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Beta thalassemia major refers to individuals who are homozygous or compound heterozygous for beta thalassemia mutations and are transfusion dependent as a result of both ineffective erythropoiesis and hemolytic anemia. Without aggressive chelation therapy, transfusion-associated hemosiderosis eventually leads to endocrine, hepatic and cardiac dysfunction.  Hb E beta thalassemia presents in infancy as a variably severe anemia with a clinical phenotype ranging from a complete lack of symptoms to transfusion dependence. Osteoporosis, iron overload, growth failure, and pulmonary hypertension are commonly reported complications in both transfused and nontransfused patients.  More than 200 beta thalassemia mutations have been described.  In many cases, the genotype may not explain the observed clinical phenotype, reflecting the complex genetic interactions between the globin genes, expression controlling regions, and a growing list of other potential genetic modifiers.  Alpha thalassemias represent a group of conditions resulting from a reduced rate of alpha globin synthesis. Hb H disease results from deletion or dysfunction of three of the four alpha globin genes.  The most common genotype leading to Hb H disease is a single alpha gene deletion coinherited with a two alpha gene deletion, e.g. - -SEA/- a4.2 in the Southeast Asian population.  Compound heterozygosity for a two alpha gene deletion and a non-deletional alpha thalassemia mutation, e.g. Hb H Constant Spring may also cause Hb H disease. Individuals with Hb H disease present with cholelithiasis, exacerbations of severe anemia, or splenomegaly arise. Intermittent transfusions are required in up to 50% of patients with Hb H disease and as early as infancy in the majority of those with the more severe HbH Constant Spring phenotype. 

Accurate diagnosis based on identification of different structural hemoglobin variants or thalassemia mutations is therefore essential for optimal management of clinically significant hemoglobin disorders.  In California, newborns are screened for sickle cell disease, HbH disease and beta-thalassemia.  The CHRCO Hemoglobinopathy Laboratory serves as the central reference laboratory for the California State Newborn Screening Program to confirm all positive primary screening results.

Most of the common Hb variants, such as Hb S, Hb C, and Hb E can be identified using a combination of chromatographic and electrophoretic methods.   
Automated cation exchange HPLC (CE-HPLC) is a rapid and sensitive technique for the presumptive identification of many abnormal hemoglobin variants and for quantitation of HbA2 and Hb F. It is the method of choice used by the California state newborn screening program to identify hemoglobin disorders.  Electrophoretic methods, including IEF and more recently, capillary IEF (cIEF), are employed by the CHRCO Hemoglobinopathy Reference Laboratory as part of a panel of complementary techniques to resolve or confirm Hb variants.
Definitive diagnosis of a hemoglobin disorder, particularly thalassemia, may require DNA analysis. The strategy used to identify uncommon Hb variants or thalassemia mutations is to first test a panel of mutations common to the population being screened, with further testing for rare mutations based on clinical suspicion, family history and ethnic background.

Alpha globin genotyping using gap-PCR is used to diagnose the most common deletional forms of alpha thalassemia and distinguish HbH disease from alpha thalassemia trait.  Direct DNA sequencing and other molecular methods such as MLPAare required to diagnose hemoglobinopathies that are suspected by laboratory or clinical findings.   

In addition to the basic tests used for detection of hemoglobin disorders, the CHRCO Hemoglobinopathy Reference Laboratory performs more specialized tests, including tests for unstable hemoglobins(heat denaturation), methemoglobin (spectrometry), oxygen carrying capacity (oxygen dissociation curve), and red cell membrane abnormalities.