the diagnosis of haemoglobinopathies from protein to - enerca

The diagnosis of haemoglobinopathies from protein to genome

Béatrice Gulbis, MD, PhD

Hôpital Erasme – Université Libre de Bruxelles

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Objectives of laboratory tests

• Prevention • Screening for haemoglobinopathies > genetic counselling

• Diagnostic purposes: symptomatic patient • Chronic haemolytic anaemia

– Sickle cell disease and syndromes

– Thalassaemia or

– Unstable Hb variant

• Polycythaemia

– Hb variants of high oxygen affinity

• Cyanosis

– M Haemoglobin

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Available tools?

History Physical examination

Laboratory

tests

Diagnosis of haemoglobinopathies

from protein to genome

First step

Clinical settings

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Clinical settings

• Diagnosis of patients

(Healthy carriers)

• Family history (recessive vs dominant)

• Population at risk (origin, inbreeding)

• Symptoms/signs

• Acute and/or chronic

• Age at first symptoms/signs

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Age at first onset

2 1 5’

3’ 16:

11: 3’

5’

Hb F

Hb A (95-98%)

Hb A2 (2-3.5%)

Haeme Haeme

Haeme Haeme

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Population at risk

Immigration and mixity?

Neonatal screening: universal or targeted?

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Physical exam(symptomatic patients)

Palor

Icterus

Splenomegaly

Hepatomegaly

Hb at high oxygen affinity Hb M Frequent …



First step

Clinical settings

Second step

First line laboratory

tests

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Laboratory tests

• Complete blood count

• Blood smear

• Reticulocytes (absolute count, index)

• Indices of haemolysis

• Separation and quantitation of Hb fractions

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Hb fractions

• Available methods

– Electrophoresis

– Chromatography

– Mass spectrometry

– Others

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Hb fractions

• Each technique is based on a different

principle of separation of Hb fractions

Variable sensitivity and specificity

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Hb fractions

Isoelectric focalisation

N

A F

S

C A2

No quantification

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Hb fractions (quantification)

Automated capillary electrophoresis

Hb A Hb X (Z5 zone)

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High performance chromatography

Hb A Hb X (S-window)

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Chromatography: HbA1c methods

Hb X Hb X (S-window)

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Hb fractions

Mass spectrometry

Trypsin

digest

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Hb fractions

• Available methods

– Electrophoresis

– Chromatography

– Mass spectrometry

– Others

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Presence of Hb S?

• Emmel test (sodium metabisulfite)

– One drop of blood and hypoxia generatted by 2% Na MB.

• Itano test (degree of solubility of Hb) – Hb S precipitates in the presence of Na dithionite – Phosphate

buffer

Characterization of the Hb variant: Why?

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A

F

X

S

A

F

X

S

Hb S Korle-Bu

Asymptomatic Hb SD Punjab

Sickle cell disorder

"A2

window"

"D-window"

• Homozygous

– Hb SS

• Heterozygous

– Hb SC

– Hb SDPunjab

– Hb SOArab

– …

– Hb Sthalassemia

• Heterozygous

– Hb ASAntilles

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Influence of HbS polymerization

• Increase

– Hb C

– Hb D Punjab

– Hb O Arab

– ...

• Decrease

– Hb F

– Hb Korle-Bu

– Hb G Philadelphia

– ...

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Quantify the variant: Why?

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A

F S

S.M. (29/07/1996): Hb AS ?

S

56%

F

25%

A

13%

Hb fractions

1 • Separation and quantification of Hb fractions

2

• Presence of Hb variant

• Using a second method of different separation principle

3 • Give a conclusion

• Interpretation of the different techniques used

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External Quality Control

• ENERCA recommendations

– Recommendations for preconceptional or

antenatal screening, prenatal diagnosis and

genetic counselling of haemoglobinopathies.

• European Molecular Genetics Quality

Network – Best practice (2002 and under

revision)

– Best Practice Guidelines for carrier

identification and prenatal diagnosis of

haemoglobinopathies -

http://www.emqn.org/emqn/Best+Practice

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First step

Clinical settings

Second step

First line laboratory tests

Third step

Specialized tests

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Laboratory tests

Additional tests

• Haemoglobinopathy

– Phenotype:

• Separation/quantification of Hb fractions

• Hb variant: characterization

• Blood smear: specific staining

• OxyHb: dissociation curve

• Hb M: absorption spectrum

– Genotype

Additional tests

• p50Hb variant

dissociative curve

p50 calculation http://www.ventworld.com/res

ources/oxydisso/oxydisso.htm

l

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Additional tests

• Unstable Hb variant?

– Isopropanol test

Unstable Hb variant: Flocculation

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Additional tests

• Presence of Hb H (alpha-thalassaemia)?

– Brilliant cresyl blue staining

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Additional tests

*

• Separation of globin chains

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Laboratory tests

Additional tests

• Haemoglobinopathy

– Phenotype:

– Genotype

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Molecular analysis

14.0 kb

10.3 kb

/ / -3.7

2

1

• Southern blot

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Molecular analysis

• MLPA (Multiplex Ligation-dependent Probe Amplification)

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Molecular analysis

• Sequencing

Next generation sequencing

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- Gene panel sequencing

- Exomes

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When to make the genotype?

• Diagnosis of

– -thalassaemia

– - thalassaemia (minor)

– Unusual Hb variant

– ALWAYS in the context of a prenatal

diagnosis

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Home message

Based on the history and physical examination,

diagnostic approach in the laboratory:

Define the type of anemia and / or corpuscular

abnormality index

Establish the differential diagnosis

Achieve adequate complementary tests

Separation and quantification of Hb fractions are requested

At least two different principle of separation or identification

External quality control requested

Interpretation of the tests is helpful for the clinician

Be careful if based on a HbA1c method

Genotype is not always requested

Ackowledgment

• Belgian HematologicalSociety – Committee Red Blood Cells Disorders

www.bhs.be

• http://www.erasme.ulb.ac.be/files/files/Chimie/bilan_depistage_neonatal_hbpathies_2012.pdf

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the diagnosis of haemoglobinopathies from protein to - enerca

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