies or incidentally when testing for some other reason.

Significant anemia, hemolysis, or pronounced microcytosis (<65 fL) with hemoglobin E should prompt further investigations for the coinheritance of another hemoglobinopathy. α-Thalassemia is relatively common with hemoglobin E, since the cis-α2 selection also occurs with high frequency in this same ethnic group. Coinheritance of ß-thalassemia, hemoglobin S, or Constant Spring produces significant disease.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

The standard hemoglobin evaluation for diagnostic purposes consists of red blood cell (RBC) indices, a sickling test, plus cation exchange high performance liquid chromatography (HPLC) or capillary electrophoresis (CEP).(Table 1)

If RBC indices are abnormal, it is appropriate to order morphology.

Assessment of iron status is important in anemia, which is usually accomplished through tests for ferritin and transferrin saturation (<20 ng/mL and <15%, respectively, in uncomplicated iron deficiency).

Some Hemoglobin E/ß-thalassemias are iron loading, and physicians should be alert to the sequelae of iron overload, which occurs when the transferrin saturation is greater than 75%.

Always attempt to obtain a transfusion history.

Obtain coagulation tests to determine if a hyoprcoagulable state exists. Postsplenectomy patients are particularly at risk because of endothelial activation from thrombocytosis and increased exposure to RBC membrane phosphatidyl serine. In these patients, physicians should be alert for sequelae, particularly pulmonary artery thrombosis and hypoxemia. Prophylactic therapy is required.

If the sickling test is positive, a variant hemoglobin is suspected from HPLC, or there is clinical suspicion of a hemoglobinopathy, isoelectric focusing (IEF) or electrophoresis (EP) of hemoglobin dimers (or less commonly free globin chains) should be ordered.

Follow-up tests on patients with known hemoglobin E may require only RBC indices. However, testing is dictated by the severity of the coinherited mutations and may include regular assessment of ferritin, the percentage of hemoglobin F and of the known variants with HPLC or CEP, plus coagulation status in patients with coinherited ß-thalassemia.

Table 1

Approximate Expected Hemoglobin Values with the most common Hemoglobin E Disorders

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications - OTC drugs or Herbals - that might affect the lab results?

Scrupulous attention to the relative percentages of hemoglobin species detected is especially important with the E disorders because of the marked differences in clinical course between patients.

The phenotype of E ß-thalassemia, the most serious of the E disorders, ranges from mild anemia to transfusion dependent thalassemia major, and there is an associated risk for thromboembolism secondary to a hypercoagulable state, which is increased by splenectomy. There are several modifiers, including the type of ß-thalassemia, the persistence of F, which accommodates excess α-chains, and coinheritance of α-thalassemia, which reduces the α-chain excess and improves the globin chain balance, and iron deficiency. All of these need assessed in each patient. Hydroxyurea therapy, with the goal of increasing the percentage of hemoglobin F, produces marked improvement in almost 40% of patients.

Iron deficiency can lower both the percentage of hemoglobin E and A2, such that a ß-thalassemia could be overlooked. An MCV/RBC less than 14 is highly suggestive of ß-thalassemia. If using the value of hemoglobin A2 as a key indicator of ß-thalassemia, it is crucial to exclude the presence of A2prime. This delta chain variant is clinically benign, but will be present at equal concentration to the A2, and must be added to it. It can be difficult to visualize on EP or IEF, since the percentage is small and it coelutes with hemoglobin S on HPLC. In addition, HPLC and IEF have difficulty determining A2 in the presence of E, but they are well separated by capillary electrophoresis (CEP).

E trait with iron deficiency looks very much like E trait α-thalassemia in laboratory tests, but there is iron-loading with the latter, so it is important to establish the true iron status of the patient before considering iron supplements or transfusion. Anemia of inflammation (anemia of chronic disease) has a normal/elevated ferritin, and further tests might be indicated to see if iron deficiency is also present. In inflammatory disease, C-Reactive Protein is elevated.

Transfused blood is always assumed to comprise 100% hemoglobin A, but this is not always the case as patients who are heterozygous for hemoglobin C or D mutations are not identified during donation, and this could alter the expected percentages of hemoglobins A and E.

What Lab Results Are Absolutely Confirmatory?

The demonstration of substitution of lysine for glutamic acid at position 26 of the ß-globin chain is diagnostic for hemoglobin E (ß26 Glu → Lys). The expense of this test is rarely justified.

In practice, however, the demonstration of a peak on HPLC in the E-elution window, together with a band eluting with hemoglobin E on EP or IEF, is considered confirmatory for the presence of hemoglobin E. Providing that the percentages of hemoglobin F and A2 are normal and the clinical severity is as expected, further testing is not usually warranted.

