Rodak: Hematology: Clinical Principles & Applications, 3rd Edition

Chapter 26: Hemoglobinopathies (Structural Defects in Hemoglobin)

Key TermsAplastic CrisisAuto-oxidationAutosplenectomyChain ExtensionChronic Hemolytic CrisisClinical FeaturesCompensatory ErythrocytosisCongenital Heinz Body AnemiaCongenital Nonspherocytic Hemolytic Anemia (CNSHA)CyanosisDeletionDouble HeterozygosityEtiologyFamilial ErythrocytosisG6PD Deficiency Gene FusionGenetic MutationsGlobin GenesHeinz BodiesHemoglobin CHemoglobin C-Harlem (C-Georgetown)Hemoglobin DHemoglobin EHemoglobin MHemoglobin SHemoglobin S/Beta-Thalassemia HemoglobinopathyHeterozygousHigh Oxygen-Affinity Hemoglobin VariantHomozygousHowell-Jolly BodiesIncidence InfarctInsertionMegaloblastic CrisisMethemoglobinNomenclatureOxygen Affinity Pappenheimer BodiesPathophysiologyPoint Mutation

Prognosis Ruddy ComplexionSequestration CrisisSickle Cell AnemiaSickle Cell Trait Splenic SequestrationStructural Hemoglobin VariantTachypneaTactoids (“Liquid Crystals”)Trait (Versus “Disease”)Unstable Hemoglobin VariantsVaso-Occlusive (Painful) CrisisZygosity

Objectives1. Compare and contrast structural hemoglobin (Hb) disorders with thalassemias.2. Briefly describe globin gene structure and the development of normal human

hemoglobins throughout prenatal and postnatal life.3. Characterize the categories of the molecular abnormalities found in the various

hemoglobinopathies.4. Differentiate between homozygous and heterozygous states.5. Define the pathologic basis of sickle cell disorder.6. Describe the inheritance pattern of Hb S.7. State the amino acid substitution found in Hb S.8. Describe the solubility of Hb S in the deoxygenated state.9. Discuss the clinical presentation of sickle cell disease including differentiation among

types of sickle cell crises.10. Locate the geographic region, define the frequency of occurrence, and describe the

impact of the incidence of Plasmodium falciparum malaria and glucose-6-phosphate dehydrogenase deficiency on hemoglobin variants.

11. Describe the peripheral blood cell profile, chemistries, and other laboratory procedures used in the diagnosis of hemoglobinopathies.

12. Define the treatment goal for Hb S disease and discuss various treatments and their purposes.

13. Identify the amino acid substitution and electrophoretic mobility of Hb C and Hb C-Harlem.

14. Describe the hemoglobin substitution in Hb E and the importance of genetic counseling.

15. Explain the importance of and methods for differentiating Hbs D and G from Hb S.16. Describe the laboratory findings in patients who are double heterozygotes for Hb S

and Hb D, Hb O-Arab, or Hb Körle Bu.17. Identify the causes of methemoglobinemia.18. Describe the inheritance patterns and causes of unstable hemoglobins.19. Discuss hemoglobins with increased and decreased oxygen affinities and explain how

they differ from unstable hemoglobins.

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20. Predict possible inheritance of hemoglobinopathies from the genotype of parents and the inheritance pattern for the gene.

21. Interpret patient hemoglobin electrophoresis patterns on cellulose acetate at pH 8.4 and citrate agar at pH 6.0 to 6.2 when given controls.

22. Correlate hemoglobin electrophoresis and solubility tests to identify hemoglobinopathies.

Study QuestionsIntroduction

What is the underlying cause of all hemoglobinopathies?

All hemoglobinopathies result from a genetic mutation in one or more genes that affect hemoglobin synthesis. All hemoglobinopathies affect hemoglobin synthesis qualitatively or quantitatively.

a disease state involving the Hgb molecule. Genetic mutation in one or more genes that affect Hgb synthesis Genes encode proteins that make up Hgb or involved in synthesizing or regulating the synthesis of globin chains

What is the difference between qualitative and quantitative hemoglobinopathies?

Qualitative—alters structure and its function Qualitative defects-

Hgb synthesis occurs at near normal rate but Hgb has structural defects with altered amino acid sequence so the function is also altered.

Only the structural defects as hemoglinopathies

Quantitative—reduced rate of hemoglobin synthesis (thalassemia)

Which group of disease conditions is defined by a structural defect in hemoglobin, qualitative hemoglobinopathies or quantitative ones?

Qualitative—structural defects

Does the term “thalassemias” refer to qualitative hemoglobinopathies or quantitative hemoglobinopathies?

Quantitative—reduced rate of hemoglobin synthesis

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Structure of Globin Genes

How many functional human globin genes are known, and on how many different chromosomes are they located?

Six functional human globin genes located on two different chromosomes.

Two of the globin genes alpha and zeta—chromosome 16

Remaining on chromosome 11: beta, gamma, delta, and epsilon

Which globin genes are referred to as “alpha-like,” and where are they located?

Globin genes alpha and zeta –alpha like—located on 16

Which globin genes are referred to as “beta-like,” and where are they located?

