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Hemoglobin & Hemoglobinopathies
beyond the peripheral slide Jane Merschen MT (ASCP), MPH"
An Emerging Global Health Burden"
Breakdown of the annual number of births with the different hemoglobin disorders
Major hemoglobin disorder No. of annual births
β-thalassemia major 22 989 Hb E thalassemia 19 128 Hb H disease 9 568 Hb Bart hydrops 5 183 Hb SS disease 217 331 Hb Sβ-thalassemia 11 074 Hb SC disease 54 736
Weatherall DJ, Blood, 2010;115:4331-4336.
Distribution of sickle cell trait Historical distribution of malaria
Modern distribution of malaria
What is hemoglobin ?"
Hemoglobin is a protein in red blood cells that carries oxygen
Hemoglobin structure"Tetrameric structure made by 2 dissimilar pairs of globin
chains each linked to a single HEME molecule"
• Alpha globin chains: (α) "
• Non-alpha globin chains:" Beta (β), gamma (γ), delta (δ) "
Normal adult Hbs consists of 2 (α) chains + another pair "
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Hemoglobin structure"Highly conserved protein""Proteins are made of amino acids""Gas transporting molecule that makes up 95% of the RBC""Carries Oxygen"
Adult Hemoglobins" HbA (α2 β2) "§ Major adult hemoglobin "§ 95% of total"
HbF (α2 γ2)"§ ‘about’ 1%"
HbA2 (α2 δ2) "§ ~ 2.5% of adult Hb "§ δ chain differs from β at ~ 10% of residues"§ function unclear"
Embryonic : Gower I (ζ2 ε2), Gower II (α2 ε2) Portland (ζ2γ2)"
Fetal Hemoglobins"
Fetal : HbF (α2 γ2)"
β, γ, δ, ε chains can all form tetramers: Hb Bart’s (γ4) HbH (β4)" α does not form tetramers"
Ø When synthesis is un-balanced:"
Globin Synthesis and Hemoglobin Assembling"
Hemoglobin HbA" HbF" HbA2"
Chains" α2 β2 α2 γ2 α2 δ2
Newborn" ~10-30%" ~70-80%" ~ 0%"
Adult" ~94-96%" ~ 1%" ~2,5%"
At birth Adult
Transition of the hemoglobin chains"
Rat
io (%
)
Antenatal Birth Post-natal
0 3 6 3 6
10
20
30
40
50
0
β
γ α
ε
δ
Newborn chromatogram on HPLC"
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ζ α2 α1
Embryonic Fetal Adult Hb Gower1 (ζ2ε2) Hb F (α2γ2) Hb A (α2β2)
Hb Gower2 (α2ε2) Hb A2 (α2δ2)
Hb Portland (ζ2γ2)
ε Aγ δ β Gγ
HEMOGLOBIN SWITCHING" Healthy adult chromatogram on HPLC"
Genetics"
Genes encoding for globin chains"
ε Gγ Αγ ψβ δ β 5`
3`
Chromosome 11
ζ ψζ ψα1 α2 α1 5` 3`
ψα2 θ
Chromosome 16
Hemoglobin gene clusters"
From Gene to Hemoglobin" We have 4 α genes and only 2 β genes "
Genotype!
Phenotype!
Presumptive or Definitive Identification ?"
ELECTROPHORESIS
CE-HPLC
IEF
MICROCOLUMNS
CE TO SCREEN HEMOGLOBINS
HPLC TO DIFFERENTIATE GLOBINS
MS TO CONFIRM AA SEQUENCE
DNA ANALYSIS TO IDENTIFY GENE MUTATIONS
ANALYTES METHODS OF ANALYSIS RESULTS
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Hemoglobinopathies"
Hemoglobinopathies"
Structural variants: § Mutation changes an amino acid in the globin chain sequence"§ Change may alter the normal hemoglobin function"§ Change may also cause thalassaemia"
The thalassemias:
§ Mutation results in either the absence or a reduction in the synthesis of one or more globin chains"§ Causes an imbalance in the ratio of the amounts of the two globin chains that make up a Hb (eg Hb A: a/b chains)"
Hemoglobinopathies"
§ More than 80% result from a substitution mutation"§ Many populations affected"§ Multiple abnormalities can be inherited"§ Less than 30% of variants result in clinical outcomes"§ Some hemoglobinopathies are ‘screen negative’"
" " " " " " " ""www.globin.cse.psu.edu!
