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. The Lepore hemoglobins are inherited mutations, resulting from δß-gene fusion products. Lepore-Boston is the only mutation to occur with any frequency and is mostly confined to persons of Mediterranean decent.
Lepore hemoglobins are stable, normally-functioning oxygen carriers that are significantly under-produced and, thus, behave as β-thalassemias. The peripheral smear of persons with Lepore trait (1 mutated gene) shows only erythrocytosis, microcytosis, hypochromia, and occasional target cells. Anemia is not expected, and carriers of the mutation are usually only revealed through family studies or incidentally when testing for some other reason.
Hemoglobin Lepore disease (2 mutated genes) is rare, except in areas where the carrier rate is high, but presents with significant anemia and clinically mimics ß-thalassemia intermedia or major. There is a wide spectrum of disease, with profound erythrocytosis, microcytosis, hypochromia, and many target cells. There is hemolysis, and most patients require transfusions and have splenomegaly and bone deformity. Age at presentation reflects the severity of the disease.
Significant anemia, hemolysis, or pronounced microcytosis (<65 fL) with hemoglobin Lepore trait should prompt further investigations for the coinheritance of another hemoglobinopathy, such as a ß-thalassemia or hemolytic disorder.
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)
|Presumptive Diagnosis||Ratio Hgb Lepore / A||Hgb A2||Other Hgb Present at > 9 months of age||Hemoglobin||MCV/Morphology|
|A/Lepore||10-15/80-90||<3%||F = 2-5%||normal||70-80 fL occasional target cells erythrocytosis|
|Lepore/Lepore||100/0||<1%||F = 5-30%||4 – 9 g/dL||50-65 fL target cells erythrocytosis|
|Lepore/A ß-thalassemia||Variable||<3%||F = 5-50%||variable||50-65 fL target cells erythrocytosis|
|Lepore/Lepore ß-thalassemia||100/0||<1%||F = 70-95%||transfusion dependent||50-65 fL target cells erythrocytosis|
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).
Hemoglobin Lepore disease and Lepore trait/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 hypercoagulable 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 Lepore may require only RBC indices. However, testing is dictated by the severity of any 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.
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?
Three different Lepore hemoglobin variants have been described, resulting from different fusion points between the δ and ß genes, but all three behave similarly on electrophoresis and isoelectric focusing and have similar clinical courses.(Table 2)
|Boston||Washington Cyprus||δ (1-87) ß (115-146)|
|Baltimore||δ (1-50) ß (86-146)|
|Hollandia||δ (1-22) ß (50-146)|
The only difference reported has been a slightly higher hemoglobin F (3-5%) in Lepore-Baltimore trait. Hemoglobin F is normal in the other 2 traits.
The phenotype of Lepore ß-thalassemia, the most serious of the Lepore disorders, ranges from moderate anemia to transfusion dependent thalassemia major, and there is an associated risk for thromboembolism secondary to a hypercoagulable state, which is increased by splenectomy. Modifiers likely include the type of ß-thalassemia, the persistence of F, which accommodates excess α-chains, coinheritance of α-thalassemia, which reduces the α-chain excess and improves the globin chain balance, and iron deficiency. All of these need to be assessed in each patient. Hydroxyurea therapy, with the goal of increasing the percentage of hemoglobin F, may have a role in treatment.
Lepore disease and Lepore with ß-thalassemia are iron-loading, so it is important to establish the true iron status of a 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.
Many Newborn Screening programs include tests for common hemoglobinopathies, but it is likely that hemoglobin Lepore will not be identified specifically. However, once the percentage of hemoglobin F is subtracted from the total hemoglobin, the same ratio of Lepore to A will be observed as in adults.
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 percentage of hemoglobin A and thus complicate the picture.
What Lab Results Are Absolutely Confirmatory?
Sequencing of the chromosome for the known specific δß-globin Lepore product is diagnostic for hemoglobin Lepore. The expense of this test is rarely justified.
In practice, however, the demonstration of a doublet peak on HPLC in the A2-elution window, together with a band eluting between F and S on IEF (or with S and A on alkaline and acid EP, respectively) is considered confirmatory for the presence of hemoglobin Lepore. 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 Lepore trait, the percentage of hemoglobin Lepore is always lower than that of hemoglobin A (typically only 10-15%), because production of the mutant “beta” chain is dependent on the much lower production rate of the delta gene.
Careful attention is required when using the percentage of hemoglobin A2 to determine the presence of a concurrent ß-thalassemia. The Lepore mutation acts as a thalassemia, but the expected increase in A2 is counteracted by the underproduction of wild type δ-globin mRNA. Thus, in Lepore trait, A2 is normal; in Lepore disease, little or none is seen. Similarly, if a ß-thalassemia occurs with A/Lepore, some A2 will be observed, but not if it occurs with homozygous Lepore. The rule of thumb that an MCV/RBC ratio less than 14 is highly suggestive of a coinherited ß-thalassemia does not hold here either, since the mere presence of Lepore trait produces the same picture.
A negative Sickling test should be observed with hemoglobin Lepore, with the exception of splenectomized patients in whom the presence of large numbers of normoblast nuclei cause strong persistent turbidity.
What Confirmatory Tests Should I Request for My Clinical Dx? In addition, what follow-up tests might be useful?
Sequencing of the chromosome for the known specific δß-globin Lepore product may be indicated if the previously mentioned techniques are unable to arrive at a definitive diagnosis because of coelution with another hemoglobin.
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). 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 the hydroxyurea increases the percentage of hemoglobin F present. Hydroxyurea is given for polycythemias, sickle disease, and as a chemotherapeutic agent.
What Factors, If Any, Might Affect the Confirmatory Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?
Lepore hemoglobins coelute with hemoglobin S on alkaline electrophoresis and with A on acid electrophoresis, but the Sickling test is negative. Hemoglobins D and G also share this pattern. However, on HPLC, Lepore runs with A2, producing a characteristic lump on the downslope. There is comigration with G (between F and S) on both IEF and capillary electrophoresis (CEP).
There are many other common causes of anemia that may need additional investigations, such as:
dietary iron deficiency or inadequate absorption (achlorhydria)
The following laboratory tests help distinguish between anemia resultant from iron deficiency (IDA), inflammation (ACI), or concurrent iron deficiency with inflammation. (Table 3)
|Lab Test||ACI||IDA||IDA and ACI|
|Soluble Transferrin Receptor (sTfR)||normal||increase||normal/increase|
|Inflammatory Markers (CRP)||elevated||normal||elevated|
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 Lepore 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
immunopathologic, such as transfusion reactions, Rhesus/ABO incompatibility, warm and cold agglutinins
Tests indicative of hemolysis include decreased or absent haptoglobin, elevated LDH and unconjugated bilirubin, and elevated serum free hemoglobin.
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- At a Glance
- What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?
- 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?
- What Lab Results Are Absolutely Confirmatory?
- What Confirmatory Tests Should I Request for My Clinical Dx? In addition, what follow-up tests might be useful?
- What Factors, If Any, Might Affect the Confirmatory Lab Results? In particular, does your patient take any medications - OTC drugs or Herbals - that might affect the lab results?