Beta, gamma, epsilon, delta—chromosome 11

Most of the globin genes occur as one copy per chromatid. Which two globin genes occur in two copies per chromatid?

There are 4 copies of the alpha and gamma genes per chromatid for a total of four genes per person. 2 from momma and 2 pappa.

Hemoglobin Development

How many globin chains are found in each hemoglobin molecule?

Each human hemoglobin molecule is composed of four globin chain

What is the globin composition of Gower-1 hemoglobin, and when in the course of human development is Gower-1 expressed?

Gower 1—2 zetas 2 epsilons –first 3 months of embryo life, only one alpha like and one beta like gene.

What are the globin chain compositions of Gower-2 hemoglobin and Portland hemoglobin, and when in the course of human development are they each expressed?

Alpha and gamma chain synthesis begins which leads to 2 alphas 2 epsilons = Gower 2

Portland- 2 zetas, 2 gammas

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What is the globin chain composition of fetal hemoglobin (Hb F), and when in the course of human development is it expressed?

Alpha 2 and gamma 2- later in fetal development

When in the course of human development does gamma chain synthesis diminish (while being replaced with beta chain synthesis), and what is the effect of this change upon the hemoglobin mix in human blood?

During the 6 months after birth, gamma chain synthesis gradually decreases and is replaced by beta chain synthesis so that adult hemoglobin is produced.

What is the globin chain composition of hemoglobin A2?

Second adult hemoglobin 2 alphas 2 deltas

What is the approximate mix of hemoglobins in adult human blood?

95% A, 3. 5% A2, (1-2%) F

Genetic MutationsHow many structural hemoglobin variants have been identified?

More than 1000 structual hemoglobin variants are known to exist.

What types of genetic mutations underlie the multitude of hemoglobin variants?

Point mutation, deletions, insertions, chain extensions, and fusions

What is the overwhelmingly common type of genetic mutation occurring in the hemoglobinopathies?

Point mutation

What is meant by the expression “point mutation”?

Replacement of one original nucleotide in the normal gene with a different nucleotide.

How do chain extension mutations occur, and what are their possible effects upon globin chains and hemoglobins?

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Chain extensions occur when the stop codon is mutates, so that translation continues beyond the typical last codon.

How do gene fusion mutations occur, and what are their possible effects upon globin chains and hemoglobins?

Gene fusions occur when two normal genes break between nucleotides, switch positions, and anneal to the opposite gene. 16 fusions gene have been identified.

Zygosity

What is the relationship between zygosity and disease severity in the case of pathologic beta chain variants?

Zygosity – in Hemoglobinopathies Heterozygous- trait- one β-chain mutated Homozygous- disease state- both β-chain mutated Because the mutations in the γ and δ chains have little effect on overall

Hgb function- transcribed and translated at very low levels. Dominant Hgb is A -so an α chain mutation would not effect the function

of Hgb as much as a β chain mutation. Severity—beta gene mutations would affect overall hemoglobin function to a greater etent

How do the terms “disease” and “trait” most often relate to the homozygous versus heterozygous inheritance of pathologic hemoglobins?

The terms disease and trait are also commonly used to refer to the homozygous (disease) and heterozygous (trait) states.

Pathophysiology

Explain why the impact of an amino acid substitution depends both upon the type of substitution and upon the location of the substitution within the amino acid sequence of the globin chain.

Location, charge and size of substituted amino acid determines its impact on the Hgb structure and function.

affects how the adjacent amino acids respond- attracted or repelled alters tertiary structure of globin chain quaternary structure of the Hgb molecule also alters how the protein folds

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What is the physicochemical effect of the amino acid substitution that results in sickle cell disease?

normal amino acid → substituted amino acid Hgb S = α2β2

6Glu → Val

Substitution of valine for glutamic acid at the 6th position of the β chain

How does the pathophysiology of (the heterozygous) hemoglobin S trait (Hb AS) compare to that of (the homozygous) sickle cell disease (Hb SS)?

On deoxygenation Hgb S is less soluble- forms long narrow tactoids–elongated liquid crystals of Hgb S

polymers distort RBC into its sickle shape

What are the four major groups into which Fishleder and Hoffman divided the structural hemoglobinopathies?

1- those that result in hemolytic anemia, Hgb S and unstable Hgbs2- methemoglobinemia, Hgb M3- those with increased or decreased oxygen affinity4- those with no clinical or functional effect

What do clinical laboratorians, hematopathologists and geneticists intend to communicate when they refer to a structural variant of hemoglobin as “clinically insignificant”?

Not clinically significant as they do not have a physiologic effect on the patient

What is the most frequently occurring structurally abnormal hemoglobin variant?

The most frequently occurring of the abnormal hemoglobins and the most severe is Hb S.

What is the most pathologically severe structurally abnormal hemoglobin variant?

And the most severe is Hb S

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Nomenclature

What is the difference between the scientific and the common designations for abnormal hemoglobins?

