D. Kieffer 2013-2014
Hemoglobinopathies: introduction
Hemoglobin
• Adult (normal) Hb = 2 x alpha + 2 x beta = α2β2
• Chromosome 11: 1 x beta globin genes
• Chromosome 16: 2 x alpha globin genes
2
Hemoglobin
3
HbA: ± 97% HbF: < 1% HbA2: 2,5-3,5%
Hemoglobinopathies
• Thalassaemia:
– Primary abnormality: reduced synthesis rate of globin chains
– Defined by imbalanced αβ ratio
• HbH = β4 (α-thalassemia)
• Hb Bart’s = γ4 (α-thalassemia)
• Insoluble aggregates of α globin chains (β-thalassemia)
– Traditionally though not invariably microcytic hypochromic anemia
• Variant hemoglobins:
– Structural abnormality in the globin chain
– Functional abnormality
Hemoglobinopathies
• Thalassaemia: – Primary abnormality: reduced synthesis rate of globin chains
– Defined by imbalanced αβ ratio
– Traditionally though not invariably microcytic hypochromic anemia
• Variant hemoglobins: – Structural abnormality in the globin chain
• (single/multiple) point mutation, deletion, fusion, chain elongation
– Functional abnormality
• Changes in quaternary structure, solubility changes, changes of accessibility of heme,….no functional changes (= silent mutants)
– Normal αβ ratio normocytic, normochromic anemia
Hemoglobinopathies
6
Thalassemia Variant hemoglobin
α-thalassemia (α+, α0) β-thalassemia (β+, β0) δβ-thalassemia/HPFH
δ-thalassemia
HbS (βS) HbE (βE) HbD (βD) HbC (βC)
HbO-arab (βO-arab) Other (α, β or δ)
Hemoglobinopathies in the world
• Most common autosomal reccessive, monogentic disorders:
– WHO: +/- 5% of world population is carrier
– Carrier status for both partners
severe hemoglobinopathy = 25%
severe hemoglobinopathy = ca. 300.000 annualy (β-thal, HbS)
• Distribution ~ due to partial protection for carriers from malaria
7
Hemoglobinopathies in the lab
• Indications for lab testing
– Screening
– Opportunistic testing
– Monitoring of known disease
8
Hemoglobinopathies in the lab
9
• Techniques
1. Routine setting 1st line (screening)
2nd line (confirmation)
presumptive diagnosis (reliable) EMQN: for all samples, screening using biochemical tests precedes genetic testing
2. Expert setting
definite diagnosis
3. Research setting
EMN 2002 Best Practice Guidelines
Hemoglobinopathies in the lab
10
• Techniques
1. Routine setting 1st line (screening)
2nd line (confirmation)
presumptive diagnosis (reliable) EMQN: for all samples, screening using biochemical tests precedes genetic testing
2. Expert setting
definite diagnosis
3. Research setting
EMN 2002 Best Practice Guidelines
• Information that should be provided
– Name
– Date of birth
– Ethnic origin
– Reason for testing
– Pregnancy status
– Recent blood transfusion (date, # units,…)
– Relevant drug therapy (Hydrea®;…)
– Family history (cave: non paternity?)
