Thalassemia Information and Courses from MediaLab, Inc.

Heterozygous states of alpha thalassemia express themselves as silent carrier (one loci deleted) thalassemia minor (two loci deleted) hemoglobin H disease (three loci deleted) The homozygous state (all four loci deleted), alpha thalassemia major, is incompatible with life.

Alpha thalassemia demonstrates problems with alpha globin chain production. One to four loci that code for the alpha chain may be deleted from chromosome 16. The greater the number of loci deleted or inactivated, the greater the severity of the anemia which develops. Many different mutations exist that result from partial deletions of alpha genes. This unit of study deals only with the forms of alpha thalassemia that have entire loci deleted.

Alpha thalassemia intermedia (Hemoglobin H Disease) results from a deletion of three out of four alpha chain loci. Infants born with alpha thalassemia intermedia appear normal at birth but often develop anemia and splenomegaly by the end of their first year. Hepatomegaly is not a common finding and there may be some association with mental retardation. Due to the hemolytic nature of this anemia, there may be an increase in respiratory infections, leg ulcers and gallstones. Skeletal changes are not commonly seen in hemoglobin H disease. Every ethnic group can have occurrences of hemoglobin H disease; but it is most often seen in Southeast Asian, the Middle East and the Mediterranean islands. Development and life expectancy are usually normal, but some affected individuals may require splenectomy and transfusion therapy.

The Silent Carrier form of alpha thalassemia results from one alpha chain loci deletion. Individuals who are silent carriers show no clinical disease and demonstrate normal results during routine laboratory testing. This form of alpha thalassemia is usually discovered upon family studies.

Chromosome 16 contains the genetic codes for the zeta and alpha hemoglobin chains.Each chromosome has two loci alpha chains 1 and 2. This equals a total of four loci of material coding for the alpha hemoglobin chain. See the image for a visual representation of these loci.In the genotypic notation of alpha thalassemia an "" represents the presence of an alpha locus. A "-" represents a deletion of a locus.The notation for the normal number of alpha loci is /. The amount of Hb A produced by this normal gene is 97-98 %.(drawing modified from Harmening, 1999)

In alpha thalassemia minor, two loci are deleted or inactive. Either homozygous or heterozygous states are possible.

The complete deletion of alpha chain loci (--/--) in alpha thalassemia major is incompatible with life. None of the vital alpha chains needed for every normal adult hemoglobin can be produced. (drawing modified from Harmening, 1999)

In the heterozygous state (--/), one parent contributes a normal gene while the other one a gene with both alpha chain loci deleted.(drawing modified from Harmening, 1999)

The normal RBC count (4.84 x 1012/L) in this case, together with the decreased hemoglobin (8.4 g/dL) and MCV (59 fl) is an indicator of ineffective erythropoeisis that often points to thalassemia.The RBC morphology shows slight hypochromic microcytosis with codocytes, schizocytes, and basophilic stippling. Schizocytes form by several mechanisms, one being the removal of RBC inclusions.This patient's elevated bilirubin correlates with her presentation of sclera icterus; her splenomegaly is consistent with increased RBC destruction.The Hb electrophoresis demonstrated a normal pattern, initially, but the unstable Hemoglobin H was revealed upon repeat electrophoresis with reduced incubation time. Hemoglobin H is the result of beta globin chain tetramer formation due to the insufficient supply of alpha globin chains in alpha thalassemia intermedia.People with Hemoglobin H disease (alpha thalassemia intermedia) usually have a normal life expectancy without treatment. However, hemolysis may lead to moderate anemia that may be treated with splenectomy.

Anemia is moderate.RBC count is increased.Hb is moderately decreased.MCV is decreased. MCHC is decreased.RDW is increased.RBC morphology shows slight hypochromic microcytosis with codocytes, schizocytes, and basophilic stippling.Reticulocytes are moderately increased.Hb electrophoresis demonstrates abnormal patterns in both adults and neonates.Adults:HbA decreasedHbA2 decreasedHbF normal to decreasedHb H -2-40% (beta chain tetramers)Neonates: 10-40% Bart's (gamma chain tetramers)Hb H inclusions are frequently seen.Bone marrow demonstrates erythroid hyperplasia.

Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of bilirubin in the serum of adults is 0.2-1mg/dl. Bilirubin levels increase with liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dl.Persons with alpha thalassemia intermedia usually have an increased bilirubin level, because of ongoing hemolysis. This bilirubin is typically the unconjugated fraction of bilirubin.