ü Hemoglobin variants: 1015 currently described ü Thalassemia mutations: 393 mutations currently described
Heterogeneous group of inherited conditions comprising of either:
Hb Variants - Types"β-chain variants 362 α-chain variants 217 γ-chain variants 70 δ-chain variants 32 They can be: • Variants with one amino acid replacement 612 • Variants with two amino acid replacements 19 • Variants with hybrid chains 10 • Variants with elongated chains 13 • Variants with deletions (17), insertions (6), • and deletions + insertion (4) 27
Common Hb Variants "
HbS is the commonest variant Exceeds 20% in parts of Africa. Also found in Turkey, Iran, Saudi Arabia, & India
HbE is the second commonest variant Exceeds 50% in the “Hb triangle” – ie parts of Thailand, Laos and Cambodia
HbC is found mainly in West Africa Found at a frequency of up to 50% on the Ivory Coast High frequencies are due to heterozygotes having increased resistance to infection by malaria (P. falciparum)
Inheritance of Hemoglobin Disorders"
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Carrier!Carrier!
25% Affected
50% Carrier
25% Unaffected
What is autosomal recessive inheritance?"
Limitations of Hemoglobin Electrophoresis"
Identification dependent on technical performance § Hemoglobin concentration"§ Amperage"§ Running temperature"§ Length of electrophoresis run""
Identical or similar migrations § Discrete differences "§ Lack of control specimens"
METHODOLOGIES"QUALITATIVE
§ Cellulose Acetate Electrophoresis (pH 8.6)"§ Citrate Agar Electrophoresis (pH 6.2)"§ Isoelectric Focusing (pH gradient)"§ Globin Chain Electrophoresis (Alkaline and Acid)""
QUANTITATIVE § Column Chromatography (Hb A2)"§ Alkali Denaturation (Hb F)"§ Radial Immunodiffusion (Hb F)"§ Densitometry (Hb A2,Hb F, and hemoglobin fractions)"§ Kleihauer-Betke Stain (Hb F)""
AUTOMATED (Qualitative and Quantitative) § Capillary Zone Electrophoresis (CZE)"§ High Performance Liquid Chromatography (HPLC)"
HELENA CELLULOSE ACETATE, pH 8.6
HELENA CITRATE AGAR, pH 6.2
A
F
S
C
A
F
S
C
D-‐Punjab, G-‐Phil, Lepore
A2, E, O-‐Arab
D, G, Lepore, A2, E, O-‐Arab
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The thalassemias"
Did you Know…"Ø Thalassemia is sometimes called Mediterranean anemia or Cooley’s anemia
Ø The name “ thalassemia “ comes from the greek thalassa which means “ sea” and haema , which means “blood” .
Ø Thalassemia is prevalent in people from the Mediterranean region and middle east
Ø India, Central Asia, and Southeast Asia also have a high prevalence of thalassemia
Ø The highest prevalence of thalassemia is geographically located in the world where there is or was malaria.
The thalassemias"
α - thalassemia types: α+ and α0 β - thalassemia types: β+ and β0 δ - thalassemia types: δ+ and δ0 δβ - thalassemia types: (δβ)0, (Aγδβ)0
γ - thalassemia type: GAγ hybrid εγδβ - thalassemia types: (εγδβ)0
Thalassemia: Quantitative defects:
Reduced synthesis of a normal globin chain § α + or β + thalassemia"
Absent synthesis of a normal globin chain § α 0 thalassaemia or β 0 thalassaemia"
Unequal α and β globin chain production
What are α- and β- thalassemias ?"