Some abnormal hemoglobins are assigned a common designation and a scientific designation. The common name is selected by the discover/ represents the geographic are. Capital letter is used to indicate a special characteristic (identical electrophoretic mobility/different amino acid substitutions) of the Hb variant

Scientific—variant chain, sequential and the helical number of the abnormal amino aicd, and the nature of the substitution [ β6 (A3) Glu-> Val) SUBSTITUTION OF VALINE FOR GLUTAMIC ACID]

How are Hb GPhiladelphia and Hb GCopenhagen alike? (In other words, why are they each designated as “G” hemoglobins?) How are these two hemoglobins likely to be different?

Identical electrophoretic mobility, containing different amino substitution, represents geographic area.

Hemoglobin S Sickle Cell Anemia Genetic Inheritance

What is the manner of inheritance of beta-hemoglobin variants? (In other words, is the inheritance of beta-hemoglobin variants X-linked, recessive, autosomal dominant, or autosomal codominant?)

Each beta like gene has two loci. Beta hemoglobin variants are inherited as autosomal codominants, with one gene inherited from each parent. Inherited a sickle (S) gene from one parent and and S, C, or Beta-Thal gene from the other.

Which results in a more severe condition, sickle cell disease (Hb SS) or Hb SC disease?

Hb SS have more severe disease than individuals who are compound heterozygotes for Hb S (Hb SC)Which results in a more severe condition, sickle cell disease (Hb SS) or Hb S/beta-thalassemia disease?

Hb SS have more severe disease than individuals who are compound heterozygotes for Hb S (Hb S beta-thal)

Incidence

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Approximately what percentage of African-Americans in the United States have inherited a hemoglobin variant?

In the US, approximately 12% of African Americans have a hemoglobin variant.

What is the incidence of sickle cell trait (Hb AS) among African-Americans in the United States?

~8%

What is the incidence of sickle cell disease (Hb SS) among African-Americans in the United States?

1 in 375

Etiology and Pathophysiology

What is the specific structural abnormality of Hb S?

Hb S is defined by the structural formula α2β2 6 GluVal

On the beta chain at the sixth position, glutamic acid is replaced by valine.

Why does the structural abnormality of Hb S affect its function?

The nonpolar valine amino acid has been placed in the position that the polar glutamic acid once held. Because glutamic acid is polar, the beta chain folds in such a way that glutamic acid extends outward from the surface of the hemoglobin tetramer to bind water and contribute to hemoglobin solubility in the cytosol.

Valine is also extended outward, but instead of binding water it seeks a hydrophobic niche with which to bind.

How is the structure of Hb S affected by its oxygen saturation?

On deoxygenation Hgb S is less soluble- forms long narrow tactoids–elongated liquid crystals of Hgb S polymers distort RBC into its sickle shape Homozygotes- sickling occurs when O2 saturation decreases to <85%

Heterozygotes- sickling does not occur until O2 saturation reaches <40%

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By what pathologic steps might the exposure of Hb Scontaining RBCs to low oxygen tensions ultimately result in infarcts within the capillaries?

Viscosity in blood increases due to polymers Slows blood flow- increasing time RBC is exposed to a hypoxic

environment pH is reduced and 2,3-biphosphoglycerate is increased – trying to get O2

into the tissues which decreases the RBC O2 affinity So… normal RBC shape when oxygenated start to sickle in ↓ O2 go into extremities where there is less O2 stay sickled and can’t get out causes vessel occlusions and infarction of tissue ones that manage to get out appear as sickle cell on PBS

What is the difference in behavior and ultimate fate between “reversible” and “irreversible” sickle cells?

Reversible sickle cells are Hb-S rbcs that change shape in response to oxygen tension. They circulate as normal when fully oxygenated. The vasoocclusive complications of SCD are thought to be due to reversible sickle cells that are able to travel into the microvasculature in the biconcave disk conformation due to their properties when oxygenated and then become distorted and viscous as they become deoxygenated.

Which are observed on peripheral blood smears, “reversible” or “irreversible” sickle cells?

Two forms Hgb S - Reversible and irreversible cells Reversible cells

ones that cause vaso-occlusive complications Travel to microvasculature in biconcave disk when oxygenated Become distorted and viscous as become deoxygenated Then convert to sickle cells in the vessel

Irreversible cells Do not change shape regardless of change in O2 tension or degree of Hgb

polymerization These are the ones seen on PBS- elongated sickle cells with point at each

end Thought that spleen removes these to keep them from entering the

microcirculation causing vaso-occlusion

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Clinical Features

Aside from the very most severe (homozygous Hb SS) form of sickle cell disease (SCD), what other three forms carry high morbidity and mortality rates?

Hundreds of hemoglobin variants are known; only four are clinically significant: 1. Hb SS, 2. Hb SC, 3. Hb S-B0-thal, 4. and Hb S-β+ -thal

At what age of an infant with Hb SS do mutated beta chains begin to replace normal gamma chains, and what is the effect of this occurrence upon the mix of hemoglobins in the infant’s blood as well as upon the infant’s physiology?

Until the second half of the first year of life because of the protective effect of Hb F. Toward the end of the first 6 months of life, mutated β chains begin to be produced and gradually replace normal gamma chains, which causes Hb S levels to increase and Hb F levels to decrease.