Hemoglobinopathies in the lab
11 Bain 2010, Variant Haemoglobins 1st edition
Routine setting
• CBC is a necessary part of diagnosis
especially in case of thalassemia, except in neonatal screening:
– Hb
– RBC (Fe deficiency vs thalassemia)
– MCV (< 79 fl, <24h)
– MCH (< 27 pg, 5d at 4°C)
– RDW (Fe deficiency vs thalassemia)
– (retics)
Hemoglobinopathies in the lab
12
Routine setting
Bain 2010, Variant Haemoglobins 1st edition Bain 2006, Haemoglobinopathy Diagnosis 2nd edition BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines
• Fe-status as a supplement to the CBC
especially in case of thalassemia (cave pregnancy)
Hemoglobinopathies in the lab
13
Routine setting
Iron Deficiency Thalassemia
Hb N or ↓ N or ↓
RBC ↓ ↑
MCV ↓ ↓
RDW ↑↑ ↑
Ferritin ↓ ↑ or N Bain 2006, Haemoglobinopathy Diagnosis 2nd edition BCSH 2010 guidelines (BJH, 2010; 149: 35-49)
• Hb analysis techniques:
Simplified interpretation
Hemoglobinopathies in the lab
14
Routine setting
Hb analysis Interpretation
HbA2 < 2,5% α-thalassemia
HbA2 > 3,5% β-thalassemia
HbF > 1% δβ-thalassemia or HPFH
• Hb analysis techniques:
Simplified interpretation
Hemoglobinopathies in the lab
15
Routine setting
Hb analysis Interpretation
HbA2 < 2,5% α-thalassemia
HbA2 > 3,5% β-thalassemia
HbF > 1% δβ-thalassemia or HPFH
= tekort α-ketens voorkeur α2β2 ipv α2δ2 verlaging HbA2
• Hb analysis techniques:
Simplified interpretation
Hemoglobinopathies in the lab
16
Routine setting
Hb analysis Interpretation
HbA2 < 2,5% α-thalassemia
HbA2 > 3,5% β-thalassemia
HbF > 1% δβ-thalassemia or HPFH
= tekort β-ketens overschot α-ketens verhoging α2δ2 (HbA2) ( verhoging α2 2 (HbF)
• Hb analysis techniques:
Simplified interpretation
Hemoglobinopathies in the lab
17
Routine setting
Hb analysis Interpretation
HbA2 < 2,5% α-thalassemia
HbA2 > 3,5% β-thalassemia
HbF > 1% δβ-thalassemia or HPFH
= tekort β- en δ-ketens overschot α- en -ketens verhoging HbF
• Hb analysis techniques:
“Complicated and CBC-integrated” interpretation
Hemoglobinopathies in the lab
Routine setting
Red cell indices Hb analysis Interpretation
Reduced Normal -Fe deficiency -α-thal trait -Mild β-thal trait - co-inheritance δ- and β-thal - γδβ-thal trait
Normal/reduced HbA2 > 3,5% Interaction α- and β-thal
Normal/reduced HbF > 1% (γ)δβ-thal or HPFH
Normal borderline HbA2 α-triplication, mild β-thal
Severely reduced HbA2 > 3.5% Multiple α-genes and β-thal trait
• Hb analysis techniques:
Hemoglobinopathies in the lab
Routine setting
HbA2 measured on Bio-Rad Variant HPLC in 100
normal non iron depleted or -thal. individuals and in
large cohorts of patients (NT = 701).
P.C. Giordano, Hemoglobinopathies Laboratory
Hemoglobinopathies in the lab
• Hb analysis techniques:
Cellulose acetate electrophoresis pH 8.4-8.6
– Identification of:
• HbA, HbF, HbS/D, HbC/E/A2/O-arab + others
• HbH, Hb Bart’s
– Advantages
• Simple, reliable, rapid, inexpensive
– Disadvantages
• No differentiation between HbS & HbD, HbC & HbE, HbC & HbO-arab
• No HbA2 quantification
• Time consuming when large number of samples
Hemoglobinopathies in the lab
BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines
Routine setting
• Hb analysis techniques:
Cellulose acetate electrophoresis pH 8.4-8.6
Hemoglobinopathies in the lab
21
Routine setting
C, A2, O, E
S,D F A
• Hb analysis techniques
Acid agarose/citrate agar electrophoresis pH 6.0
– Only useful in selected cases
