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At a Glance
Alpha-thalassemia should be suspected in a patient with microcytic anemia and erythrocytosis.
There are 4 α chain genes, α1 and α2 on each copy of chromosome 16. As outlined in the subsequent table, the severity of the anemia depends on the number of mutated or deleted alpha chain genes. Deletion of 1 gene on a chromosome is designated α+-thalassemia, whereas deletion of 2 adjacent genes is considered αo-thalassemia.
A heterozygote for α+-thalassemia is asymptomatic with a mild anemia and microcytosis. Homozygotes for α+-thalassemia may have more significant anemia with microcytosis. The αo-thalassemia trait may have an increased red blood cell (RBC) count, and hemoglobin electrophoresis performed on cord blood at birth would demonstrate 2-20% hemoglobin Barts (gamma4).
Hemoglobin H disease is usually due to compound heterozygosity for α+-thalassemia and αo-thalassemia. The significant reduction in α chain synthesis and resultant β chain excess results in the hypochromic, microcytic anemia. The hemoglobin H β tetramers are prone to oxidation and precipitation, resulting in ineffective erythropoiesis, as well as membrane damage that causes hemolysis. Erythrophagocytosis of the damaged erythrocytes as they circulate through the spleen results in the characteristic splenomegaly.
In hemoglobin H disease, guidelines for characteristic RBC indices are a markedly reduced mean cell volume (MCV) of 50-65, hemoglobin of 7-10 g/dL, mean cell hemoglobin (MCH) of 15-20 pg, and mean cell hemoglobin concentration (MCHC) between 25 and 30 g/dL. The peripheral blood smear will show marked anisopoikilocytosis, hypochromia, and microcytosis.
In hydrops fetalis, four of four α chain genes are mutated and deleted. Since gamma4 (hemoglobin Barts) has a high affinity for oxygen but will not let it go in the peripheral tissue, this condition is not usually compatible with extrauterine life.
What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?
Tests to rule out an iron deficiency anemia should be performed, as this is the most common cause of a microcytic anemia. Serum iron, total iron binding capacity (TIBC), percent saturation, and ferritin should be checked.(Table 1)
Examination of the automated RBC indices can be helpful as an initial screen to determine if the patient has iron deficiency anemia versus beta-thalassemia. Patients with an erythrocytosis (RBC count > 5.5 mil/μL) and microcytosis (MCV < 80 fL) should be suspected of having thalassemia. If the MCV/RBC ratio is less than 13, additional testing for thalassemia should be performed.
If the RBC indices suggest thalassemia, then high pressure liquid chromatography (HPLC) should be performed on lysed RBCs to measure hemoglobin A2 (α2delta2) concentration and hemoglobin F (α2gamma2) concentration. As these 2 forms of hemoglobin do not utilize beta chains, they are elevated in beta-thalassemia but not in alpha thalassemia. If hemoglobin A2 and hemoglobin F are not elevated, then the possibility of alpha-thalassemia should be addressed. The following tests address the presence of hemoglobin Barts (gamma4) and Hemoglobin H (beta4), unstable tetramers that form because of the lack of alpha globin chains:
Hemoglobin electrophoresis: Isoelectric focusing is the best electrophoretic method for detecting the often faint hemoglobin H and hemoglobin Barts bands. These are both fast moving hemoglobin bands that migrate in front of (anodal) of hemoglobin A, toward the positive terminal (anode). Hemoglobin electrophoresis is not very sensitive for detecting hemoglobin H, because hemoglobin H is synthesized in low quantities and is unstable during the preparation of the red blood cell lysate.
Supravital dye staining: Incubation of peripheral blood with brilliant cresyl blue or methyl violet for 1-2 hours at 37oC, followed by smear preparation and examination under the microscope demonstrates a pattern of precipitated denatured hemoglobin H (beta4) that forms inclusion bodies evenly spaced over the red blood cell. This characteristic “golf ball” pattern of precipitation is pathognomonic of Hemoglobin H disease.
Table 1
| Syndrome | α globin output % | Genotype | Clinical features | Hgb pattern-new born | Hgb pattern-after 1st year |
|---|---|---|---|---|---|
| Normal | 100 | αα/αα | Normal | Hb Barts 0-trace | None |
| Silent carrier | 75 | -α/αα | Normal | Hb Barts 1-2% | None |
| Thalassemia minor | 50 | -α/-α or–/αα | Mild microcytic hypochromic anemia | Hb Barts 2-20% | None |
| Hb H Disease | 25 | -α/– or –/ααcs | Chronic hemolytic anemia | Hb Barts 20-40% (HbCS present) | HbH 5-30% |
| HbBarts trace (HbCS 2-3%) | |||||
| Hydrops fetalis | 0 | –/– | Fetal or neonatal death with severe anemia | Hb Barts >80% | —– |
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?
Other unstable hemoglobin variants will precipitate on incubation with methyl violet or brilliant cresyl blue. However, only hemoglobin H results in the evenly distributed “golf ball” distribution of precipitated hemoglobin.
A concomitant megaloblastic anemia may raise the MCV over what is expected in α-thalassemia.
What Lab Results Are Absolutely Confirmatory?
Globin chain synthesis analysis can identify alpha-thalassemia carriers without clear-cut test results using hemoglobin electrophoresis or supravital dye staining. Red blood cells are incubated with radiolabeled amino acids, and then alpha and beta globin chains are separated by urea carboxymethylcellulose chromatography. Comparison of the radioactivity incorporated in each chain gives the relative rate of synthesis. An α:β ratio of less than 0.9 is diagnostic of an alpha-thalassemia trait.
Detection of deletions or mutations in the α chain genes is absolutely confirmatory of an alpha-thalassemia. Alpha-thalassemias often have larger deletions than are commonly seen in beta-thalassemias. Therefore, Southern blot analysis or gap-PCR (polymerase chain reaction) with a mix of primers determined by the common alpha chain mutations in the geographical region of origin of the patient may be used to detect the alpha chain defect.
In addition, point mutations may be detected by several PCR-based methods relying on allele-specific primers.
The most common assay used to detect known mutations is the amplification refractory mutation system (ARMS) assay, which relies on the principle that perfectly matched primers will amplify best. To detect a specific mutation, two parallel reactions are run, both with a common forward primer. One reaction has a reverse primer complementary to the mutant sequence, and 1 reaction has a reverse primer complementary to the wild type sequence. If the mutation is present, there will be a product in the reaction using the mutant primer.
A heterozygote will have products in each reaction. Variations of this assay have been created so that multiplex reactions can be run to detect the mutations common in the patient’s population of origin. Another version of this assay is reverse dot-blotting with allele-specific oligonucleotides (ASOs) in which wild type and mutant oligonucleotides are blotted onto a nylon membrane, and labeled PCR products amplified from the patient’s genomic DNA are hybridized to the membrane.
What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?
The patient may have an as yet undescribed mutation. Denaturing gradient gel electrophoresis (DGGE) and single-stranded conformation polymorphism (SSCP) assays can be used to detect the presence of a mutation that alters the migration of the DNA. Then sequencing of the implicated region would then be performed to determine the sequence of the mutant DNA.
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