Hemoglobinopathy Flashcards
1
Q
What are the broad classifications of Hb disorders?
A
-
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
Q
- What are the normal hemoglobins?
- Which one dominates during fetal life?
- What are the expected values for Hb in an adult?
A
- 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%
3
Q
Abnormal hemoglobins
A
- 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
Q
Hb Structural Abnormalities:
- Depends On:
- **Potential Consequences: **
A
-
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
- What globin gene is affected
-
Potential consequences:
- Sickling
- Instability
- Altered oxygen affinity (increased or decreased)
- Increased susceptibility to oxidation to methemoglobin
- Under-production
- Various combinations of the above
5
Q
What lab techniques are used to diagnose hemoglobinopathies?
A
-
Hemoglobin electrophoresis
- Gel
- Capillary
- High performance liquid chromatography (HPLC)
- Other advanced techniques
- Isoelectric focusing
- Globin chain electrophoresis
- Gene mutation analysis
6
Q
- How is routine electrophoresis typically performed?
- What is the isoelectric point of HbA?
- What does the migration of other hemoglobins depend on?
A
- 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
7
Q
What are the two methods of HPLC?
A
- 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
8
Q
- Define sickle cell disease:
- What causes SCD?
- Which type of HbS protects against malaria?
- What is the frequency of homozygous S?
A
- 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
Q
Sickle Cell Disease (SS)
Pathophysiology
A
-
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
Q
What factors affect HbS concentration?
A
-
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
Q
How is sickling of RBCs time dependent?
A
- 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
Q
What clinical settings are predisposing to sickling of RBCs?
A
- 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
Q
SCD Pathophysiology:
- At what pressure do RBCs begin to sickle?
- Is sickling reversible?
- What happens to RBC lifespan?
A
- 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
Q
- What are the long term effects of sickling?
- Which of these correlates with irreversibly sickled cells?
- Which of these correlates with “stickiness”?
A
-
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
Q
When do clinical manifestations of SCD begin to appear?
A
- 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
Q
What are the major complications of SCD?
A
- 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
Q
What are other potential complications of SCD?
A
- Aplastic crisis
- Infections
- Liver damage (multifactorial)
- Splenic sequestration crisis
- Megaloblastic anemia
- Growth retardation
- Bony abnormalities
- Renal dysfunction
- Leg ulcers
- Cholelithiasis
18
Q
What causes auto-spleenectomy in sickle cell patients?
A
- 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
Q
What is acute chest syndrome?
A
- 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
Q
- What causes aplastic crisis in sickle cell patients?
- Why is this particularly dangerous for sickle cell patients?
A
- 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
Q
What is splenic sequestration crisis?
A
- Acute pooling of blood in spleen
- Precipitous drop in hemoglobin
- Potential for hypovolemic shock
22
Q
What causes megaloblastic anemia?
A
Folate consumption because of chronic erythroid hyperproliferation
23
Q
Sickle Cell Disease
Laboratory Findings
A
-
Chronic anemia
-
steady state hemoglobin from 5-11 g/dl
- most commonly about 7
-
steady state hemoglobin from 5-11 g/dl
- Increased bilirubin
- Sickled cells, target cells, and polychromasia
- Increased reticulocytes
- Normal MCV
- Post-splenectomy changes in adults
24
Q
What do you expect to see on HPLC for a sickle cell disease patient?
A
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**?
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_**
37
What can be seen on peripheral blood smear in a patient with HbC disease?
* **Numerous target cells**
* Mild microcytosis
* **Spherocytes**
* Occasional **C crystals**
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
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%
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