– Identification of:
• Differentiation of HbC, HbE & HbO-arab from each other
• Differentiation of HbS & HbDPunjab
Hemoglobinopathies in the lab
22
Routine setting
BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines
• Hb analysis techniques
Acid agarose/citrate agar electrophoresis pH 6.0
Hemoglobinopathies in the lab
23
Routine setting
C S
F
A, D, E, A2
• Hb analysis techniques
CE-HPLC
– Identification + quantification of:
• HbA, HbF, HbS, HbA2, HbC, HbD, HbG, HbO-Arab + others
– Advantages
• HbA2 quantification
• Quantification of all fractions on every sample
• Automation and small sample volumes
• Provisional identification of many variant Hbs
• δ-chain variant detection possible
– Disadvantages
Hemoglobinopathies in the lab
Routine setting
BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines
• Hb analysis techniques
CE-HPLC
– Identification + quantification of:
• HbA, HbF, HbS, HbA2, HbC, HbD, HbG, HbO-Arab + others
– Advantages
– Disadvantages
• Co-elution of HbE, HbA2 and Hb Lepore
• Less reliable results for HbH and Hb Bart’s
• Separation of glycosylated & derivative forms: difficult interpretation
• Careful examination of each chromatogram (column T°C or flow rate)
• Daily check of screening windows
• Daily calibration + controls
Hemoglobinopathies in the lab
Routine setting
BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines
• Hb analysis techniques
CE-HPLC
Hemoglobinopathies in the lab
26
Routine setting
F
A
A2
• Hb analysis techniques
CZE
– Identification + quantification of:
• HbA, HbF, HbS, HbA2, HbE, HbC, HbD, HbG + others
– Disadvantages
• No detection of HbO-arab
• If HbA is not present (homozygous HbS, HbC, HbE): no zones
• HbA2% not reliable when HbC present
Hemoglobinopathies in the lab
Routine setting
BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines
• Hb analysis techniques
CZE
Hemoglobinopathies in the lab
28
Routine setting
• UZ Leuven Practice
Analyse op maandag en donderdag
– praktische uitvoering en ingave resultaten door MLT
– preliminaire interpretatie GASO • nazicht chromatogrammen (correct %, elutietijd,…)
• Interpretatie ikv
– klinische inlichtingen
– historiek
– leeftijd
– supervisie D. Kieffer • aanwezig op maandag (preliminaire protocols rond 15u te bespreken)
Hemoglobinopathies in the lab
Routine setting
Hemoglobinopathies in the lab
• cases
30
HbC
• UZ Leuven Practice
Hemoglobinopathies in the lab
31
Routine setting
blanco (2x) kalibratie
• UZ Leuven Practice
Hemoglobinopathies in the lab
32
Routine setting
lage controle hoge controle
• UZ Leuven Practice
Hemoglobinopathies in the lab
33
Routine setting
normaal
HbA: ± 97% HbF: < 2% HbA2: 2,5-3,8%
Ref. waarden UZ Leuven
• UZ Leuven Practice
Hemoglobinopathies in the lab
34
Routine setting
HbA2: < 2,5%
Weatherall, DJ. Nature Reviews Genetics 2001;2(4):245-55.
normaal
-α/αα (α+ -heterozygoot): geen symptomen
--/αα (αo -heterozygoot): milde anemie
-α/-α (α+ -homozygoot): geen symptomen
--/-α (HbH-ziekte): microcytaire anemie
--/-- (Hb Bart’s Hydrops fetalis) †
• UZ Leuven Practice
Hemoglobinopathies in the lab
Routine setting
• UZ Leuven Practice
Hemoglobinopathies in the lab
36
Routine setting
HbA2: > 3,8%
Weatherall, DJ. Nature Reviews Genetics 2001;2(4):245-55.
• UZ Leuven Practice
Hemoglobinopathies in the lab
37
Routine setting
HbA2: > 3,8% HbF: > 2,0%
Weatherall, DJ. Nature Reviews Genetics 2001;2(4):245-55.
