Hemoglobins - What the results mean PDF Print E-mail
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By :James R. Eckman, M.D., SERGG Workshop 1992

Normal Hemoglobins, Hemoglobin S, Hemoglobin C, Barts and Alpha Thalassemia, Beta Thalassemias, Hemoglobin F, Hemoglobin E, Hemoglobin D, Hemoglobin J, Hemoglobin G- Philadelphia, Hemoglobin Constant Spring, Hemoglobin N, Hemoglobin O-Arab

See a chart of hemoglobin electrophoresis patterns at http://family.georgetown.edu/welchjj/netscut/heme_onc/hemoglobin_electrophoresis.html

 

Normal Hemoglobins

Normal hemoglobins in the red cell consist of Hb A, Hb F, and Hb A2. The protein sequences are DNA coded on Chromosome 11 for the beta, delta and gamma chains. The alpha chains are coded on Chromosome 16. The beta variants such as Hb S, Hb C, and Hb D all occur from a mutation on Chromosome 11.

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SICKLE HEMOGLOBINS

Hemoglobin S (Hb S) has a substitution of valine for glutamic acid in the sixth position of the Beta globin chain. Hb S occurs in high frequency in populations previously exposed to falciparum malaria including those from Africa, India, the Mediterranean area, and Saudi Arabia. Sickle hemoglobin crystallizes with deoxygenation causing distortion of the erythrocyte and many clinical problems. Sickling of erythrocytes is facilitated by increased temperature (fever), decreased pH (acidosis), and high MCHC (dehydration).

The common sickle cell syndromes result when the gene for sickle hemoglobin is inherited from both parents (Sickle Cell Anemia), when a gene for sickle hemoglobin is inherited from one parent and a gene for hemoglobin C is inherited from the other (Hemoglobin SC Disease), or when a gene for sickle hemoglobin is inherited from one parent and a gene for beta thalassemia is inherited from the other (Hemoglobin S Beta Thalassemia). There are some differences between these syndromes, but all have similar clinical manifestations. Past statements stressing the benign nature of Hb SC disease and Hb S Beta thalassemia are generally incorrect.

Clinical manifestations of all syndromes may include moderate to severe hemolytic anemia, increased severity of certain infections, tissue infarction with organ damage and failure, and recurrent pain episodes. The hemolytic anemia is generally well tolerated but does lead to premature gall stones in many patients. The anemia may become life-threatening during aplastic crises or splenic sequestration crises. Early recognition and treatment with transfusions are important in both. Splenectomy is done for splenic sequestration in older children and with recurrence. Children and some adults have increased incidence of sepsis, meningitis, and other serious infections with Streptococcus pneumoniae, Hemophilus influenzae, Salmonella species, and Mycoplasma pneumoniae. Tissue infarction may cause significant morbidity and mortality. Strokes occur in children secondary to brain infarctions. Involvement of the bone may cause pain episodes, predispose to osteomyelitis, and lead to aseptic necrosis of the femur and humerus. Obstruction of retinal vessels may lead to vitreous hemorrhage or retinal detachment with resulting loss of vision. As patients age, cumulative damage to the lungs and kidneys make pulmonary and renal insufficiency common problems. Pain episodes cause life-long morbidity, however, usually are not associated with mortality.

Individuals who inherit a gene for sickle hemoglobin from one parent and one for hemoglobin A from the other are genetic carriers of sickle syndromes. This is often called sickle trait and is not associated with any hematological abnormalities. Carriers may have episodes of hematuria (blood in the urine) and may have more urinary tract infections. Rarely, pain episodes or splenic infarctions have been seen with extreme lack of oxygen. Sudden death may be slightly more common at the extremes of human endurance.

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HEMOGLOBIN C

Hemoglobin C (Hb C) has a substitution of lysine for glutamic acid in the sixth position of the Beta globin chain. Hb C occurs in higher frequency in individuals with heritage from Western Africa, Italy, Greece, Turkey, and the Middle East. Association of Hb C with the erythrocyte membrane causes red cell dehydration and resulting increase in MCHC. This leads to a shortened red cell survival in Hb C homozygotes and sickling complications in compound heterozygotes for Hb S and Hb C.