Haptoglobin is the plasma protein responsible for binding free hemoglobin during episodes of hemolysis and would normally demonstrate decreased levels during a hemolytic crisis.The normal level of haptoglobin is 40-330mg/dl. Individuals who are in hemolytic crisis demonstrate greatly reduced levels to an absence of haptoglobin.In alpha thalassemia, however, haptoglobin levels remain normal or only slightly decreased, even during hemolytic events.The reason for this is that haptoglobin functions by binding the alpha chain portion of hemoglobin. With the absence of these chains in alpha thalassemia major and intermedia, haptoglobin cannot bind free hemoglobin. Therefore it is not consumed.

Beta thalassemia demonstrates problems with beta globin chain production. One or two loci that code for the beta chain may be deleted from chromosome 11. The greater the number of loci deleted or inactivated, the greater the severity of the anemia which develops. Many different mutations exist that result from partial deletions of beta genes. This unit of study deals only with the forms of beta thalassemia that have entire loci deleted. Deletions of additional globin genes coded for on chromosome 11 can result in such combinations as delta-beta thalassemia.

In thalassemia, there is often an excess production or accumulation of globin chains whose genes are not affected by the deletion.In beta thalassemia, this may be seen as an increase in gamma chain and delta chain production, leading to increased levels of hemoglobin F and A2 respectively.Excess alpha chains may also form tetramers which often lead to red cell membrane damage and decreased red cell deformability. This leads to a hemolytic anemia. Adding to the anemia is a decrease in the total amount of hemoglobin produced in spite of the erythroid hyperplasia of the bone marrow.

Children with beta thalassemia major, also called Cooley's anemia, usually develop clinical signs during their first year of life. They appear to be malnourished and may exhibit abdominal girth expansion. They show skeletal deformations, which are a result of increased erythropoiesis. A common finding is facial bone changes. Other clinical signs include frequent infections, hepatomegaly, splenomegaly, cardiomegaly, gall stones, leg ulcers, and poor growth and sexual development. Death usually occurs by the time these patients are in their early twenties unless treated with blood transfusions along with iron-chelating agents. If no chelating agent is used during treatment life will only be prolonged by about a decade.Beta thalassemia is found most often in populations of people from the Mediterranean, southern China, and India.

Persons with beta thalassemia minor rarely have physical signs or symptoms caused by this disorder and usually do not require any treatment. Beta thalassemia minor is most common in Thailand and among the American Black population.

Delta-beta thalassemia exists in both heterozygous and homozygous forms. The symptoms are mild to moderate depending on the severity of the disease.This form of beta thalassemia can be found in many ethnic groups, but is most common in persons from Greece and Italy.

Beta thalassemia intermedia (homozygous or combined heterozygous for mild gene deletions) displays a level of beta chain production midway between beta thalassemias minor and major.Beta thalassemia intermedia exists in similar states as that of beta thalassemia minor.The following pages illustrate each of these possible states.

The silent carrier state of beta thalassemia, B++s/B, involves one minor beta chain deletion or mutation. This state produces such a small drop in the level of beta chain synthesis that the alpha to beta chain ratio remains at a near normal state.Hemoglobin A levels remain normal (98% or higher).(drawing modified from Harmening, 1999)

In Beta thalassemia minor B+/B, one beta gene locus is partially deleted or inactive. With this deletion, only 85% to 95% of the normal level of Hb A is made.(drawing modified from Harmening, 1999)

Occasionally, the beta chain gene deletion extends to include the locus for the delta chain gene. If this deletion occurs on only one chromosome of the pair, it creates delta-beta thalassemia minor. Delta-Beta 0/ BetaHb A and A2 will both be decreased and Hb F will be increased.(drawing modified from Harmening, 1999)

In Beta thalassemia intermedia B0/B+s, there is one completely deleted or inactive beta chain gene, while the other is partially deleted or inactive.This state also results in Hb A production of 55%-75% of normal.(drawing modified from Harmening, 1999)