• UZ Leuven Practice
Hemoglobinopathies in the lab
38
Routine setting
D. Rund & E. Rachmilewitz, NEJM, 353, 1135-1146 (2005)
• UZ Leuven Practice
Hemoglobinopathies in the lab
39
Routine setting
HbS: 33,4%
HbS: • most commmon Hb variant • position 6 β-chain: Glu Val • polymerisation at low PO2
• RBC deformation ~ sickle cells – vaso-occlusion, infarctions – shortened RBC lifespan – anemia
o destruction RBC o reduced O2-affinity
• UZ Leuven Practice
Hemoglobinopathies in the lab
40
Routine setting
HbS: 33,4%
• UZ Leuven Practice
Hemoglobinopathies in the lab
41
Routine setting
HbS: 33,4%
HbS-trait: • +/- 35-40% HbS • normal Hb • normal MCV and MCH
HBS-trait + alfa-thalassemia • < 35% HbS • Hb slightly reduced • MCV and MCH reduced
– cave: Fe-deficiency
• UZ Leuven Practice
Hemoglobinopathies in the lab
42
Routine setting
HbS: 33,4%
HbS and HbA2%: • HbA2% not reliable on HPLC • post-translational HbS-derivatives • correction: (HbA2%-(2,3% of HbS))
• UZ Leuven Practice
Hemoglobinopathies in the lab
43
Routine setting
HbS: 33,4%
Confirmation: • HbS solubility or “Itano” test • > 10-20% HbS
• UZ Leuven Practice
Hemoglobinopathies in the lab
44
Routine setting
HbS: 74,6%
HbS homozygosity: = sickle cell anaemia = sickle cell disease
• UZ Leuven Practice
Hemoglobinopathies in the lab
45
Routine setting
HbS: 74,6%
HbS homozygosity: • symptoms as early as 6 months old • bone infarctions • infarction internal organs (lungs) • cerebral haemorrhage/infarctions • splenomegaly/hypersplenism • hyposplenism: cave infections! • jaundice, gallstones • frontal bossing • … • median life expectancy: 42-48 y
• UZ Leuven Practice
Hemoglobinopathies in the lab
46
Routine setting
HbS: 74,6%
HbS homozygosity: • hydroxy-urea: increased HbF • regular/intermittent transfusions • stem cell transplantation
• UZ Leuven Practice
Hemoglobinopathies in the lab
47
Routine setting
HbS: 74,6%
HbS homozygosity: • normal blood count at birth (HbF) • Hb 6-10 g/dL (HbF) • reticulocytosis (inefficient) • normal MCV and MCH (HbF) • RDW increased • sickle cells, target cells, HJ-bodies
• UZ Leuven Practice
Hemoglobinopathies in the lab
48
Routine setting
HbS: 74,6%
HbS homozygosity: • absence of HbA • variable % of HbF:
• normal: 5-10% • haplotype
• Arab-Indian: 10-25% • Senegal: 7-10% • Bantu & Benin: 6-7% • Cameroon: 5-6%
• therapy-induced: up to 20-25% • coinheritance of HPFH: 20-30% • remains high in infancy (3 years) • higher in women
• UZ Leuven Practice
Hemoglobinopathies in the lab
49
Routine setting
HbS: 74,6%
S/beta thalassemia: • S/beta0 thalassemia
• milder syndrome • Hb higher than HbSS • MCV, MCH reduced • elevated HbA2
• family study or DNA study
• UZ Leuven Practice
Hemoglobinopathies in the lab
50
Routine setting
HbS: 62,7%
S/beta thalassemia: • S/beta+ thalassemia
• milder symdrome than S/beta0 • Hb higher than S/beta0 • HbF: 5-15% • MCV, MCH reduced (<S/beta0) • elevated HbA2 • HbA present (up to 45%) • HbS > 50%
• cave: transfusion!