Individuals with Hb CC disease inherit a Hb C gene from each parent. They have a mild hemolytic anemia, microcytosis, and target cell formation. There may be very occasional episodes of joint and abdominal pain which are attributed to Hb CC disease. Splenomegaly is common. Aplastic crises and gall stones may occur.

Compound heterozygotes with Hb SC disease inherit a Hb C gene from one parent and a Hb S gene from the other. In general, they have a sickle syndrome which is very similar to sickle cell anemia. The hemolysis is usually less severe so the hemoglobin level is higher. Splenomegaly is much more common in older children and adults even though function is lost. There may be more problems with retinal disease and aseptic necrosis. Other manifestations are similar.

Hemoglobin C carriers inherit Hb C from one parent and Hb A from the other. They have no anemia but will usually have target cells on blood smear and may have a slightly low MCV. There are no other clinical problems.

Individuals who are compound heterozygotes for Hb C and Beta thalassemia inherit a Hb C gene from one parent and a Beta thalassemia gene from the other. If they inherit B+ thalassemia there is 65 - 70% Hb C, 20 - 30% Hb A, and increased Hb F. If they inherit Beta 0 thalassemia on electrophoresis there is no Hb A and increased Hb F with Hb C. Individuals with Hb C Beta + thalassemia have a mild anemia, low MCV, and target cells. Individuals with Hb C Beta 0 thalassemia have a moderately severe anemia, splenomegaly, and may have bone changes.

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BARTS HEMOGLOBIN AND ALPHA THALASSEMIA

The carrier states for alpha thalassemia are very common in the African, Mediterranean and Asian populations. Cord blood testing may detect individuals who have inherited alpha thalassemia because.small amounts of hemoglobin Barts will be present.

The alpha thalassemias result from the loss of alpha globin genes. There are normally four genes for alpha globin production so that the loss of one to four genes is: possible. The lack of all four genes causes hydrops fetalis and is usually fatal in utero. The loss of three genes causes hemoglobin H disease which is a moderately severe form of thalassemia. Alpha thalassemia minor results from loss of two genes and causes a mild anemia which resembles iron deficiency anemia. Finally, loss of one gene causes no clinically detectable problems but may cause small amounts of hemoglobin Parts to be present in cord blood samples. In general, only the loss of one or two genes, is seen in African Americans. Individuals from Southeast Asia and the Mediterranean area may have all four types of alpha thalassemia.

The percentage of hemoglobin Barts in the cord blood sample may indicate the number of alpha genes that have been lost. If the percentage of hemoglobin Barts is small (less than 5 %), the infant most likely has lost one gene and will be a normal carrier for alpha thalassemia minor. Hemoglobin Barts between 5 to 10% indicates the presence of alpha thalassemia minor with loss of two alpha genes. If the hemoglobin Barts is greater than 10% (usually 15-20%), a more severe form of alpha thalassemia may be present and further testing is indicated.

Individuals with alpha thalassemia minor (hemoglobin Barts 5 to 10%) will have a very mild anemia with microcytosis (small red blood cells) and no other clinical problems. This anemia is, however, frequently confused with iron deficiency anemia. Parents of infants with hemoglobin Barts should be told their child has alpha thalassemia minor and this disorder will have no effect on the child's health. They should be told it is inherited so others in the family may have a similar disorder. They should be instructed to tell health professionals that alpha thalassemia runs in their family to prevent unnecessary tests or treatment with iron. If alpha thalassemia minor is detected in Oriental or Mediterranean infants, family studies should be initiated to detect the presence of more serious forms of alpha thalassemia. Infants with greater than 10% Barts hemoglobin should be referred to tertiary care centers for further evaluation.

Long term treatment of infants with alpha thalassemia with supplemental iron will not correct the anemia and may be harmful. Therefore, iron deficiency in children with hemoglobin Barts should be documented by iron studies (serum iron and total iron binding capacity) or by serum ferritin determination. If iron deficiency is also present, then the child should be treated for six months and the iron supplements discontinued. W.I.C. approved formulas, which contain dietary amounts of iron, can be given to infants with alpha thalassemia without causing any problems.

