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Flashcards in Hemoglobinopathy Deck (51):
1

What are the broad classifications of Hb disorders?

  • Structural variants
    • Abnormal globin chain structure due to globin gene mutation
    • Varied clinical effects depending on location and nature of mutation in globin chains
  • Thalassemias
    • Under-production of structurally normal globin chains
    • Generally microcytic/hypochromic anemias of varying severity

2

  • What are the normal hemoglobins?
  • Which one dominates during fetal life?
  • What are the expected values for Hb in an adult?

  • Three normal hemogobin species in fetal and postnatal life
    • Hemolobin A: (α2β2)
    • Hemoglobin F: (α2γ2)
    • Hemoglobin A2: (α2δ2)
  • Hemoglobin F dominates during fetal life
  • Normal adult complement (achieved after 6 months to 1 year of age)
    • HbA: 96%
    • Hb F: 1%
    • Hb A2: 3%

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3

Abnormal hemoglobins

  • More than 500 structural hemoglobin variants have been described
    • Most are single amino acid replacements in globin molecules (due to single base pair substitutions in globin genes)
    • Any globin gene may be affected
    • Occasional other types of mutations
  • 15 variants with 2 amino acids replaced
    • Deletions
    • Insertions
    • Chain elongation
    • Fusion genes
  • Most clinically silent

4

Hb Structural Abnormalities:

  • Depends On:
  • Potential Consequences: 

  • Depends on:
    • What globin gene is affected
      • e.g, delta gene mutations are clinically inconsequential
    • Location of substitution in the tertiary structure and/or quaternary stuctures of the globin or hemoglobin molecules
  • Potential consequences:
    • Sickling
    • Instability
    • Altered oxygen affinity (increased or decreased)
    • Increased susceptibility to oxidation to methemoglobin
    • Under-production
    • Various combinations of the above

5

What lab techniques are used to diagnose hemoglobinopathies?

  • Hemoglobin electrophoresis
    • Gel
    • Capillary
  • High performance liquid chromatography (HPLC)
  • Other advanced techniques
    • Isoelectric focusing
    • Globin chain electrophoresis
    • Gene mutation analysis

6

  • How is routine electrophoresis typically performed?
  • What is the isoelectric point of HbA?
  • What does the migration of other hemoglobins depend on?

  • Typically performed in parallel with alkaline and acid buffers
  • HbA has isoelectric point of 6.8
    • Negative charge in alkaline buffers ⇒ migrates toward anode (+)
    • Positive charge in acid buffers ⇒ migrates toward cathode (-)
  • Migration of other hemoglobins depends on:
    • Net charge in alkaline electrophoresis
    • Net charge and interaction with components of media in acid electrophoresis

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7

What are the two methods of HPLC?

  • Fully automated cation exchange chromatography method
  • Whole blood method (whole blood hemolysate)
    • Hemoglobins adsorbed onto resin particles in column
    • Different species differentially eluted based on affinity for resin by gradually changing ionic strength of elution buffer
    • Hemoglobins come off the column at highly predictable retention times
      • Some correlation with migration on alkaline electrophoresis

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8

  • Define sickle cell disease:
  • What causes SCD?
  • Which type of HbS protects against malaria?
  • What is the frequency of homozygous S?

  • Homozygous abnormality of the beta globin chain
  • Glu to Val substitution at amino acid 6 of β-chain (β6Val)
  • Heterozygous HbS (S-trait) confers protection against malaria
    • 4% allelic frequency for Hb S gene among African-Americans
    • Rare in other ethnic groups
  • Homozygous S occurs at a frequency of 1 in 600 in African Americans

9

Sickle Cell Disease (SS)

Pathophysiology

  • Deoxygenated HbS forms long polymers that distort the shape of the cell into an elongated, sickled form
    • Intermolecular contacts involve abnormal valine at amino acid 6
  • Extent of HbS polymerization is time and concentration dependent

10

What factors affect HbS concentration?