Hemoglobinopathies in the lab
• cases
51
HbC
• UZ Leuven Practice
Hemoglobinopathies in the lab
52
Routine setting
HbA2: 85,5%
2,5-3,8%: normal 3,8-8,0%: beta thalassemia 8-15%: Hb-Lepore? 15%-30%: HbE-trait 85-99%: HbEE
• UZ Leuven Practice
Hemoglobinopathies in the lab
53
Routine setting
HbA2: 85,5%
• UZ Leuven Practice
Hemoglobinopathies in the lab
54
Routine setting
HbA2: 85,5%
HbE: • beta-chain variant: Glu Lys • second most important variant Hb
• UZ Leuven Practice
Hemoglobinopathies in the lab
55
Routine setting
HbA2: 85,5%
HbE trait • beta-chain variant: Glu Lys • asymptomatic • normal to slightly reduced MVC/MCH • normal to slightly anemic • = normal or thalassemic indices
• HbE is unstable: < 30% • HbE/beta thal: > 39% • HbE/alpha thal: < 25%
• positive heat/isopropanol test
• UZ Leuven Practice
Hemoglobinopathies in the lab
56
Routine setting
HbA2: 85,5%
HbE-homozygosity (HbEE) • asymptomatic (HbE disease obsolete) • thalassemic indices and blood count • resembles beta-thalassemia trait
HbE/beta thalassemia • more severe clinical picture • beta thalassemia intermedia/major • MCV, MCH more reduced than HbEE
Hemoglobinopathies in the lab
• cases
57
HbC
• UZ Leuven Practice
Hemoglobinopathies in the lab
58
Routine setting
HbC: 27,5%
HbC: • beta chain variant: Glu Lys • third most common Hb variant
• UZ Leuven Practice
Hemoglobinopathies in the lab
59
Routine setting
HbC: 27,5%
HbC-trait (HbAC) • no clinical significance • genetic counseling (HbC/S) • normal blood count and Hb • microcytosis?
• HbC +/- 45% • HbC/alpha: < 35%
• UZ Leuven Practice
Hemoglobinopathies in the lab
60
Routine setting
HbC: 27,5%
HbC-homozygosity (HbCC) • HbC +/- 95% • clinically mild
• increased frequency of gall stones • splenomegaly
• chronic haemolytic anemia • Hb: 8 g/dL-normal • often microcytosis and MCHC
• activation K/Cl cotransporter • dehydration • irregularly contracted cells
• UZ Leuven Practice
Hemoglobinopathies in the lab
61
Routine setting
HbC: 27,5%
HbC/beta talassemia • resembles thal intermedia
• splenomegaly • hypersplenism • moederately severe anemia
• Hb: 7-10 g/dL • microcytosis
• difficult DD HbCC
Hemoglobinopathies in the lab
• cases
62
HbC
• UZ Leuven Practice
Hemoglobinopathies in the lab
63
Routine setting
HbD: 37,7%
HbD-Punjab/D-Los Angeles: • beta chain variant: Glu Gln • Fourth most important Hb variant • Sikhs population in the Punjab • only importance: interaction HbS • normal stability
• UZ Leuven Practice
Hemoglobinopathies in the lab
64
Routine setting
HbD: 37,7%
HbD trait: • clinically normal • normal blood count/blood film • HbD: slightly less than 50%
• UZ Leuven Practice
Hemoglobinopathies in the lab
65
Routine setting
HbD: 37,7%
HbD homozygosity: • clinically mild • mild haemolytic anemia • Hb: 9-10 g/dL – normal • thalassemic indices
• cave DD HbD/beta thalassemia
HbD/beta thalassemia: • mild thalassemic condition • microcytosis • Hb: 10,5 g/dL - normal
• UZ Leuven Practice
Hemoglobinopathies in the lab
66
Routine setting
HbD: 37,7%
Hemoglobinopathies in the lab
• cases
67
HbC
• UZ Leuven Practice
Hemoglobinopathies in the lab
68
Routine setting
HbA2’ (HbB2): 1,3%
HbA2’: • 1% of black population
• elution in HbS window
• +/- equal amount as HbA2
• clinically not significant
Hemoglobinopathies in the lab
• cases
69
HbC
• UZ Leuven Practice
Hemoglobinopathies in the lab
70
Routine setting
cord blood
Variant detection • HbS, HbC, HbD, HbE, HbO-arab
• maternal contamination?
• HbF < 65%
• HbA > 30%
• HbA2 > 0,5%
maternal contamination
• UZ Leuven Practice
Hemoglobinopathies in the lab
71
Routine setting
• UZ Leuven Practice
Analyse op maandag en donderdag
– praktische uitvoering en ingave resultaten door MLT
– preliminaire interpretatie GASO • nazicht chromatogrammen (correct %, elutietijd,…)
• Interpretatie ikv
– klinische inlichtingen
– historiek
– leeftijd
– supervisie D. Kieffer • aanwezig op maandag (preliminaire protocols rond 15u te bespreken)
Hemoglobinopathies in the lab
Routine setting
Top Related