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BETA THALASSEMIAS

Beta thalassemias are inherited disorders of Beta globin synthesis. In most, globin structure is normal but the rate of production is reduced because of decrease transcription of DNA, ·abnormal processing of pre-mRNA, or decreased translation of mRNA. Decreased hemoglobin -synthesis causes microcytosis and unbalance synthesis of alpha and beta globin leads to ineffective erythropoiesis and hemolysis. Beta thalassemia interacts with structurally abnormal hemoglobins to produce significant diseases..

Individuals who are homozygous for a beta thalassemia may have no production of Beta globin (Beta 0 thalassemia) or markedly reduced 13 globin production (Beta + thalassemia). Clinical manifestations vary from Beta thalassemia major which is fatal in early childhood without transfusion to beta thalassemia intermedia where anemia is severe but transfusions are not required. On electrophoresis, Hb F is the major hemoglobin, Hb A2 is increased, and Hb A is absent (Beta 0) or markedly reduced (Beta +)

Heterozygotes for a normal and beta thalassemia gene have beta thalassemia minor. They have mild anemia, microcytic erythrocytes, and splenomegaly. Hemoglobin electrophoresis shows Hb A with slight increase in Hb A2 with normal or slightly increased Hb F. The anemia is often confused with iron deficiency anemia.

Compound heterozygotes of beta thalassemia and Hb S have very significant clinical problems. In Hb S Beta 0 thalassemia, no Hb A is made so hemoglobin electrophoresis shows Hb S, increased Hb A2, and increased Hb F. In Hb S Beta+ thalassemia, Hb A is reduced so hemoglobin electrophoresis shows Hb A, 5 - 25%, Hb F, Hb S, and increased Hb A2.

The severity of the clinical manifestations show great variation between patients. Most individuals with Hb S Beta+ thalassemia have preservation of splenic function and less problems with infection, fewer pain episodes, and less end-organ damage. Individuals with Hb S Beta 0 thalassemia may have very severe disease identical to homozygous sickle cell anemia. Hemoglobin levels may be higher on average, splenic function is lost later in childhood, and splenomegaly is common into adulthood. Pain episodes, end-organ damage, and prognosis may be similar or perhaps even worse for bone and retinal disease when compared to homozygous sickle anemia.

When doing genetic counseling and prenatal diagnosis for structurally abnormal hemoglobins one must always consider that one partner may be a carrier of a beta thalassemia gene. These carriers are very easily missed because hemoglobin levels may be normal or near normal and hemoglobin electrophoresis will only show subtle increase in Hb A, and Hb F which is often overlooked. A complete blood count with mean corpuscular volume (MCV) and red cell count should always be obtained before counseling couples if one has "normal" electrophoresis. he MCV is almost always low in beta thalassemia and the red cell count is often elevated. Quantitation on Hb A, and Hb F is often diagnostic.

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HEMOGLOBIN F

Hemoglobin F (fetal hemoglobin) is the predominant hemoglobin before birth and a normal minor hemoglobin during adult life composing less than 2 percent of total hemoglobin. This percentage is based on a heterogeneous distribution of Hb F with most erythrocytes having no Hb F and a small percentage having high percentages. Hb F is elevated in a number of anemias, sickle cell anemia, and thalassemias with a heterogeneous distribution. High levels with homogeneous distribution are found .with Hereditary Persistence of Fetal Hemoglobin (HPFH) inherited alone or in combination with Hb S. HbS-HPFH compound heterozygotes have levels of 15 - 30% and generally mild clinical disease.

Fetal hemoglobin declines over the first six months of life to near adult levels. Decline may be slowly in individuals with sickle syndromes. Increases can be seen during pregnancy, severe.anemia, and with leukemia. Individuals with Beta thalassemias usually have persistent elevations for life. Those with sickle cell anemia have levels from 2% to 20% with some suggestion that higher levels are associated with reduction in some complications.