  • Percentage of hemoglobin S of total hemoglobin
    • Homozygous versus heterozygous
    • Presence of other hemoglobin species (e.g., Hb F)
  • Total hemoglobin concentration in the red cells (MCHC)
    • Increased in states of cellular dehydration
    • Decreased when there is co-existent thalassemia

11

How is sickling of RBCs time dependent?

  • Importance of transit time of red cells through low oxygen tension microvasculature
  • Sickling enhanced in anatomic sites with sluggish flow (e.g., spleen and bone marrow)
  • Blood flow through microvasculature retarded in certain pathologic states
    • Inflammation

12

What clinical settings are predisposing to sickling of RBCs?

  • Hypoxia
  • Acidosis
    • Shift of oxygen dissociation curve to right, causing increased deoxygenation of Hb S
  • Dehydration
    • Hypertonicity causing RBC dehydration
  • Cold temperatures
    • Probably as a result of peripheral vasoconstriction with resultant sluggish flow
  • Infections (multiple mechanisms)

13

SCD Pathophysiology:

  • At what pressure do RBCs begin to sickle?
  • Is sickling reversible?
  • What happens to RBC lifespan?

  • SS cells begin to sickle at ~40mm Hg
  • Sickling is initially a reversible process, but after multiple sickling/unsickling cycles, membrane damage produces an irreversibly sickled cell
  • RBC lifespan decreased to 20 days

14

  1. What are the long term effects of sickling?
  2. Which of these correlates with irreversibly sickled cells? 
  3. Which of these correlates with "stickiness"?

  • Chronic hemolysis
    • Correlates with the number of irreversibly sickled cells
  • Microvascular occlusion with resultant tissue hypoxia and infarction
    • Does not correlate with irreversibly sickled cells
    • Related to increased “stickiness” of SS red cells because of membrane damage

15

When do clinical manifestations of SCD begin to appear?

  • Newborns clinically fine because of high HbF levels
  • Hematologic manifestations begin by 10-12 weeks of age
  • Clinical severity variable from patient to patient

16

What are the major complications of SCD?

  • Severe anemia
  • Acute pain crises
    • Result from vaso-occlusion, particularly in marrow
    • Major cause of ED visits and hospital admissions
  • Auto-splenectomy
  • Acute chest syndrome
  • Strokes
    • Risk of stroke of 11% by age 20
    • First clinical stroke most frequently occurs between 2 and 8 years of age

17

What are other potential complications of SCD?

  • Aplastic crisis
  • Infections
  • Liver damage (multifactorial)
  • Splenic sequestration crisis
  • Megaloblastic anemia
  • Growth retardation
  • Bony abnormalities
  • Renal dysfunction
  • Leg ulcers
  • Cholelithiasis

18

What causes auto-spleenectomy in sickle cell patients?

  • Repeated episodes of splenic infarction, resulting in shrunken, fibrotic, non-functional spleen
  • Seen in essentially all adults with SS disease
  • Increased risk for infection by encapsulated bacteria

19

What is acute chest syndrome?

  • Severe complication, major cause of death
  • Result from pulmonary infections or fat emboli from infarcted marrow
  • Sluggish blood flow from inflammation causes sickling and vaso-occlusion, triggering vicious cycle

20

  • What causes aplastic crisis in sickle cell patients? 
  • Why is this particularly dangerous for sickle cell patients?

  • Caused by acute decrease in RBC production
  • Usually due to parvovirus B19 infection
    • Common childhood virus (“Fifth’s disease”)
    • Infects erythroid precursors and cause red cell aplasia with absent erythropoiesis for 7-10 days

21

What is splenic sequestration crisis?

  • Acute pooling of blood in spleen
  • Precipitous drop in hemoglobin
  • Potential for hypovolemic shock

22

What causes megaloblastic anemia?

Folate consumption because of chronic erythroid hyperproliferation

23

Sickle Cell Disease

Laboratory Findings

  • Chronic anemia
    • ​steady state hemoglobin from 5-11 g/dl
      • most commonly about 7
  • Increased bilirubin
  • Sickled cells, target cells, and polychromasia
  • Increased reticulocytes
  • Normal MCV
  • Post-splenectomy changes in adults

24

What do you expect to see on HPLC for a sickle cell disease patient?