What is meant by the term “sickle cell crisis”?

SCD undergo episodes of recurring pain termed crises: “any new syndrome that develops rapidly in patients with SCD owing to the inherited abnormality” Various crises may occur:

Episodes of recurring pain termed crisis.

What are the five different types of sickle cell crises?

1. vasoocclusive or “painful”2. aplastic,3. megaloblastic,4. Splenic sequestration,5. and chronic hemolytic.

Which type of sickle cell crisis is the hallmark feature of SCD?

Vasooclusion

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What are the most common triggers of vaso-occlusive crises in SCD patients?

Triggered by acidosis, hypoxia, dehydration, infection, fever and exposure to extreme cold

What organs and tissues are most affected by vaso-occlusive crises?

Painful episodes occur most often in these tissues

Lungs, Bones, liver, spleen, penis, eyes, central nervous system and urinary tract

Besides Hb S polymerization and RBC sickling, what other risk factors are known to contribute to vaso-occlusive crises?

Possible risk factors includes 1. cell adherence, 2. endothelial cell activation, 3. white blood cell (WBC) and platelet activation,4. hemostatic activation, and vascular tone.

Crises are often associated with respiratory tract infections. Gradual loss of splenic function

How frequent and persistent do vaso-occlusive crises tend to be among Hb SS patients?

The frequency of painful episodes varies from none to six per year. On average, each episode persists for 4-5 days.

What is autosplenectomy?

Gradual loss of splenic function is referred to as autosplenectomy and is evidenced by the presence of Howell-Jolly and Pappenheimer bodies on the peripheral blood film.

What are the effects of autosplenectomy as manifested on the peripheral blood smears of SCD patients?

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Presence of Howell Jolly and pappenheimer bodies.

How is autosplenectomy similar to other organ failures in SCD patients?

What is the significance of splenic sequestration in the pathology of SCD?

What are some common species of bacteria that tend to cause life-threatening infections in SCD patients?

Susceptible to life threatening infection from

Staphylococcus aureusStreptococcus pneumoniaeHaemophilus influenzae

What are the key features of the chronic hemolytic anemia that is characteristic of SCD?

Chronic hemolytic anemia is characterized by shortened RBC survival with a corresponding decrease in hemoglobin and hematocrit (Hct), an elevated reticulocyte count, and jaundice.

What is the most common cause of aplastic crises in SCD patients, and why are these crises so very dangerous?

Aplastic episodes (bone marrow failure) most common life-threatening hematologic complications and are usually associate with infection particularly parvovirus infection.

Laboratory Diagnosis

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What are the key features of peripheral blood smears of patients with SCD?

Poikilocytosis and anisocytosis with normal RBCs, sickle cells, target cells, nucleated RBCs along with a few spherocytes, basophilic stippling,

What level of reticulocytosis is typical of SCD patients?

10%-25%

What are the effects of chronic hemolysis upon the appearance of SCD patients?

Chronic hemolytic anemia is characterized by shortened RBC survival with a corresponding decrease in hemoglobin and hematocrit and elevated retic and jaundice. Continuous screening and removal of sickle cells by the spleen perpetuate the chronic hemolytic anemia and autosplenectomy effect.

jaundice from unrelenting severe hemolytic anemia

What clinical chemistry test results result from the chronic hemolysis occurring in SCD patients?

bilirubin - ↑ total and indirect

What are the two fundamental steps most often used in the laboratory diagnosis of SCD? (Step one is a screening test and step two is a confirmatory test.)

The diagnosis of SCD is made by demonstrating the insolubility of Hb S in the deoxygenated form using a screening test, followed by confirmation of its presence using hemoglobin electrophoresis, high-performance liquid chromatography (HPLC), or capillary electrophoresis.

What is the principle behind the hemoglobin solubility test?

Solubility test Sodium dithionite reagent – lyses RBCs and Hgb released

deoxygenated Hgb is insoluble precipitates in solution -becomes turbid False positive results occur in lipemic sample False negative when using inadequate number of RBCs or low Hct

What are the key reagents used in the hemoglobin solubility test?

Sodium dithionite reagent – lyses RBCs and Hgb released 2% Sodium metabisulfite

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What is the difference in appearance between a positive and a negative hemoglobin solubility test?\

What are two causes of false-positive results when performing the hemoglobin solubility test?

False positive results occur in lipemic sample Few rare hemoglobinopathies (Hb C-Harlem, Hb S-Memphis) Heavily inoculated test solutions.

What are two causes of false-negative results when performing the hemoglobin solubility test?

False-negative results can occur because of :1. an inadequate number of RBCs or2. a low hematocrit.

What type of electrophoresis should be performed first in clinical laboratory testing of structural hemoglobinopathies?

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Alkaline hemoglobin electrophoresis is a common first step in the definitive diagnosis of hemoglinopathies. Hemoglobins that exhibit an abnormal electrophoretic pattern at an alkaline pH may be subjected to electrophoretic pattern at an acid pH definitive separation.

What procedure is most often carried out on hemoglobins that exhibit an abnormal electrophoretic pattern in an alkaline pH when attempting to diagnose structural hemoglobinopathies?