Hb F is formed from two alpha and two gamma chains. The gene is duplicated with the product of each differing in a single amino acid glycine or alanine in position 136. Gamma chain hemoglobin variants may be present at birth and rapidly disappear in the first months of life. Because these hemoglobins are not present long after birth, they are of no clinical importance to the individual or family. Differentiation of homozygous Hb SS, Hb S-HPFH, and Hb S-Beta thalassemia has implications in genetic counseling and may be important to the education and management of the affected individual.

Hb F can be quantitated by alkaline denaturation, high performance liquid chromatography, and radial immunodiffusion. Distribution can be determined by acid elution of a smear (Kleihauer - Betke test) or by immunofluorescent techniques using Hb F specific antibodies.

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HEMOGLOBIN E

Hemoglobin E is a structurally abnormal hemoglobin caused by substitution of lysine for glutamic acid at the 26 position of the B globin chain which has a B+ thalassemia phenotype. The substitution causes abnormal processing of pre-mRNA to functional mRNA, resulting in decreased synthesis of Hb E. The Hb E gene is very common in many areas of Southeast Asia, India, and China.

Heterozygotes for Hb E and Hb A have no anemia, a low MCV, and target cells on blood smear. Hemoglobin electrophoresis will show about 75 % Hb A and 25% Hb E.

Homozygotes for Hemoglobin E may have normal hemoglobin levels or slight anemia. The MCV is low and many target cells are present on blood smear. There is a single band in the Hb C / A, position on cellulose acetate electrophoresis and increased Hb F (10 - 15 %). There are no significant clinical problems.

Individuals who are compound heterozygotes for Hb E and B" thalassemia have a severe disease with severe anemia, microcytosis, splenomegaly, jaundice, and expansion of marrow space. Hemoglobin electrophoresis will show Hb E and significant increase in Hb F (30 - 60%). Treatment is similar to homozygous beta thalassemia.

Individuals who are compound heterozygotes for Hb E and B+ thalassemia have a moderate disease with anemia, microcytosis, splenomegaly, and jaundice. Hemoglobin electrophoresis will show Hb E (40%), Hb A (1 - 30%), and significant increase in Hb F (30 - 50%).

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HEMOGLOBINS "D"

There are a number of hemoglobins termed Hb D based on migration on hemoglobin electrophoresis. In general, they are of significance because they migrate in the same position as Hb S on cellulose acetate, alkaline electrophoresis. They move with Hb A on citrate agar, acid electrophoresis. The mobility on isoelectric focusing is variable.

Heterozygotes for Hb D and Hb A are normal. Homozygosity for Hb D is associated with normal hemoglobin levels, decreased osmotic fragility, and some target cells. Double heterozygotes for Hb D and B thalassemia have mild anemia and microcytosis.

There are D hemoglobins that interact with Hb S. Hb D-Los Angeles (also called D-Punjab has a substitution of glycine for the glutamic acid at B 121. Individuals who are compound heterozygotes for Hb S and Hb D-Los Angeles have moderately severe hemolytic anemia and occasional pain episodes.

Hb D-Ibadan has a beta Beta7 substitution of lysine for the normal threonine. Compound heterozygotes for Hb S and Hb D-Ibadan have less anemia and usually do not have other complications.

Hb D will migrate with Hb S on cellulose acetate electrophoresis, however, a solubility test will be negative. Confirmation of suspected Hb S requires electrophoresis with citrate agar, isoelectric screening, other techniques before counseling is offered because of this potential for false positive initial testing result.

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Hemoglobins "J"

There are 58 hemoglobins designated Hb J by electrophoretic mobility (fast band) on cellulose acetate eiectrophoresis. The vast majority of these are of no clinical significance. There are 6 that are unstable and Hb J-Cape Town has increased oxygen affinity. The potential for interaction with Hb S is not defined.

The electrophoretic pattern observed may vary significantly depending on whether the a or p chain is involved in the mutation and with the stability of the J variant. Hb J-Baltimore is the most common found in Northern European and some African Americans.