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25

Describe Hemoglobin SC disease:

  • Compound heterozygous state
  • Hemoglobin C results from glu to lys substitution at the 6th amino acid of the beta globin chain
    • Does not apparently co-polymerize with HbS, but causes cellular dehydration and consequent sicking
  • Generally milder disease than SS, but highly variable
  • Hb levels usually 10 to 12 g/dl

26

Describe Hb S/Beta thalassemia:

  • Heterozygous Hb S with trans beta thalassemia allele 
    • resulting in decreased or absent production of normal beta chains
  • Ranges from asymptomatic to a disorder nearly identical to SS disease, depending on output of normal beta chains from thalassemia allele
  • Lab findings:
    • Hb S > Hb A

27

How is sickle cell disease managed?

  • Newborn screening
  • Infection prophylaxis
  • Supportive care for acute manifestations
  • Hydroxyurea (most commonly used Tx for chronic disease management)
  • Regular red cell transfusions
  • Allogeneic stem cell transplant

28

What is the benefit of using hydroxyurea for sickle cell patients?

  • Chemotherapy agent used to reduce blood cell counts in myeloproliferative neoplasms
  • Inhibits ribonucleotide reductase and causes cell cycle arrest
  • Increases erythrocyte levels of HbF, ameliorating the sickling manifestations
  • Dramatically reduces frequency of pain crises, as well as significantly decreased transfusion requirements, hospital admissions, incidence of acute chest sydrome

29

What is the only cure for SCD?

Allogeneic stem cell transplant

30

What is the prognosis/outcome for SCD?

  • Median age of death of 42 for males and 48 for females with SS disease
  • Gains mainly seen due to decreased mortality rates in children due to aggressive infection prophlaxis and comprehensive care approaches
  • No apparent decrease in mortality rates in adults over last several decades

31

What are the major causes of death in sickle cell patients?

  • Liver dysfunction
  • Pulmonary hypertension
  • Stroke
  • Vaso-occlusive crisis
  • Acute chest syndrome

32

S-Trait

  • How does it compare to sickle cell disease?
  • What is the major complication?
  • What are the Hb lab findings?

  • 8% of African Americans
  • Clinically benign
    • No anemia
    • Normal RBC survival
    • No crises or other complications in vast majority of patients
    • Normal peripheral blood smear
  • May be mild, sub-clinical kidney damage
    • Impairment of urine concentration
    • Microhematuria
  • Laboratory
    • 60% Hb A, 40% HbS

33

What do you expect to see on HPLC for a sickle cell trait patient?

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34

What are the manifestations of HbC disease?

  • Mild to moderate hemolytic anemia
  • Often asymptomatic
  • Splenomegaly
    • May cause occasional abdominal pain
  • 1/6000 African Americans

35

  • What is the pathophysiology of HbC disease?
  • How does it differ from SCD?

  • Glu to Lys substitution of amino acid 6 of β chain (β6Lys)
  • Cells abnormally rigid and dehydrated
  • RBC life span shortened to 30-35 days
  • Not a sickling disorder

36

What are the expected CBC and Hb findings for HbC disease?

  • Hemoglobin levels range from 8-12 g/dl
  • >90% HbC
  • No HbA

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37

What can be seen on peripheral blood smear in a patient with HbC disease?

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  • Numerous target cells
  • Mild microcytosis
  • Spherocytes
  • Occasional C crystals

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38

What are the manifestations of HbC trait?

  • 2% of African-Americans
  • No anemia
  • Few target cells
  • 50-60% HbA, 30-40% HbC

39

  • Define thalassemias:
  • Distribution of β-thal and α-thal:

  • Group of inherited disorders characterized by decreased production of structurally normal globin chains
  • Highly heterogeneous both clinically and genetically
  • Distribution
    • β-thal: Mediterranean, Middle East, parts of India and Pakistan, and Southeast Asia
    • α-thal: Africa, Mediterranean, Middle East, and Southeast Asia

40

  • What is the typical RBC morphology for thalassemia?
  • What determines the severity of hematologic manifestations?

