Hemoglobinopathies: introduction

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

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Hemoglobin

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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

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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

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Hemoglobinopathies in the lab

• Indications for lab testing

– Screening

– Opportunistic testing

– Monitoring of known disease

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• 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


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• 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?)


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)


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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)


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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


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Routine setting

Hb analysis Interpretation

HbA2 < 2,5% α-thalassemia

HbA2 > 3,5% β-thalassemia

HbF > 1% δβ-thalassemia or HPFH




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Routine setting





= tekort α-ketens voorkeur α2β2 ipv α2δ2 verlaging HbA2




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Routine setting





= tekort β-ketens overschot α-ketens verhoging α2δ2 (HbA2) ( verhoging α2 2 (HbF)




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Routine setting





= tekort β- en δ-ketens overschot α- en -ketens verhoging HbF


“Complicated and CBC-integrated” interpretation


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



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



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


BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines

Routine setting


Cellulose acetate electrophoresis pH 8.4-8.6


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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


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Routine setting



Acid agarose/citrate agar electrophoresis pH 6.0


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Routine setting

C S

F

A, D, E, A2


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


Routine setting



CE-HPLC


• 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


Routine setting



CE-HPLC


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Routine setting

F

A

A2


CZE


• 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


Routine setting



CZE


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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)


Routine setting


• cases

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HbC



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Routine setting

blanco (2x) kalibratie



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Routine setting

lage controle hoge controle



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Routine setting

normaal

HbA: ± 97% HbF: < 2% HbA2: 2,5-3,8%

Ref. waarden UZ Leuven



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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) †



Routine setting



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Routine setting

HbA2: > 3,8%




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Routine setting

HbA2: > 3,8% HbF: > 2,0%




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Routine setting

D. Rund & E. Rachmilewitz, NEJM, 353, 1135-1146 (2005)



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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



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Routine setting

HbS: 33,4%



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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



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Routine setting

HbS: 33,4%

HbS and HbA2%: • HbA2% not reliable on HPLC • post-translational HbS-derivatives • correction: (HbA2%-(2,3% of HbS))



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Routine setting

HbS: 33,4%

Confirmation: • HbS solubility or “Itano” test • > 10-20% HbS



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Routine setting

HbS: 74,6%

HbS homozygosity: = sickle cell anaemia = sickle cell disease



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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



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Routine setting

HbS: 74,6%

HbS homozygosity: • hydroxy-urea: increased HbF • regular/intermittent transfusions • stem cell transplantation



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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



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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



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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



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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!


• cases

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HbC



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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



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Routine setting

HbA2: 85,5%



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Routine setting

HbA2: 85,5%

HbE: • beta-chain variant: Glu Lys • second most important variant Hb



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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



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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


• cases

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HbC



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Routine setting

HbC: 27,5%

HbC: • beta chain variant: Glu Lys • third most common Hb variant



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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%



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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



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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


• cases

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HbC



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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



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Routine setting

HbD: 37,7%

HbD trait: • clinically normal • normal blood count/blood film • HbD: slightly less than 50%



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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



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Routine setting

HbD: 37,7%


• cases

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HbC



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Routine setting

HbA2’ (HbB2): 1,3%

HbA2’: • 1% of black population

• elution in HbS window

• +/- equal amount as HbA2

• clinically not significant


• cases

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HbC



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Routine setting

cord blood

Variant detection • HbS, HbC, HbD, HbE, HbO-arab

• maternal contamination?

• HbF < 65%

• HbA > 30%

• HbA2 > 0,5%

maternal contamination



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Routine setting


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)


Routine setting

Hemoglobinopathies: introduction

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