Hemoglobins that exhibit an abnormal electrophoretic pattern at an alkaline pH may be subjected to electrophoretic pattern at an acid pH definitive separation.

What are the two predominant hemoglobins detected in patients with Hb SS SCD?

If have S/S → no Hgb A A2 -2-5% Hgb F 1-20% Hgb S >85%

What are two examples of abnormal hemoglobins that migrate to the same position as Hb S on cellulose acetate electrophoresis, yet are negative if screened with the hemoglobin solubility test?

Hb G, Hb D do not give a positive result on the tube solubility test

Treatment

What types of supportive care have provided the mainstay of SCD therapy for many years?

Adequate hydrationVitamin therapyAvoidance of low oxygen environment Analgesia for painAntibiotic therapy Avoidance of strenuous exercise, high altitudes. Immunization and prophylactic antibiotics

What three new therapies have recently been shown able to improve the quality and extend the quantity of life for SCD patients?

Hydroxyurea to ↑ Hgb F levels

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does not copolymerize with Hgb S severity of disease proportional to Hgb F levels

Non steroidal anti-inflammatory Hypertransfusion during aplastic and splenetic crises and before surgery to- ↑ Hgb

A levels and ↓ viscosity BM transplant- usually for severe disease in children Prophylactic antibiotics- decrease infections in children

What are the five key components of supportive therapy for SCD patients?

Adequate hydrationVitamin therapyAvoidance of low oxygen environment Analgesia for painAntibiotic therapy Avoidance of strenuous exercise, high altitudes. Immunization and prophylactic antibiotics

What has been the effect of immunization and prophylactic antibiotics upon the impact of pneumococcal disease in SCD patients?

Pneumococcal disease has been a leading cause of morbidity and mortality in children. With immunization and prophylactic antibiotics, however, this is now a preventable complication. Heptavalent conjugate pneumooccal vaccine at 2, 4, and 6 months of age.

What is the effect of hydroxyurea and/or butyrate therapy upon SCD patients?

Hydroxyurea to ↑ Hgb F levels does not copolymerize with Hgb S severity of disease proportional to Hgb F levels

Arginine butyrate administered for a short period increased the synthesis of gamma globin chains and Hb F in a small number of patients with sickle cell disease and beta thalassemia syndromes.

Why do Hb SS SCD patients benefit when the percentage of Hb F is increased in their RBC pool?

Hgb F does not polymerize

Sickle Cell Trait

What are the most typical symptoms and hematologic manifestations of patients with sickle cell trait (Hb AS)?

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Individual with sickle cell trait are generally asymptomatic and present with no significant clinical or hematologic manifestations.

By what means is sickle cell trait (Hb AS) diagnosed?

The hemoglobin solubility screening test yields positive test results, and sickle cell traits is diagnosed by detecting the presence of Hb S and Hb A on hemoglobin electrophoresis or HPLC.

Hemoglobin C

What is the most common non-sickling hemoglobin variant encountered in the United States?

Hb C. It is the most common nonsickling variant encountered in the United States and the third most common in the world.

How does the severity of Hb CC disease compare to that of Hb SS disease?

Hb C is inherited in the same manner as Hb S but manifests as a milder disease. Similar to Hb S, Hb C polymerizes under low oxygen tension, but the structure of the polymers differs.

What is the appearance of polymerized Hb C on peripheral blood smears?

A mild to moderate, normochromic, normocytic anemia occurs in homozygous Hb C disease. Occasionally, some microcytosis and mild hypochromia may be present. There is a marked increase in the number of target cells

What peripheral blood smear observations are most typical of patients with Hb CC disease?

A mild to moderate, normochromic, normocytic anemia occurs in homozygous Hb C disease. Occasionally, some microcytosis and mild hypochromia. Increase in target cells, increase in retics and NRBcs.

What is the appearance of Hb C crystals on peripheral blood smears?

The crystals are densely stained and vary in size and appear oblong with pyramid-shaped or pointed ends. These crystals may be seen on wet preps by washings RBCs and resuspending them in a solution of sodium citrate.

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What are the hemoglobin solubility test results of Hb CC patients?

Hb C yields a negative result on the hemoglobin solubility test. No hemoglobin A is present in Hb CC disease.

What is the migratory behavior of Hb C during alkaline electrophoresis? During acid electrophoresis?

On cellulose acetate electrophoresis at an alkaline pH, Hb C migrates in the same position as Hb A2, Hb E, and Hb O-Arab

A2, C, E, O

Hb C is separated from these other hemoglobins on citrate agar electrophoresis at an acid pH.

C on its own.

When are Hb CC patients most likely to need medical support (treatment)?

No specific treatment is required. This disorder becomes problematic only if infection occurs or if mild chronic hemolysis leads to gallbladder disease.

Hemoglobin E

In what human subpopulations (ethnicities) is the Hb E gene variant enriched?

The variant occurs with an incidence of 30% in Southeast Asia.

What are the hematologic features of patients with Hb EE? (Be sure to include any of the following that are relevant).