In Hemoglobin E trait, the percentage of Hemoglobin E is always lower than that of hemoglobin A (typically 30%), because the mutation is near the end of the first exon and it creates a cryptic splicing site that sometimes fails to produce full length functional mRNA. The mutation also results in a weakened α/ß chain interface, leading to slight stability during conditions of increased oxidative stress.

Many Newborn Screening programs include tests for common hemoglobinopathies. Once the percentage of hemoglobin F is subtracted from the total hemoglobin, the same ratio of E to A will be observed as in adults.

A negative Sickling test should be observed with hemoglobin E, with the exception of E trait ß0 patients post-splenectomy, where the presence of large numbers of normoblast nuclei cause strong persistent turbidity.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

Sequencing of the chromosome for the known specific point mutations for ß-globin E may be indicated if these techniques are unable to arrive at a definitive diagnosis because of coelution with another hemoglobin.

Sequencing of the chromosome for common point mutations or deletions in the ß-globin gene is only rarely indicated in the event of the appearance of a previously unknown hemoglobin entity.

If the severity of the clinical presentation does not match the initial diagnosis, sequencing of the α- and/or ß-globin transcription regulator genes or sequencing of the gene in its entirety may be necessary to arrive at a definitive diagnosis. The presence of hemoglobin H may indicate a 3-gene α-thalassemia (or a 2-gene deletion in a neonate). Because of the geographic distribution of α-thalassemia, the cis combination is more likely. An elevated percentage of hemoglobin A2 is indicative of a ß-thalassemia. An elevated percentage of hemoglobin F is suggestive of a ß0 thalassemia, hemoglobin S E disease, or hereditary persistence of fetal hemoglobin.

Treatment with hydroxyurea increases the percentage of hemoglobin F present. Hydroxyurea is given for polycythemias, sickle disease, and as a chemotherapeutic agent.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications - OTC drugs or Herbals - that might affect the lab results?

Hemoglobins C, O-Arab, and C-Harlem all migrate with hemoglobin E on alkaline electrophoresis, but none coelutes with hemoglobin E on acid EP. On HPLC, hemoglobin E migrates with A2, which poses problems when assessing for the presence of ß-thalassemias, but they are well separated on capillary electrophoresis (CEP).

The absence of hemoglobin H in both E trait/H disease and homozygous E/H disease is due to the fact that the ßE tetramers are thought to comigrate with hemoglobin E. Hemoglobin Bart's (γ-chain tetramers) appears in both of these conditions and also in E trait/Constant Spring/H disease, where there is additionally 3 other small bands attributable to Constant Spring on alkaline EP).

The current generation of hemoglobin A1C (glycated hemoglobin) assays has eliminated previously observed unreliability in the presence of hemoglobin C and S trait but may still give unreliable results with hemoglobin E trait. Immunoassay-based tests perform better than HPLC, but results of diabetes tests should be interpreted with caution in these patients.

The Sickling test is a screening test that detects any hemoglobin that polymerizes under reduced oxygen tension and cannot differentiate between Homozygous S disease or one of the sickle traits or the presence of a doubly substituted S mutation, such as hemoglobin C-Harlem. All results should be confirmed by additional testing, especially if they do not agree with the clinical picture.

The Sickling test may give a false negative if the hemoglobin S concentration is below 1 g/dL (typically <10-15% of the total hemoglobin), after transfusion, or in cases where the F is greater than 90% (neonates and hereditary persistence of fetal hemoglobin.

The Sickling test may give a false positive if there are nucleated RBCs in the peripheral blood or the patient has a marked hypergammaglobulinemia, such as multiple myeloma.

There are many causes of hemolysis other than hemoglobinopathies, some of which are:

• RBC enzyme deficiencies, such as G6PD, Pyruvate Kinase, Glucose Phosphate Isomerase, or NADH reductase

• mechanical destruction from artificial valves or burns

• infection

• immunopathologic, such as transfusion reactions, Rhesus/ABO incompatibility, or warm and cold agglutinins

Tests indicative of hemolysis include decreased or absent haptoglobin, elevated LDH and unconjugated bilirubin, and elevated serum free hemoglobin.

There are many other common causes of anemia that may need additional investigations, such as:

• dietary iron deficiency or inadequate absorption (achlorhydria)

• pregnancy

• chronic disease

• malignancy

• malnutrition

• GI bleeding

The following laboratory tests help distinguish between anemia resultant from iron deficiency (IDA), inflammation (ACI), or concurrent iron deficiency with inflammation. (Table 2)

Table 2

Distinguishing Test Results