Most have no clinical significance and extensive testing and counseling is not generally indicated. Extended testing at a reference laboratory may be indicated if erythrocytosis (high hemoglobin) or anemia is present.

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HEMOGLOBIN G - PHILADELPHIA

Hemoglobin G Philadelphia (Hb G-Phil) is an a chain variant which is often associated with deletion a thalassemia of the cis (linked) a gene. The frequency is increased in African Americans, making this the most common a gene variant in this population. The electrophoretic mobility is the same as Hb S on cellulose acetate, causing occasional misdiagnosis of sickle trait.

This hemoglobin variant has no clinical consequences. Individuals should be reassured that there are no clinical problems.

Numerous electrophoretic bands can occur when Hb G-Philadelphia is present. When Hb AA is present, there is usually 25 to 40 % Hb G-Phil and a faint band of G, in the carbonic anhydrase location. Abnormal density of the Hb S band is present with Hb AS and Hb G-Phil. Four bands are seen with Hb AC and G-Phil. In newborns, hybrids with Hb F are also observed providing potential for multiple bands when other variants are also present.

Rare examples of hemoglobin H disease have been described in association with Hb G-Phil. In these two families, the G-Phil link to a deletion alpha thalassemia was inherited with a two gene deletion alpha thalassemia. Genetic counseling is probably not indicated unless the family is known to carry a two gene deletion, cis alpha thalassemia 1 phenotype.

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HEMOGLOBIN CONSTANT SPRING

Hemoglobin Constant Spring (Hb CSpr) is an a chain variant with an elongated alpha globin chain of 28 to 31 amino acids. This is caused by a mutation that alter the mRNA termination codon.

Electrophoretic: mobility is between carbonic anhydrase and Hb A, on cellulose acetate. The percentage is usually about 1% in heterozygote carriers, 5 to 7% in homozygotes, and 3 to 5% in hemoglobin H disease caused by Hb CSpr with trans two gene deletion a thalassemia.

Heterozygotes with Hb CSpr and two normal trans at genes are hematologically normal. Homozygotes for Hb CSpr have a mild hemolytic anemia and may have splenomegaly. Individuals with hemoglobin H disease from Hb CSpr and two gene deletion alpha thalassemia have a more severe disease with more Hb H and Bart's than three gene deletion alpha thalassemia.

Hb CSpr is common in Southeast Asia and found in high frequency in American immigrants from some of those areas. Hb CSpr also has been found in Creeks, Counseling is indicated in individuals from these geographic areas because of the high incidence of cis alpha thalassemia 1 which puts couples at risk far having infants with Hb H disease (see a thalassemia) .

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HEMOGLOBINS "N"

This group of 6 fast hemoglobins has electrophoretic mobility between Hb J and Hb H on cellulose acetate electrophoresis. Hb N-Baltimore is the most prevalent N hemoglobin in African Americans. There are no associated hematological abnormalities and counseling is not indicated.

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HEMOGLOBIN O-ARAB

Hemoglobin O-Arab has significance in sickle syndromes because it interacts with Hb S to produce clinical manifestations approaching the severity of Hb SS disease. The animo acid substitution is lysine for glutamic acid in the Beta 121 position.

Heterozygote carriers have no clinical manifestations. Compound heterozygotes for Hb S and HbO-Arab have hemoglobins in the 7 - 8 gm/dl range with reticulocytosis, jaundice, splenomegaly, episodes of pain, and many other complications seen in Hb SS disease. Compound heterozygotes for Hb O-Arab and Beta thalassemia have manifestations similar to thalassemia intermedia.

Electrophoretic mobility is in the Hb A, / C position on cellulose acetate and between Hb S and Hb A on citrate agar, pH 6,2. Migration on isoelectric focusing is with Hb E and Hb C Harlem.

These hemoglobins occur in individuals from North Africa, Arabia, Bulgaria, and the eastern Mediterranean area. Counseling of carriers is indicated because of the potential for interaction with Hb S and Beta thalassemia producing significant disease.

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Last Updated on Tuesday, 22 June 2010 09:37
 
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