Anemia—mild anemia Abnormalities of RBC indices—low MCV (55-65 fL) RBC morphology—few to many target cells Reticulocytosis—normal retic count Hb E—90%

The heterozygous state…

Anemia—slight erythrocytosis Abnormalities of RBC indices—MCV 65 fL RBC morphology—target cells Hb E—30-40%

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Do Hb EE patients typically have jaundice? Do they typically have splenomegaly?

Hb EE not associated with clinically observable icterus, hemolysis, or splenomegaly.

Why is it important to not misidentify patients with Hb EE? (In other words, what other conditions might Hb EE mimic, and how might it be harmful for a patient to be misdiagnosed?

The main concern in identifying homozygoys Hb E is differentiating it from iron deficiency, β-thalassemia trait, and Hb E-β-thal. The disease, Hb EE resembles thalassemia trait

What heterozygotic Hb E condition closely resembles beta-thalassemia major? (Patients with this condition may require transfusion support.)

When Hb E is combine with β-thalassemia, however, the disease becomes more severe than Hb EE and more closely resembles β-thalassemia major, requiring regular blood transfusions.

Does Hb E produce a positive hemoglobin solubility test result?

Hb E does not produce a positive hemoglobin solubility test result and must be confirmed using electrophoresis.

What is the migratory behavior of Hb E during alkaline electrophoresis? During acid electrophoresis?

On cellulose acetate electrophoresis at an alkaline pH, Hb E migrates with Hb C, Hb O, and Hb A2. On citrate agar electrophoresis at an acid pH, Hb E can be separated from Hb C, but it co-migrates with Hb A and Hb O.

Double Heterozygosity: Hb S With an Alternate Beta-Hemoglobin Variant

Which four alternate beta-hemoglobin variants may produce hemolytic anemia of variable severity when coupled with Hb S as a “double heterozygosity”?

Hemoglobin SC (Double Heterozygosity)

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What is the most common double heterozygosity of structural hemoglobinopathies?

Hb SC is the most common compound heterozygous syndrome that results in a structural defect in the hemoglobin molecule in which different amino substitutions are found on each of two β-globin chains. At the sixth position, glutamic acid is replaced by valine (Hb S) on one β-globin chain and by lysine (Hb C) on the other β-globin chain.

How does the severity of the clinical features of Hb SC disease compare to that of Hb SS disease?

Hb SC disease resembles a mild sickle cell disease. Unlike Hb SS, Hb SC usually does not produce significant symptons until the teenage years.

How do the vaso-occlusive complications of Hb SC disease compare to those of Hb SS disease?

What are some other pathologic features that Hb SC disease patients manifest in a milder form than do those with Hb SS disease?What are the hematologic features of Hb SC patients? (Be sure to include any of the following that are relevant).

Anemia Abnormalities of RBC indices RBC morphology Reticulocytosis

Does Hb SC produce a positive hemoglobin solubility test result? Why or why not?What band pattern is produced by Hb SC during alkaline electrophoresis? During acid electrophoresis?

Hemoglobin S/Beta-Thalassemia (Double Heterozygosity)

What is the most common cause of sickle cell syndrome in patients of Mediterranean descent?

Hb S and β-thalassemia is the most common cause of sickle cell syndrome in patients of Mediterrannean descent and is second to Hb SC disease among all compound heterozygotes.

What is the second most common “doubly heterozygous” sickle cell disorder?

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Hb S and β-thalassemia is the most common cause of sickle cell syndrome in patients of Mediterrannean descent and is second to Hb SC disease among all compound heterozygotes.

What would suffice as an apt and brief description of the clinical syndrome of hemoglobin S/beta-thalassemia patients?

Hb S-β-thal usually causes a clinical syndrome resembling that of mild or moderate sickle cell anemia. The severity of this compound heterozygous condition depends on the β chain production of the affected β-thalassemia gene.

If S-β0-thal clinical course similar to homozygous sickle cell anemiaIf there is production of β-globin (S-β+ -thal), patients tend to have a milder condition that patient with Hb SC.

How may beta-thalassemia genes vary in the amount of beta-globin produced, and how does this influence the clinical syndrome of hemoglobin S/beta-thalassemia patients?

What hematologic test results distinguish hemoglobin S/beta-thalassemia patients from sickle cell trait (Hb AS) patients?

These patients can be distinguished from individuals with sickle cell trait because of the presence of greater amounts of Hb S than of Hb A, increased levels of Hb A2 and Hb F, microcytosis from the thalassemia, hemolytic anemia, abnormal peripheral blood morphology, and splenomegaly.

What is the relationship between the percentage of Hb A2 in a patient’s blood and the presence of heterozygous beta-thalassemia?

Hb disorder

AbnormalHb

Structural defect

Groups 1°

affected

Hb solubility

Hbs present

RbcMorph.

Symptoms Treatment

Hb-S-βThal

Hb S+ α2β2 6Glu->Val

andβ0 or β+

Same as Hb S

Positive Hb S variable, Some Hb A in β+ ,

increased Hb A2

and Hb F

Sickle cells, target cells,

microcytes

Hemolytic anemia,

splenomegaly

Similar to that for Hb S; varies

depending on amount of Hb A present.

