1. Hematology 425 Hemoglobinopathies
Russ Morrison
November 13, 2006
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2. Hemoglobin – NL review
A review of normal adult Hgb production reminds us that
Heme is a protoporphyrin ring with Fe2+ (ferrous iron)
Globin is a polypeptide chain
Hgb molecule has 2 alpha and 2 non-alpha globin molecules
Globin chains bind heme after release from the ribosomes and then pair off
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3. Hemoglobin – NL review
An alpha chain and a non-alpha chain pair to form a dimer
Two dimers combine to form tetramers completing formation of the Hgb molecule
NL Adult Hgbs:
HgbA (2 alpha and 2 beta chains)
HgbA2 (2 alpha and 2 delta chains)
HgbF (2 alpha and 2 gamma chains)
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4. Hemoglobinopathies
Hemoglobinopathies are inherited disorders where genetic mutations alter either the structure or the rate of synthesis of a particular globin chain
Divided into 2 categories
Hemoglobinopathies, amino acid sequence is altered
Thalassemias, reduce or completely eliminate the production of one or more globin chains (Chapter 25)
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5. Hemoglobinopathies There are approximately 700 Hgb variants
Molecular abnormalities may cause mutant hemoglobins in any adult hemoglobin chain (alpha, beta, gamma, or delta)
Molecular abnormalities are characterized as follows:
Substitution of one amino acid for another (valine for glutamic acid in HgbS
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6. Hemoglobinopathies
Substitution of more than one amino acid (glutamic acid for valine and aspartate for asparagine in HgbC-Harlem)
Deletion of one or more amino acids (glutamic acid deletion in Hgb Leiden)
Fusions of Hgb chains (δβ in Hgb Lepore)
Extension of an amino acid chain (Hgb Constant Spring with 31 additional amino acid residues)
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7. Hemoglobinopathies The impact of the amino acid changes is variable
Many of the changes are clinically insignificant because there is no detectable physiologic effect
Most are associated with beta-chain abnormalities and more patients with beta-chain abnormalities demonstrate clinical disease
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8. Hemoglobinopathies
Table 24-2 in the text lists clinically significant abnormal hemoglobins
The most frequently occurring, as well as the most severe, of the abnormal Hgbs is hemoglobin S
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9. Hemoglobinopathies
Abnormal Hgbs may demonstrate heterozygous or homozygous inheritance patterns
In homozygous beta hemoglobinopathies, the variant Hgb is the major component and normal Hgb (HgbA) is absent. Examples include sickle cell disease (HgbSS) and Hgb C disease (HgbCC)
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10. Hemoglobinopathies
In heterozygous beta-hemoglobinopathies, the variant hemoglobin is usually present in lesser amounts than normal HgbA
Some cases demonstrate equal amounts of HgbA and the abnormal Hgb, examples are HgbC trait (HgbAC) and Hgb S trait (HgbAS)
In both cases, Hgbs F and A2 are usually normal structurally
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11. Hemoglobinopathies
In heterozygous alpha- hemoglobinopathies, an abnormalityin the alpha chain will affect all three normal adult hemoglogin types (A, F, A2) and 6 different hemoglobin types are found, three of which are normal and three abnormal
Examples of heterozygous alpha types include HgbD-Baltimore and HgbM-Boston
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12. Hemoglobinopathies
Hemoglobinopathies are genetic disorders in which a structural abnormality results from the alteration of DNA genetic code for one or more globin chain producing a qualitative difference
Thalassemias are similar except that the genetic alteration inhibits or completely eliminates production of a globin chain, hence a quantitative difference
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13. Hemoglobinopathies
Results of Hgb changes depends on the amino acids involved and their position in the molecule
Hemoglobinopathies are divided into 4 groups:
Abnormal hemoglobins that result in hemolytic anemia, such as HgbS and the unstable Hgbs
Methemoglobinemia, such as HgbM (ferric iron, increased affinity for oxygen)
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14. Hemoglobinopathies
Hemoglobins with either increased or decreased oxygen affinity
Abnormal Hgbs with no clinical or functional effect
Functional classification of Hgb variants is summarized in Table 24-3 in the text
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15. Hemoglobinopathies
As hemoglobins were discovered and reported, they were designated by letters of the alphabet (HbA, HbF)
Soon, it was discovered that there would not be enough letters and a geographic notation was added (HbGPhiladelphia)
Designation may also include the variant chain, sequential number and nature of the substitution (examples in Table 24-3)
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16. Hemoglobinopathies – Hgb S
Sickle Cell anemia is a relatively recent discovery having first been described in 1910 with a student suffering from severe anemia
Sickle cell diseases are the most comon hemoglobinopathies and include a group of hereditary disorders characterized by the presence of only HbSS or HbS in combination with another Hgb beta-chain mutation
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17. Hemoglobin S
The most common variants of the disease are HbSC and HbS-β-thalassemia
Genes that carry information for globin chain synthesis are located at specific loci on C16 and C11
Alpha genes are located on C16, while Beta chain coding is located on the short arm of C11
Each chain has 2 loci resulting in Hgb variants that are inherited as autosomal codominants with one gen coming from each parent
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18. Hemoglobin S
Patients with Sickle Cell disease (SS, SC or S/β-thal) have inherited a sickle (S) gene from one parent and either an S, C or β-thalassemia gene from the other.
Individuals who are homozygous have more severe disease than those who are hetorzygous for HgbS
Inheritance is typical as deomonstrated by Pnnnett square predictions
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19. Hemoglobin S
Hgb S is defined by a β chain substitution of glutamic acid by valine at the 6th position
The carrier rate of Hgb S is between 20 and 40% in Africa
Approximately 12% of African Americans carry the gene
The homozygous state (HbSS) is the most severe with an incidence of 1 in 375 African-American births
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20. Hemoglobin S
Sickle cell trait occurs at the rate of 8% in the African-American population
HbSC occurs at a rate of 0.12% and HbS/ β-thal occurs in 0.16% of African Americans
Sickle cell anemia may also be found in persons from the Middle East, India and the Mediterranean (distribution fig 24-3)
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21. Hemoglobin S
Hgb S can also be found in persons from the Caribbean and from Central and South America
HbS is soluble in the RBC and as long as the cell is oxygenated it remains a biconcave disk
Upon deoxygenation, the Hgb becomes less soluble, liquid crystals of HbS form and cellular sickling occurs
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22. Hemoglobin S
In homozygotes, the sickling begins when Oxygen saturation drops below 85%
In heterozygotes, oxygen saturation must be below 40% for sickling to occur
Upon sickling, the blood becomes more viscous, pH is reduced and there is an increase in 2,3-BPG
Sickling results in occlusion of capillaries and arterioles and infarction of surrounding tissues
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23. Hemoglobin S Sickle cells come in two types
Reversible, cells that undergo hgb polymerization, increased viscosity and change shape upon deoxygenation; the cells are normal in shape, viscosity and presence of polymers when oxygenated
Vaso-occlusive complications are blamed on these cells who travel into the microvasculature when oxygenated and then become distorted while becoming deoxygenated in the periphery
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24. Hemoglobin S The second type of sickle cell is termed irreversible.