Range of Clinical Symptoms § β-thalassemia minor"
-Asymptomatic to mild microcytic anemia
§ β-thalassemia intermedia"Range of symptoms
§ β-thalassemia major"Fatal if untreated Transfusion Dependency Hepatosplenomegaly Severe Anemia Skeletal Deformities
§ α-thalassemia trait (-α/αα) (-α/-α) (--/αα)"Asymtomatic
§ Hemoglobin H Disease (--/-α)"Chronic hemolytic anemia
§ Hydrops Fetalis"Stillbirth
Clinical Information: thalassemias " Transfusion therapy"
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β-thalassemia"
: α-Thalassemia : β-Thalassemia
: A group of inherited disorders in which globin chain synthesis is impaired.
Thalassemia
α β
δγ
Normal
~97% Hb A=α2β2 ~2.5% HbA2=α2δ2 ~0.5% Hb F=α2γ2
α β
δγ
α-Thalassemia
β4=HbH γ4=HbBart’s
α β
δγ
β-Thalassemia
Increased HbA2 (4-6%) Increased HbF (10-70%)
PATHOPHYSIOLOGY OF THALASSEMIA "
Thalassemia genes!Defective α- or β-chain synthesis, Excess α- or β-chains !
Inclusion bodies!Membrane damage!
Increased RBC destruction!Iron excess!Increased bilirubin!production
Hyperurcemia
RE hyperplasia!
Hepato-splenomegaly!
Anemia!
Massive erythropoiesis!
Hypersplenism!Extramedullary hematopoiesis!Jaundice!
Gallstones!Bone changes!Inadequate nutrition!
Defective development!
Iron overload! Increased infection!
Cardiac failure, Cirrhosis!
Blood transfusion!
Beta thalassemia "
§ Inherited mutation defect on beta gene § Mutation results in an either the absence or a reduction in the synthesis of b-globin chains:
β+ type: reduced expression of b-globin β0 type: no expression of b-globin
Causes an imbalance in the ratio of the amounts
of the two globin chains that make up a Hb"
β-thalassemia Genotypes"
Ø β-thalassemia TRAIT (β0 or β+ type) both types can be mild or severe
Ø β-thalassemia MAJOR (β0/β0 or β+/β+)
β+ is a severe type
Ø β-thalassemia Intermedia (β0/β+ or β+/β+)
β+ is a mild type (β+ +)
Phenotypes of β-thalassemia"
β+-thalassemia (trait)
Silent (normal MCH & normal HbA2) normal HbA2 (reduced MCH & normal HbA2) Mild (reduced MCH & raised HbA2) Severe (reduced MCH & raised HbA2)
β0-thalassemia (trait)
Mild (reduced MCH & raised HbA2) Severe (reduced MCH & raised HbA2) Dominant (reduced MCH & raised HbA2 , plus anemia and splenomegaly)
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There is great variety in β Thal alleles- and approx 20 diff alleles comprise 80% of the mutations worldwide.