Compared to Sickle cell

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Hb disorder

AbnormalHb

Structural defect

Groups 1° affected

Hb solubility

Hbs present

RbcMorph.

Symptoms Treatment

Sickle cell anemia

Hb S

α2β

2 6Glu->Val African

American, Middle Eastern, Indian, Mediterranean

Positive 0% Hb A, >80% Hb S, 1-20% Hb F, 2-5% Hb A2

Sickle cells, target cells, nucleated RBCs, polychromasia, Howell-Jolly bodies, baso stippling, spherocytes

Vasoocclusion,Bacterial infections, hemolytic anemia, aplastic episodes; bones, lungs, liver, spleen, penis

Transfusions,Antibiotics, analgesics, bone marrow transplants, hydroxyurea.

Hemoglobin M

Why was Hb M given the designation “M”?

Hb M is caused by a variety of mutations in the α, β, and γ genes, all of which result in the production of methemoglobin ---hence the Hb M designation .

What is the effect upon heme iron of inheriting Hb M?

Genetic mutations above result in a structural abnormality in the globin portion of the molecule. Most M hemoglobins involve a substitution of a tyrosine amino acid for either the proximal (F8) or the distal (E7) histidine amino acid in the α,β,γ chains. These substitutions cause heme iron to autooxidize, which results in methemoglobinemia. Hb M has iron in the ferric state (Fe 3+) and is unable to carry oxygen, which produces cyanosis.

What is the consequence of auto-oxidation upon the redox state of heme iron?

These substitutions cause heme iron to autooxidize, which results in methemoglobinemia. Hb M has iron in the ferric state (Fe 3+) and is unable to carry oxygen, which produces cyanosis.

What is the consequence of auto-oxidation upon hemoglobin’s ability to carry oxygen?

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Is unable to carry oxygen.

What scientific term describes the bluish color of the tissues of patients whose blood is enriched with methemoglobin?

Cyanosis.

What is the effect of certain oxidants, such as sulfonamides or phenacetin, upon the blood and appearance of Hb M patients?

When Hb M is exposed to certain oxidants, such as sulfonamides or phenacin, a chocolate brown color develops. Methemoglobin causes the blood sample to appear brown.

Why are Heinz bodies sometimes seen on wet preparations of Hb M patients’ blood?

Methemoglobin causes globin chains to precipitate.

Why is it important to diagnose Hb M patients and by what laboratory testing means is diagnosis accomplished?

Diagnosis is made by spectral absorption of the hemolysate or by hemoglobin electrophoresis. Before electrophoresis, all hemoglobin types are converted to methemoglobin by adding potassium cyanide to the sample so that any migration differences observed are obly due to an amino acid substitution, not differences in iron states. Use HPLC, or DNA globin gene analysis. Diagnosis is essential to prevent inappropriate treatment for other conditions, such as cyanotic heart disease.

Unstable Hemoglobin Variants

Why do unstable hemoglobin variants result in Heinz body formation?

Unstable hemoglobin variant result from genetic mutations to globin genes creting hemoglobin products that precipitate, producing Heinz bodies and causing a hemolytic anemia.

Approximately how many unstable hemoglobin variants have been identified, and what is the range of their clinical significance?

More than 200 variants of unstable hemoglobin exist. The majority of these are β chain variants.

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Most unstable hemoglobin variants have no clinical significance, although the majority have an increased oxygen affinity

About 25% of unstable hemoglobins are responsible for hemolytic anemia, which varies from compensated mild anemia to severe hemolytic episodes.

What are two archaic synonyms for unstable hemoglobin disease?

Congenital nospherocytic hemolytic anemiaCongenital Heinz body anemia.

What are the characteristic clinical features of unstable hemoglobin disease?

Detected in early childhood in patients with hemolytic anemia accompanied by jaundice and splenomegaly. Fever or ingestion of an oxidant exarcebates the hemolysis. The severity of the anemia depends on the degree of instability of the hemoglobin molecule. Hemoglobin precipitates in vivo and in vitro in response to factors that do not affect normal hemoglobins, such as drug ingestion and exposure to heat or cold.

What two risk factors may exacerbate the hemolysis experienced by patients with unstable hemoglobin disease?

Fever or ingestion of an oxidant exarcebates the hemolysis. The severity of the anemia depends on the degree of instability of the hemoglobin molecule. Hemoglobin precipitates in vivo and in vitro in response to factors that do not affect normal hemoglobins, such as drug ingestion and exposure to heat or cold

What form does precipitated hemoglobin take in patients with unstable hemoglobin disease?

The hemoglobin precipitates in the RBC as Heinz bodies.

What is the pathologic effect of denatured hemoglobin upon the cell membrane in patients with unstable hemoglobin disease?

Attaches to the cell membrane, causing clustering of band 3, attachment of autologous immunoglobulins, and macrophage activation. In addition, Heinz bodies can be trapped mechanically in the splenic sieve, which shortens RBC survival.

How is the large variability in the degree of instability among unstable hemoglobin variants reflected in terms of clinical presentation?

Because of the large variability in the degree of instability in these hemoglobins, the extent of hemolysis varies greatly. For some of the variants, the presence of an oxidant is required for any significant hemolysis to occur.