Irreversible cells do not change their shape regardless of the change in environment and hemoglobin polymerization
Irreversible cells are seen on the PB smear as elongated sickle cells with a point at each end
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25. Hemoglobin S- clinical features
Symptoms can vary from asymptomatic to potentially lethal
Symptoms characteristically develop during the second half of the first year of life as patients are protected by HgbF
As beta chains of HbS replace gamma chains of HbF, the progressive problems related to increasing HbS develop
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26. Hemoglobin S- clinical features
Only 4 variants of HgbS inheritance are known to be clinically significant (HbSS, HbSC, HbS-β0thal and HbS-β+thal)
These 4 forms carry high morbidity and mortality
Average life expectancy has improved and is now 45 years for the HbSS patient and 65 years for the HbSC patient
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27. Hemoglobin S- clinical features
Many victims of sickle cell disease undergo episodes of recurring pain termed sickle crises, the pathogenesis of which is not understood
The hallmark of sickle cell disease is vaso-occlusion, with acute, severe pain resulting in visits to the emergency room
Triggers of crises include acidosis, hypoxia, dehydration, infection and fever, exposure to cold
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28. Hemoglobin S- clinical features
Pain most often occurs in the bones, lungs, liver, spleen, penis, eyes and central nervous system
Frequency of painful symptoms varies from none to as many as 6 per year and last for 4-5 days
Repeated infarcts of the spleen result in the spleen’s diminishment due to scarring
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29. Hemoglobin S- clinical features
Chronic hemolytic anemia is characterized by
Shortened RBC survival
Decreased hgb and hct
Elevated reticulocyte count
Jaundice
Clinical features of sickle cell disease are summarized in table 24-4 of the text
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30. Hemoglobin S- Incidence With Malaria and G6PD Deficiency
The frequency of the sickle gene occurs in parallel to the incidence of Plasmodium falciparum and appears to offer protection from the parasite
One explanation is that the infected cell is quickly sickled and destroyed, reducing the number of organisms and increasing available time for immunity to develop
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31. Hemoglobin S- Incidence With Malaria and G6PD Deficiency
It has also been suggested that G6PD deficiency plays some type of protective role with malarial parasites
This correlation has not been confirmed by scientific study
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32. Hemoglobin S – Lab Diagnosis
Anemia of SCD is that of a chronic hemolytic anemia with normocytic, normochromic RBCs
PB smear shows marked poikilocytosis and anisocytosis with normal, sickled, target and nucleated RBCs, a few spherocytes, basophilic stippling, Pappenheimer bodies and HJ bodies
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33. Sickle Cell - EM
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34. Sickle Cell - EM
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35. Hemoglobin S – Lab Diagnosis
The diagnosis is made by demonstration of the insolubility of S Hgb in the deoxygenated form with a sickle cell screen
If the screening test is positive, the presence of hemoglobin S is confirmed using electrophoresis
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36. Hemoglobin S – Treatment
Supportive care (transfusion, analgesics) has been the hallmark of treatment
Current research aimed toward modifying the genetic pathogenesis of sickle cell disease looks promising
Neonatal screening, childhood prophylactic penicillin, bone marrow transplantation and other treatments may extend the life of the sickle disease patient
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37. Hemoglobin S – Treatment
Transfusion should only be used for prevention of the complications of sickle cell disease (increasing the RBC mass)
Red cell exchange processes with HbAA blood may be used when compications arise during surgery
Bone marrow transplantation is generally performed in children with severe complications
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38. Hemoglobin S – Prognosis
Median life expectancy for men and women with homozygous SCD is 42 and 48 years, respectively
Heterozygote SC disease imparts a life expectancy of 60 and 68 years for men and women, respectively
Individuals with HbSS are discouraged from jobs that require strenuous physical exertion, exposure to high altitudes or extreme environmental temperature changes
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39. Sickle Cell Trait
Heterozygotes (HbAS) are described as having sickle cell trait
Individuals are generally asymptomatic, having no significant clinical or hematologic manifestations
However, under extreme hypoxic conditions, sickling and the associated complications may occur
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40. Sickle Cell Trait
Conditions such as severe respiratory infections, unpressurized flights at high altitude and anesthesia may trigger these complications
PB smear is normal (maybe a few target cells)
Sickle cell screen (solubility test) will be positive
Electrophoretograms demonstrate approximately 40% HgbS
No treatment is required for this condition and life expectancy is not affected
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41. Hemoglobin C & E
Hemoglobins C and E are the second most common hemoglobinopathies