Frequencies of common β-thalassemia mutations"
UK Romania Spain Greece Egypt Tunisia
Arab Penins
ula India China African
IVS110 (G→A) 5.5 34.7 10.2 42.5 28.5 20.0 CD 39 (C→T) 3.4 13.7 35.5 19.6 2.1 45.4 IVSI-6 (T→C) 2.2 17.0 9.4 8.4 19.7 1.7 IVSII-745 (C→G) 1.7 12.1 1.5 4.8 3.7 3.1 IVS1-1 (G→ A) 1.9 7.2 25.0 13.4 15.1 3.9 CD 8/9 (+G) 9.8 14.1 12.0 IVSI-5 (G→C) 22.5 32.4 619 bp deletion 1.7 13.1 IVSI-1 (G→T) 1.6 10.4 1.3 CD41/42 (-TCTT) 9.0 6.3 42.3 -28 (A→G) 0.7 12.4 CD17 (A→T) 1.0 16.6 CD 71/72 (+T) 0.1 5.8 IVSII-654 (C→T) 1.6 12.1 -29 (A→G) 2.3 2.3 68.4 -88 (C→T) 4.8 24.3 CD 24 (T→G) 0.2 2.7
α-thalassemia"
Alpha thalassemia"§ Inherited deletion or mutation defect on alpha genes "§ Deletion or mutation results in an either the absence or a reduction in the synthesis of α-globin chains:""
α + type: reduced expression of α-globin genes α 0 type: no expression of α-globin genes
§ Causes an imbalance in the ratio of the amounts of the two globin chains that make up a Hb (eg Hb A: α/β chains):"§ Excess of γ chains in the fetal stage can generate γ4 tetramers: Hb Bart’s"§ Excess of β chains in the adult stage can generate β4 tetramers: HbH""
Genetics of α-thalassemia "
Caused by the loss of both the α1 and the α2 genes on the same chromosome
5 common mutations: --SEA, --FIL, --THAI, & --MED, -α20.5
Two rare deletions have been described in Asian-Indian population
αo-thalassemia "
Genetics of α-thalassemia "
Ø Single gene deletion: 3.7kb (- α3.7) Mediterranean, Middle east, Africa, India 4.2kb (- α4.2) India, Southeast Asia, Pacific populations
Ø Non-deletion mutation on either α1 or α2 gene 60 mutations – some result in hyper-unstable globins
α+-thalassemia "
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Hb Constant Spring"
Ø non-deletional form of α-thalassemia"Ø α2 globin allele contains a point mutation "Ø the globin chains αCS is elongated by 31 amino acids"Ø Hb Constant Spring is highly unstable"Ø HbCS in heterozygotes constitutes only 1-2% of the " total hemoglobin"Ø HbCS interacting with deletional αo-thalassemia can " cause a severe HbH disease ( --/ αCS α)"
Alpha Thalassemia genetics "
• 4 alleles = Normal α
α α
α
1 gene deletion = silent carrier
α
α α
- • asymptomatic • may see small amount of Hb Bart’s (γ4) at birth • normal red cell indices – normal looking morphology • detected only by DNA methods
Alpha Thalassemia genetics "
2 gene deletion = α-thalassemia trait
• mild anemia • small red blood cells (low MCV) • Hb Bart’s at birth • difference between cis and trans becomes important when considering inheritance
α
α -
-
α
-
α
-
CIS
TRANS
Alpha Thalassemia genetics "
3 gene deletion = hemoglobin H disease
• moderate to marked anemia • very small red blood cells • Hb Bart’s (γ4) present at birth, replaced by HbH (β4)
4 gene deletion = hydrops fetalis
α
- -
-
-
-
-
-
• extreme anemia produces congestive heart failure, edema in utero • stillbirth or early neonatal death
Risk for HbH"
A pregnancy is at risk for HbH disease (3 gene deletion) if:
a) One parent has 1 α gene deletion and the other parent has 2 α gene deletion (CIS or α°-thalassemia heterozygote)
α
α -
-
α
α α
- +"
or"b) One parent has 1 α gene deletion and the other parent has HbH disease
+"α
- -
-
α
α α
-
α
α -
-
Risk for Hydrops Fetalis"
A pregnancy is at risk for Hydrops Fetalis if:
a) Both parents have a 2 α gene deletion (CIS or α°-thalassemia heterozygote)
α
α -
- +"
or"
b) One parent has 2 α gene deletion (CIS)and the other parent has HbH disease
+"α
- -
-
α
α -
-
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Alpha thalassemia hematology "
Red Blood Cell Index
Normal Affected Carrier
Male Female Hb Bart’s Hydrops Fetalis
HbH disease α-thalassemia trait (-α/-α)1
α-thalassemia silent carrier
Mean Corpuscolar volume (MCV,fL ) 89.1+/-5.01 87.6+/-5.5 136+/-5.1
56+/-5 (children) 61+/-4 (adults) 71.6+/-4.1 81.2+/-6.9
Mean Corpuscolar hemoglobin (MCH, pg)
30.9+/-1.9 30.2+/-2.1 31.9+/-9 18.4+/-1.2 22.9+/-1.3 26.2+/-2.3
Hemoglobin (Hb,dL) 15.9+/-1.0 14.0+/-0.9 3-8
M10.9+/-1.0 F9.5+/-0.8
M13.9+/-1.7 F12.0+/-1.0
M14.3+/-1.4 F12.6+/-1.2
§ Galanello R. ; Cao A. ; Last update : Nov 2005 ; Alpha-Thalassemia In: GeneReviews at GeneTests: Medical Genetics Information Resource (database online).