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Laboratory Diagnosis

How may the RBC morphology vary among patients with unstable hemoglobin disease?

The RBC morphology varies. It may be normal or show slight hypochromia and prominent basophilic stippling, which possibly is caused by excessive clumping of ribosomes.

What are typical ranges for hemoglobin concentration and reticulocyte percentage among patients with unstable hemoglobin disease, prior to splenectomy?

Before splenectomy, the hemoglobin level ranges from 7 to 12 g/dL with a 4% to 20% retic count.

What are typical ranges for hemoglobin concentration and reticulocyte percentage among patients with unstable hemoglobin disease, postsplenectomy?

(see above) After splenectomy, anemia is corrected, but reticulocytosis persists.

By what laboratory means are Heinz bodies demonstrated, and what is the effect of splenectomy upon their occurrence in patients with unstable hemoglobin disease?

Heinz bodies can be shown using a supravital stain. After splenectomy, Heinz bodies are larger and more numerous.

Are unstable hemoglobins typically detected and identified via hemoglobin electrophoresis?

No. Many unstable hemoglobin migrate in the normal AA pattern and thus are not detected on electrophoresis.

What two means can be used to demonstrate the presence of unstable hemoglobins? (Both methods cause the hemoglobins to denature and precipitate out of a hemolysate solution.)

Other test used to detect unstable hemoglobins include the isopropanol precipitation test, The heat denaturation test also can be used.

What steps are involved in performing an isopropanol precipitation test, and what constitutes a positive result?

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Based on the principle that an isopropanol solution at 37 °C weakens the bonding forces of the hemoglobin molecule. If unstable hemoglobins are present, rapid precipitation occurs in 5 minutes, and heavy flocculation occurs after 20 mins.

What steps are involved in performing a heat denaturation test, and what constitutes a positive result?

When incuabated at 50 °C for 1 hour, heat-sensitive unstable hemoglobins show a flocculent precipitation , whereas normal blood shows little or no precipitation.

Hemoglobins With Increased and Decreased Oxygen Affinity

Among the more than 60 hemoglobin variants that have been discovered to have abnormal oxygen affinity, which are more commonhigh-affinity or low-affinity variants?

Most are high affinity.

Why would it be correct to refer to patients with high oxygen-affinity hemoglobin variants as having “familial erythrocytosis”?

Hemoglobins With Increased Oxygen Affinity

Why do patients with high oxygen-affinity hemoglobin variants have compensatory erythrocytosis?

More than 115 variant hemoglobin with high oxygen affinity have been discovered. Such hemoglobins fail to release oxygen on demand, and hypoxia results. The kidneys sense the hypoxia and respond by increasing the release of erythropoietin, which leads to a compensatory erythrocytosis. These variants differ from unstable hemoglobin, which also may have abnormal oxygen affinity, in that they do not precipitate in vivo to produce hemolysis and there is no abnormal RBC morphology.

In what important ways are high oxygen-affinity hemoglobin variants similar to unstable hemoglobin variants?

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The high-affinity variants, like other structurally abnormal hemoglobins, show an autosomal dominant pattern of inheritance. Affected individuals have equal volumes of Hb A and the abnormal variant.

In what ways do high oxygen-affinity hemoglobin variants differ from unstable hemoglobin variants?

These variants differ from unstable hemoglobin, which also may have abnormal oxygen affinity, in that they do not precipitate in vivo to produce hemolysis and there is no abnormal RBC morphology.

What is the most notable aspect of the appearance of patients with high oxygen-affinity hemoglobin variants?

Most individuals are asymptomatic and show no physical symptoms except a ruddy complexion.

Patients with high oxygen-affinity hemoglobin variants typically have erythrocytosis. What results are therefore expected when their RBC counts, hemoglobins, and hematocrits are measured? What are the typical WBC count results of patients with high oxygen-affinity hemoglobin variants?

Erythrocytosis is usually detected during routine examination, because the patent generally has a high RBC count, hemoglobin, and hematocrit. The WBC count, platelet count, and peripheral blood film findings are generally normal.

What are the typical platelet count results of patients with high oxygen-affinity hemoglobin variants?

Normal.

What are the typical peripheral blood smear results of patients with high oxygen-affinity hemoglobin variants?

Normal

What type of laboratory testing is required to definitively diagnosis a patient as having inherited a high oxygen-affinity hemoglobin variant?

In some cases, hemoglobin electrophoresis may establish a diagnosis. An abnormal band that separates from the A band is present on cellulose acetate in some variants; however

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if a band is not found, the diagnosis of increased oxygen affinity cannot be ruled out. In some cases, the abnormal hemoglobin can be separated using citrate agar (pH 6.0) or by gel electrophoresis.

Measurement of oxygen affinity is required for definitive diagnosis.

“Patients with high-oxygen-affinity hemoglobins live normal lives and require no treatment.” Why, then, is it important to diagnosis this inherited condition?

Diagnosis should be made to avoid unnecessary treatment of the erythrocytosis as a myeloproliferative neoplasm or a secondary erythrocytosis.

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