They cause mild hemolysis in the homozygous state
In heterozygous inheritance patterns, they are asymptomatic
HgbCC patients will often demonstrate tetragonal crystals that may be seen inside and outside the RBC membrane on a PB smear
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42. Hemoglobin C & E HbEE results in a mild microcytic anemia
No treatment is usually required for patients homozygous for HbC or HbE as the patient’s health is usually not affected
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43. Hemoglobin C Disease
                   
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44. HbS in Combination With Other Hgb Variants
In the double heterozygous state, where a different abnormal chain is inherited from each parent, varying severity of HA may occur
HbSC is a double heterozygous syndrome where both beta-globin chains are affected by amino acid subsititutions
Incidence of HbSC is 1 in 833 births in the US
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45. HbSC HbSC disease resembles mild SCD
Childhood growth and development are delayed compared to normal children
Significant symptoms usually do not occur until the teenage years
May exhibit all of the vaso-occlusive symptoms of sickle cell disease
Episodes are less frequent and damage is less disabling
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46. HbSC
Life expectancy for men in the US is 60 years and for women, 68 years
Sickle screen is positive (solubility test)
Confirmation is by electrophoresis
Treatment is similar to those patients with sickle cell disease
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47. HbS/β-Thalassemia
This is the most common form of double heterozygosity in patients of Mediterranean descent and is second most frequent after HbSC
Syndrome is similar to mild SCD
If there is no production of beta globin (S-β0-thal), clinical course is similar to HbSS anemia
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48. HbS/β-Thalassemia
If there is production of beta globin (S-β+-thal) patients tend to have a milder disease than those with HbSC
These patients have more HbS than HbA, microcytosis, hemolytic anemia, abnormal peripheral blood morphology and splenomegaly
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49. Unstable Hemoglobin Variants
Approximately 200 variants of unstable hemoglobin have been described
Most are beta-chain variants, while some are alpha-chain variants
Few gamma-and delta-chain variants exist
Most of the unstable Hgb variants have no clinical significance, though the majority have increased oxygen affinity
About 25% are responsible for a variable hemolytic anemia
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50. Unstable Hemoglobin Variants
This syndrome, unstable hemoglobin disease, occurs at or just after birth
Inherited as an autosomal dominant
All patients are heterozygous as the homozygous condition results in death
Instability of the Hgb molecule has several causes:
Substitution in the interior of the molecule of the polar for a nonpolar pocket
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51. Unstable Hemoglobin Variants
Causes of Hgb Instability (continued)
Substitution around the heme pocket
Substitution of the alpha and beta chains at the contact point
Replacement with proline
Deletion or elongation of the primary structure
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52. Unstable Hemoglobin Variants
Clinical features include presentation in early childhood in patients with HA concurrent with jaundice and splenomegaly
Fever or ingestion of an oxidant exacerbates the HA
Severity of the anemia depends on the Hgb variant present
Examples include HbZurich, HbKoln, etc.
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53. Unstable Hemoglobin Variants
Laboratory diagnosis relies on observation of precipitated Hgb in the RBC as Heinz bodies
The precipitated Hgb attaches to the cell membrane, decreasing flexibility and shortening RBC survival
Oxygen affinity of these RBCs is abnormal
Patients are treated to prevent hemolytic crises, sometimes the spleen is removed
Oxidant drugs are not advised
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54. Hgbs with Abnormal Oxygen Affinity
More than 60 Hgb variants have been described which have abnormal oxygen affinity
Majority are high affinity variants and have been associated with familial erythrocytosis
Others are low affinity variants and may be associated with mild to moderate anemia
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55. Hgbs with Increased Oxygen Affinity
Inherited through an autosomal dominant pattern
Hgb with high oxygen affinity produce increased erythropoietin and a compensatory erythrocytosis
Most individuals are asymptomatic and have no symptoms (ruddy complexion)
Detected during routine physicial exam when CBC demonstrates high RBC count, high Hgb and high Hct
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56. Hgbs with Increased Oxygen Affinity
Patients with high-oxygen-affinity hemoglobins live normal lives and require no treatment
Diagnosis should separate these patients from those with increased RBC counts caused by myeloproliferative disorders or secondary erythrocytosis
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57. Hgbs with Decreased Oxygen Affinity
These Hgbs quickly release oxygen to the tissues, resulting in normal to decreased Hgb concentration and slight anemia
Best known example is HbKansas
Patients may be discovered when cyanosis and normal arterial oxygen tension exist at the same time
Most may be detected by Hgb electrophoresis
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