Imaging Studies
In patients with beta thalassemia major who are not regularly transfused, plain radiographs reveal classic changes in the bones. The striking expansion of the erythroid marrow widens the marrow spaces, thinning the cortex and causing osteoporosis. In addition to the classic "hair on end" appearance of the skull (shown below), which results from widening of the diploic spaces and is observed on plain radiographs, the maxilla may overgrow, which results in maxillary overbite, prominence of the upper incisors, and separation of the orbit. These changes contribute to the classic "chipmunk" facies observed in patients with thalassemia major. [10] Other bony structures, such as the ribs, long bones, and flat bones, may also be sites of major deformities. Plain radiographs of the long bones may reveal a lacy trabecular pattern with osteopenia and osteoporosis. Changes in the pelvis, skull, and spine become more evident during the second decade of life, when marrow activity shifts primarily to those bones, and compression fractures may be noted in the vertebrae. [22]
The classic "hair on end" appearance on plain skull radiographs of a patient with Cooley anemia.
Laboratory Studies
CBC results and red cell indices, along with peripheral blood film examination outcomes, are usually sufficient to suspect a diagnosis of thalassemia. Laboratory results are as follows:
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In severe forms of thalassemia, the Hb level ranges from 2-8 g/dL
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Mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) are significantly low, but, unlike thalassemia trait, thalassemia major is associated with a markedly elevated red cell distribution width (RDW), reflecting extreme anisocytosis
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The white blood cell (WBC) count is usually elevated in beta-thalassemia major; this is due, in part, to a miscount of the many nucleated RBCs as leukocytes; leukocytosis is usually present, even after excluding the nucleated RBCs, with a shift to the left
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The platelet count is usually normal, unless the spleen is markedly enlarged, in which case it may be diminished
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Peripheral blood film examination reveals marked hypochromasia and microcytosis, hypochromic macrocytes that represent the polychromatophilic cells, nucleated RBCs, basophilic stippling, and occasional immature leukocytes, as shown below
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Contrast this with the peripheral blood film shown below, which on supravital staining shows intracellular Heinz body inclusions (β4) associated with HbH, a severe alpha thalassemia
Supra vital stain in hemoglobin H disease that reveals Heinz bodies (golf ball appearance).
Hb electrophoresis can usually confirm the diagnosis of beta thalassemia, HbH disease, and HbE/β-thalassemia. The electrophoresis usually reveals an elevated HbF fraction, which is distributed heterogeneously in the RBCs of patients with beta thalassemia. HbH is usually found in patients with HbH disease (but it is unstable), while Hb Bart is found in newborns with alpha-thalassemia trait. In β0 thalassemia, no HbA is usually present; only HbA2 and HbF are found.
In cases that are less clear-cut, checking both biologic parents' CBC, reticulocyte count, and red cell indices and performing Hb electrophoresis on the parents can be helpful in arriving at a diagnosis.
A complete RBC phenotype assessment, a hepatitis screen, folic acid level evaluation, and human leukocyte antigen (HLA) typing are recommended before initiation of blood transfusion therapy.
Prenatal diagnosis
Globin chain synthesis, which was once used in postnatal diagnosis, has also been used on fetal cells obtained by fetoscopy to screen the fetus. This test reveals imbalanced production of certain globin chains that are diagnostic of thalassemia.
Since PCR assay techniques became available, several new methods have come into use to identify affected babies or carrier individuals accurately and quickly. The DNA material is obtained by chorionic villus sampling (CVS), and mutations that change restriction enzyme cutting sites can be identified.
Because many of the mutations that cause alpha and beta thalassemia have become known, identifying such mutations on the amplified β-globin gene region is now possible with specific labeled oligonucleotide probes. Moreover, the sensitivity of next-generation sequencing (NGS) has allowed noninvasive screening to be done on fetal DNA obtained from maternal plasma. While highly promising, such techniques should be used with caution and need to be validated for each laboratory, given the consequences of the results obtained. [23]
Other Tests
The following tests may be indicated:
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HLA typing is performed in patients for whom bone marrow transplantation is being considered
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Eye examinations, hearing tests, renal function tests, liver function tests, and frequent blood counts are required to monitor the effects of chelating agents routinely used (see Treatment, Medication)
Procedures
Bone marrow aspiration is rarely needed but may be used in certain patients at the time of the initial diagnosis to exclude other conditions that may manifest with signs and symptoms similar to those of thalassemia major.
Staging
A classification system introduced by Lucarelli is used for patients with severe thalassemia who are candidates for hematopoietic stem cell transplantation (HSCT). This predicts outcome based on:
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Degree of hepatomegaly
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Presence of portal fibrosis in liver biopsy sample
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Effectiveness of chelation therapy prior to transplantation
Class 1 patients lack any of these risk factors, and their event-free survival (EFS) rate after allogeneic HSCT is 90%. In contrast, those who have all 3 risk factors (class 3) have an EFS rate of only 56%. Class 2 patients have 1-2 risk factors, and their outcome lies between those for class 1 and class 3. [4]
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Alpha chain genes in duplication on chromosome 16 pairing with non-alpha chains to produce various normal hemoglobins.
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Alpha and beta globin genes (chromosomes 16 and 11, respectively).
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Various mutations in the beta gene that result in beta thalassemia.
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Supra vital stain in hemoglobin H disease that reveals Heinz bodies (golf ball appearance).
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Peripheral blood film in Cooley anemia.
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Peripheral blood film in thalassemia minor.
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Peripheral blood film in hemoglobin H disease in a newborn.
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Peripheral blood in iron deficiency anemia.
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The classic "hair on end" appearance on plain skull radiographs of a patient with Cooley anemia.
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Excessive iron in a bone marrow preparation.