Beta Thalassemia Silent Carrier Beta Thalassemia Minor Beta Thalassemia Intermedia Beta Thalassemia Major Delta-Beta Thalassemia Anemia Absent Mild to absent Moderate Severe Mild Red blood cell (RBC) count Normal Increased Decreased to normal Decreased Decreased to normal Hemoglobin(Hb) Normal Decreased to normal (10 - 12 g/dL) Decreased (7 - 10 g/dL) Marked decrease (<7 g/dL) Decreased to normal (8 - 13 g/dL) Mean corpuscular volume (MCV) Slight to no decrease Marked decrease Marked decrease Marked decrease May be slightly decreased Mean corpuscular hemoglobin concentration (MCHC) Slight to no decrease Marked decrease Marked decrease Marked decrease May be slightly decreased Red blood cell distribution width (RDW) Normal Normal to slightly increased Increased Increased Normal RBC morphology Normal Marked hypochromia and microcytosis Codocytes (target cells) Possible basophilic stippling Nucleated RBCs are usually not present Marked hypochromia and microcytosis Codocytes (target cells) Possible basophilic stippling Nucleated RBCs are usually not present Marked hypochromia and microcytosis Codocytes (target cells) schistocytes ovalocytes basophilic stippling polychromasia nucleated RBCs Possible hypochromia and microcytosis Codocytes (target cells) Basophilic stippling Reticulocyte count Normal May be slightly increased Slightly increased (<5%) Mildly increased (5 - 10%) Mildly increased Hb electrophoresis Normal pattern Decreased amount of Hb A Variable amounts of Hb A2 and Hb F Decreased amount of Hb A Variable amount of Hb A2 Hb F is usually increased Severly decreased amount of Hb A Variable amount of Hb A2 Usually an increased amount of Hb F Decreased amount of Hb A and Hb A2 Increased amount of Hb F (15 - 20%) If red blood cells are normochromic and normocytic, the RBC, Hb, and Hematocrit (HCT) test values follow in three-fold progression (i.e., RBC x 3 = Hb and Hb x 3 = HCT). This is sometimes referred to as "the rule of threes." This rule will usually not apply in cases of beta thalassemia, particularly beta thalassemia minor where the RBCs are not normochromic and are microcytic, and where there is a disproportionate number of RBCs for the amount of hemoglobin that is present.

Persons with beta thalassemia may have a slightly increased level of serum iron with a slightly decreased iron binding capacity. The percent of iron saturation is normal to slightly increased.An iron stain of bone marrow usually demonstrates increased levels of hemosiderin. Sideroblasts may be present.

Hemoglobin electrophoresis is the movement of hemoglobin proteins in an electric field at a fixed pH.Because the various hemoglobins are comprised of different combinations of globin chains (normal or abnormal), they will demonstrate different degrees of mobility. Typically, when a thalassemia or hemoglobinopathy is suspected, an alkaline electrophoresis is performed which may be confirmed with acid electrophoresis.For an alkaline hemoglobin electrophoresis, a hemolysate is applied to cellulose acetate which is electrophoresed in a buffer at pH 8.4-8.6. At this pH hemoglobin proteins move from cathode to anode. The proteins are visualized by the application of a dye which also makes them measurable by densitometry.

The laboratory findings in this case represent classic findings seen in beta thalassemia minor including: erythrocytosis, decreased hemoglobin, normal hematocrit, normal RDW, and the presence of codocytes (target cells). This patient does have a mild anemia, but some patients with beta thalassemia minor have no anemia. Hemoglobin electrophoresis confirms this diagnosis, showing an increased Hb A2 level and decreased Hb A.In addition, the slightly increased iron and slightly decreased TIBC contradict a suspicion of iron deficiency. These chemistry results are typical for beta thalassemia, even though the red blood cells are microcytic and hypochromic.

Fine basophilic stippling is associated with increased red cell production and is commonly seen when there is increased polychromatophilia. Coarse basophilic stippling is seen in megaloblastic anemia and other forms of severe anemias, lead poisoning, and thalassemia. Coarse basophilic stippling indicates impaired hemoglobin synthesis, probably due to the instability of RNA in the young cell.

Pappenheimer bodies are iron-containing granules that aggregate with mitochondria and are deposited in RBC or normoblast cytoplasm. Small and irregular, they are found only in pathological states as thalassemia and sideroblastic anemias(upper image). Wright-Giemsa stain defines the cytoplasmic content (protein), but Prussian blue staining is necessary to define the iron content, the essence of the Pappenheimer body (lower image). Pappenheimer bodies lie typically in small clusters (upper image) and tend to locate at the periphery of the red cell cytoplasm. A cluster is typically smaller than a single Howell-Jolly body.

As previously mentioned, tear drop cells are present in disorders with altered splenic or bone marrow structure. Disrupted splenic cords and myelofibrosis with myeloid metaplasia are examples. Tear drop cells appear in the peripheral blood as a response to red cell alterations by thalassemia when red cell inclusions are expelled by a stripping process through splenic cords. A marrow disrupted by malignant cells may also set the stage for release of teardrop cells into the peripheral blood. Importantly, teardrop cells may arise as an artifact of improper smear preparation, identified by their uniformity in pointing in the same direction. In contrast, teardrops noted in the photograph are irregularly arranged and oriented in various directions. Teardrops always have pointed ends and disappear after splenectomy.