Copyright, University of Washington, Seattle. 1997-2008. Available at http://www.genetests.org. Accessed [5-26-08].
Red Blood Cell Indices in Adults with Alpha-Thalassemia§
(1) α-thalassemia carriers with the ( - -/ α α ) genotype have slightly lower RBC indices.
Alpha thalassemia hemoglobin types "
Hemoglobin Type Normal
Affected Carrier
Hb Bart’s Hydrops Fetalis
HbH disease α-thalassemia trait or α°-thalassemia
α-thalassemia silent carrier or α+-thalassemia
HbA 96-98% 0 60-90%) 96-98% 96-98%
HbF < 1% 0 <1% <1% <1%
Hb Bart’s 0 85-90% 2-5% 0 0
HbH 0 0 0.8-40% 0 0
HbA2 2-3% 0 <2.0% 1.5-3% 2-3%
§ Galanello R. ; Cao A. ; Last update : Nov 2005 ; Alpha-Thalassemia In: GeneReviews at GeneTests: Medical Genetics Information Resource (database online). Copyright, University of Washington, Seattle. 1997-2008. Available at http://www.genetests.org. Accessed [5-26-08].
Hemoglobin Patterns in Alpha-Thalassemia (After 12 Months of Age) §
Newborns chromatograms on HPLC"
Affected"Healthy"
Hb Bart’s"
HbH" δβ-thalassemia & HPFH phenotypes"
δβ – thalassemia" Reduced MCH Hb F 4-20% Heterocellular
Deletion HPFH" Normal MCH Hb F 20-35% Pancellular
Non - deletion HPFH" Normal MCH Hb F 3-35% Heterocellular
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HPFH - Hereditary Persistance of Fetal hemoglobin § Deletional and non-deletional "
"(deletional can have a Hb F of 25-35%)"§ Cellular distribution (pancellular or homocellular)"§ D-10 (A1c) can report A1c in the presense of Fetal hemoglobin up to 10%. Using the Dual Mode, the c/o for Fetal is 17%."