  • Typically are microcytic, hypochromic anemias of varying severity
    • Decreased hemoglobin production produces hypochromia and microcytosis
      • “Cytoplasmic maturation defect”
  • Severity of hematologic manifestations is directly related to the degree of chain imbalance
    • Excess normally produced globin chains accumulate and cause intramedullary cell death and/or decreased RBC survival

41

  • What is β thalassemia?
  • What mutations can cause it?

  • Decreased beta globin chain production from affected alleles
  • More than 250 mutations described:
    • Mutations causing splicing errors (most common)
    • Mutations in promoters causing decreased transcription
    • Translation errors (frameshift or nonsense codons)
    • Gene deletion rare

42

How are β thalassemias classified?

Classified clinically because of extreme genetic heterogeneity

  1. β-thal major (Cooley’s anemia)
  2. β-thal intermedia
  3. β-thal minor

43

Define β-thal major:

  • Absence or marked decrease in beta-chain production on both beta alleles
    • Excess of normal alpha chains, which are unable to form tetramers, and precipitate in normoblasts and erythrocytes
    • Intramedullary cell death and decreased RBC lifespan
      • Hybrid of hemolytic anemia and ineffective erythropoiesis

44

What is the clinical progression of β-thal major?

  • Infants well at birth--anemia develops over the first few months of life
  • Severe anemia-baseline Hb of 2-3 g/dL
    • Virtually all Hb F
    • Bizarre red cell morphology (hypochromia, targeting, erythroblastosis)
  • Transfusion dependence
  • Severity of clinical effects depends on:
    • adequacy of transfusion program 
    • efficacy of iron chelation

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45

Consequences of Inadequately Transfused β-Thal Major:

  • Stunted growth
  • Frontal bossing
  • “Mongoloid” facies
  • Increased skin pigmentation
  • Death in childhood
  • Characteristic bony abnormalities
  • Folate deficiency
  • Fever
  • Wasting
  • Hyperuricemia
  • Spontaneous fractures
  • Hepatosplenomegaly
  • Infections

46

Consequences of Adequately Transfused β-Thal Major:

  • Without adequate iron chelation therapy
  • With adequate iron chelation therapy

  • Essentially normal early development
  • Avoidance of classic complications
  • Without adequate iron chelation therapy
    • Absence of pubertal growth spurt and menarche
    • Endocrine disturbances such as DM, adrenal insufficiency
    • Death from cardiac disease by end of third decade
  • With aggressive iron chelation therapy
    • Less severe cardiac disease and endocrine disturbances
    • Significantly improved life-span

47

Define β-Thal Minor:

  • Heterozygous form
  • Asymptomatic
  • Discovered incidentally
  • Incidence
    • Common in Mediterranean and Asian populations
    • 1.5% of African Americans

48

What are the lab findings for β-Thal Minor?

  • Mild or no anemia (Hb>~10g)
  • Microcytosis (50-70 fl)
  • Mild anisopoikilocytosis
    • Scattered target cells
  • Basophilic stippling
  • Elevated HbA2: 3.5-7%

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49

What is β-Thal Intermedia?

  • Heterogeneous group
  • Intermediate severity between beta thalassemia major and minor

50

What causes α Thalassemia?

Usually a result of gene deletion

  • in contrast to the beta-thalassemias
     

51

α-Thalassemia Clinical Subtypes:

  1. 1 α gene deleted:
  2. 2 α genes deleted:
  3. 3 α genes deleted:
  4. 4 α genes deleted:

  1. Silent carrier (1 alpha gene deleted)
  2. Alpha-thal trait (2 genes deleted)
    • Mild microcytic anemia similar to beta-thal minor, discovered incidentally
  3. Hemoglobin H disease (3 genes deleted)
    • Mild to moderate, chronic hemolytic anemia
    • Hb H represents beta tetramers
      • Does not effectively transfer oxygen
    • Hb H soluble, so does not initially precipitate in normoblasts (no intramedullary cell death)
      • Unstable over time, so precipitates in circulating red cells, causing hemolysis
  4. Hydrops fetalis (4 genes deleted)
    • Infants either stillborn or die within first few hours of life

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