The patient whose blood smear is shown in the photograph was a 32-year-old female from Virginia who came to the high country of Colorado to ski. The day after arrival, she experienced shortness of breath, fatigue, and upper abdominal pain. She was seen in a medical center in the mountains where a working diagnosis of altitude sickness was made. A CBC revealed RBCs 5.1 x 1012/L, hemoglobin 12.8g/dL, MCV 60fL, hematocrit 40.9%, and normal total WBC, differential, and platelet count. The RDW was normal. Further questioning revealed a previous diagnosis of heterozygous beta-chain thalassemia. No other abnormal hemoglobins were found on hemoglobin electrophoresis, but HbA-2 was elevated to 5%, supporting the diagnosis of beta thalassemia. The patient's poikylocytosis and anisocytosis may be a clue to an underlying erythrocyte abnormality. Persons with iron deficiency anemia may experience various degrees of hypoxia upon arriving at high altitudes. Those with sickle cell disease and thalassemia minor (as in this case) may experience bone pain or other symptoms of "crisis" and/or alteration in the appearance of their erythrocytes upon sudden high altitude exposure. The classic teaching is that in differentiating iron deficiency anemia from thalassemia, increased RDW would favor iron deficiency; normal RDW favors thalassemia.

The family (cited in the previous case history) was from a region of Thailand where the physician knew HbE carriers are prevalent. Homozygous hemoglobin E is common in Southeast Asia and presents with very mild anemia and seldom requires transfusion. Over 30 million people in the world are HbE carriers, making this abnormal hemoglobin almost as common as HbS. Hemoglobin E is uncommon in North America and in Europe, but with changing immigration patterns, hemoglobinopathy E cannot be ignored. Peripheral blood smear findings of target cells, microspherocytes, red cell hypochromia, a few red blood cell fragments, and nucleated red blood cells require evidence from hemoglobin electrophoresis to establish a diagnosis. Clinically, a very important and severe syndrome is hemoglobin E/beta thalassemia in which there is hemolysis requiring repeated transfusions. The patient has a severe anemia, low MCV (50's), and high RBC. This is characteristic of Hgb E/beta thalassemia.

The peripheral blood smear of HbH disease presented before is reviewed in the upper photograph.As mentioned, these leptocytes are pale-staining with hemoglobin confined to a thin, flat, cell membrane.Illustrated in the lower photograph are target cells or codocytes (a term derived from a Greek word for hat)Membrane accumulations of phospholipids and cholesterol (particularly in obstructive jaundice) promote target cell formation.When these cells are spread out on a glass slide, a central bump of hemoglobin appears to produce the target, a manifestation of excess cellular membrane compared to the amount of hemoglobin inside.The early descriptions of thalassemias, then called hereditary leptocytosis (Mediterranean anemia, Cooley's anemia), include description of leptocyes, which may have represented HbH disease.

Laboratory data must be presented to clinicians in a user friendly way to promote effective decision making. Databases must be designed to provide clear information that leads quickly to the best patient care outcome. We continue learning how to collect and retrieve laboratory data from our machines, but we are not always in tune to how entry and retrieval of data is geared to and, more directly, influences patient care outcomes. Examples of blood cell abnormalities on a peripheral blood smear that may immediately direct the physician to a specific diagnosis are: (1) presence of target cells as found in thalassemia or hemoglobinopathies and target cells in liver disease, particularly with obstructive jaundice; (2) burr cells as a signal of chronic renal disease and uremia; and (3)atypical neutrophil inclusions relating to genetic disorders. Critical appraisal of such observations could add valuable clues for a diagnosis. Laboratory professionals must establish a set of principles for orderly observation of blood cell morphology, have a clear vision of the applications of their work, and understand the potential clinical implications of their reports and interpretations. Emphasis on values and relevance focuses on patient care outcomes and their dependency on prompt availability of results and contextual interpretations.

Conditions in which teardrop cells can be found include myelofibrosis/myeloid metaplasia, bone marrow metastases, thalassemias, and anemias causing Heinz body formation. Dacryocytes are not diagnostically indicative of any specific condition.

Another example of elliptocytes as seen in hereditary elliptocytosis. Other conditions which may have varying numbers of elliptocytes include thalassemias, iron deficiency, megaloblastic anemia and anemia associated with leukemia.