HPFH"Location and size of deletion mutants of "
hereditary persistence of fetal hemoglobin (HPFH)
5’ 3’
HPFH-1 HPFH-2 HPFH-3 HPFH-4 HPFH-5 HPFH-6
-20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
ε Gγ Aγ ψβ δ β
Prenatal Diagnosis"Significant hemoglobinopathies that require prenatal diagnosis by DNA analysis:
Sickle cell diseases: Hb SS, SD-Punjab, SC, SO-Arab β-thalassemia major β-thalassemia intermedia (severe genotypes) HbE/ β-thalassemia HbS/ β-thalassemia α-thalassemia
– Hb Bart’s hydrops fetalis syndrome – Hb H hydrops fetalis syndrome
Alpha Thalassemia genetics "
4α genes Normal
α
α α
α - α / - α
2α genes deleted α+-thal homozygote
- α/αα 1α genes deleted
α+-thal heterozygote
- - / - α 3α genes deleted Hb H disease
- - / α α 2α genes deleted
αo-thal heterozygote
- - / - - 4 genes deleted Hydrops fetalis
Alpha thalassemia main deletions:
α -3.7 Kb
α -4.2 Kb α+ thalassemias αο thalassemias del SEA
del MED
α
α α
-
α
-
α
-
α
α -
-
α
- -
-
αα/αα
- α / α α
- α / - α
-- / α α
- α / --
-- / --
4
3
2*
2*
1
0
Normal
Normal
MCV, MCH
MCV, MCH
HbH disease
Hb Barts hydrops fetalis
α-thalassemia"
Genotype No. Genes Functional
Phenotype
Normal RBCs Alpha-thal RBCs Beta-thal RBCs
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Normal Thalassemia
RED BLOOD CELLS" THALASSEMIA "RED BLOOD CELLS"
Normal RBCs
Beta-thal minor RBCs
Beta-thal major RBCs
• Separates Hbs based on charge differences • Positively charged Hbs are separated by their absorption on a negatively charged stationary phase in a column • The cations (positive charge) in the mobile phase (buffers with increasing ionic strength) compete with the absorbed Hbs eluting them off • The fractions are detected optically by a spectrophotometer that measures the concentration of Hgb in each fraction which is quantified by calculating the area under the peaks
High Performance Liquid Chromatography (HPLC)"
High Performance Liquid Chromatography (HPLC)"
Polyaspartic acid column bound to a silica gel support, producing a weak cation exchange resin Two mobile phases (buffers) produce gradient of increasing ionic strength Blending of the phases, the flow rate and the physical characteristics of the hemoglobin determine the retention time on the column Elution from column measured at 415 nm. The resulting chromatograph is integrated and the % hemoglobins calculated
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HPLC controls
A S F A2
Manufacturer-Assigned Windows"
P1-Window 0.63 - 0.85 minutes F-Window 0.98 - 1.20 minutes P2-Window 1.24 - 1.40 minutes P3-Window 1.40 - 1.90 minutes A0-Window 1.90 - 3.10 minutes A2-Window 3.30 - 3.90 minutes D-Window 3.90 - 4.30 minutes S-Window 4.30 - 4.70 minutes C-Window 4.90 - 5.30 minutes
Healthy Adult
αAγA αAδA αAβA
Dx: Hb S trait
67 y.o. M RBC 4.67 MCV 87.2
HB 13.6 MCH 29.2
HCT 40.7 RDW 14.8
αAγA αAδA
αAβA αAβS
2 d.o. F RBC 4.89 MCV 112.2
HB 18.0 MCH 36.9
HCT 54.9 RDW 17.8
6 m.o. F RBC 4.38 MCV 96.7
HB 13.6 MCH 31.0
HCT 42.4 RDW 16.1
Dx: Healthy Newborn Dx: Newborn with Hb SS disease
αAγA
αAβS αAβA
αAγA
Dx: Hb E trait
18 y.o. F RBC 5.0 MCV 74.0
HB 12.4 MCH 24.8
HCT 37.0 RDW 12.8
αAγA
αAδA
αAβA αAβE
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26 M RBC 3.77 MCV 97.4
HB 12.8 MCH 29.4
HCT 36.8
Infant Father Mother
Dx: Hb E trait
αAγA αAβE
αAγA
αAβA
αAβE αAγA αAβA
αAβE
Dx: Hb D-‐Punjab Trait
26 y.o. F RBC 3.81 MCV 83.8
HB 11.2 MCH 29.3
HCT 32.0 RDW 13.0
αAγA αAδA
αAβA αAβD
Dx: Hb D-‐Punjab trait + α-‐thal
1 y.o. M RBC 4.95 MCV 60.6
HB 10.5 MCH 19.7
HCT 32.0 RDW 12.8
αAγA αAδA
αAβA
αAβD
α+ + β+ slow α+ + β-‐ normal α+ + β2-‐ fast
Bunn and Forget, Hemoglobin: Molecular, GeneYc and Clinical Aspects, 1986
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