hemoglobinopathies: routine lab work-up and beyond - … · 2013-09-04 · nhs sickle cell &...

D. Kieffer 16-04-2013

Hemoglobinopathies: routine lab work-up and beyond

Outline

• Introduction: hemoglobin and its disorders

• Routine diagnostic setting

• Expert diagnostic setting

• Research diagnostic setting

Hemoglobin

• Quaternary structure = 4 x heme + 4 globin chains • Heme = Fe2+ + protoporfyrin IX

Hemoglobin

HbA: ± 97% HbA2: 2,5-3,5% HbF: < 1%

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

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 HbVar: A database of Human Hemoglobin Variants & Thalassemias http://globin.cse.psu.edu/globin/hbvar/menu.html

Hemoglobinopathies • α-Thalassemia:

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) †

Hemoglobinopathies

• β-Thalassemia:

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

Hemoglobinopathies in the world

• Most common autosomal recessive, monogenetic disorders: – WHO: +/- 5% of world population is carrier – Carrier status for both partners

severe hemoglobinopathy = 25% severe hemoglobinopathy = ca. 300.000 annually (β-thal major, HbS)

• Distribution ~ due to partial protection for carriers from malaria

Hemoglobinopathies in the world

Lopez at al. Gene 2010; 467: 1-12

Hemoglobinopathies in common practice

• N-E patients: ca. 1/1300 carrier

• N-E patients with distant foreign ancestry: ca. 1/50 carrier

• Recent immigrants: ca. 1/15 carrier – Mediterranean, Turkey, Morrocco, Africa, Far-Middle-East,… – α-thal, β-thal, HbS, HbC, HbE, HbD,...

• Chinese/E-A patients: ca. 1/30 carrier

– α-thal, β-thal

www.hbpinfo.com – LUMC Leiden

Hemoglobinopathies in the lab

• Indications for lab testing – Screening – Opportunistic testing – Monitoring of known disease

BCSH 2010 guidelines (BJH, 2010; 149: 35-49)

• Indications for lab testing – Screening

• Pre-conceptual • Antenatal • Neonatal • Pre-operative/pre-anaesthesia

– Opportunistic testing – Monitoring of known disease

~ risk group vs universal

• Pre-conceptual – When?

» Best option, less time dependence – Who?

» Recommended in risk groups, infertility, assisted conception – How?

» Minimal analysis: not specified » If abnormality is found partner testing (cfr. NHS guidelines)

• Antenatal • Neonatal • Pre-operative/pre-anaesthesia

• Pre-conceptual • Antenatal NHS Sickle Cell & Thalassemia screening programme 2009

– When? » Before week 11-12

– Who? » All, but different stratification according to risk group (FOQ)

– How? » Low prevalence: CBC + FOQ ↔ High prevalence: CBC + Hb-analysis + FOQ » If abnormality is found partner testing (maternal/paternal/fetal DNA testing)

• Neonatal • Pre-operative/pre-anaesthesia

• Pre-conceptual • Antenatal • Neonatal

– Who? » UK: All newborn babies » Other: selective » Universal: > 16 carriers/1000 births » Universal: > 0.5 SCD/1000 births

– What? » Focus on SCD and β-thal major. Full list:

– How? » Dried blood spot at 5-8d post partum » Cord blood also possible

• Pre-operative/pre-anaesthesia – Opportunistic testing – Monitoring of known disease BCSH 2010 guidelines (BJH, 2010; 149: 35-49)

B. Gulbis. Training course on hemoglobin disorders 2011

– Who? » All pts from high prevalence HbS origin

– When and How? » Routine operations: CBC + Hb-analysis during pre-assessment visit » Emergency : CBC + sickle solubility test

• Indications for lab testing – Screening – Opportunistic testing

• Red cell indices or morphological appearances suggestive of Hbpathy – Ic microcytosis: tests for Fe-deficiency, anemia of chronic disease and Hbpathy (if risk

group) – Unexplained microcytosis even if red cell indices are not typical (comorbidity?).

• GP, medical specialist or lab (reflex testing) – Monitoring of known disease

• Indications for lab testing – Screening – Opportunistic testing

• Indications for lab testing – Screening – Opportunistic testing – Monitoring of known disease

• SCD patients receiving blood transfusion % HbS • SCD or β-thal intermedia patients receiving Hydrea® % HbF

• Techniques

1. Routine setting = hematological/biochemical testing

2. Expert setting = molecular/DNA analysis

3. research setting = protein/(tandem)MS analysis

• 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

EMQN 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?)

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 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)

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:

“Complicated and CBC-integrated” interpretation

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

• Morphology (desirable):

– Sickle cells (HbS) – HbC crystals (HbC) – Irregularly contracted cells (HbC, HbE, unstable Hb) – Basophilic stipling (thalassemia, HbCs) – Polychromasia (HbH, unstable Hb) – Striking poikilocytosis (thalassemia, HbH disease) – …

Bain 2010, Variant Haemoglobins 1st edition Bain 2006, Haemoglobinopathy Diagnosis 2nd edition BCSH 2010 guidelines (BJH, 2010; 149: 35-49)

• Morphology (desirable):

HbC crystal Sickle cell β-thalassemie major - target cells - teardrop cells - aniso/poikilo: +++

What do we want to know? – Variant Hb?

• HbS, HbC, HbE, HbD-Punjab, Hb-O-arab – % of HbF? – % of HbA2?

Hb analysis Interpretation

HbA2 < 2,5% α-thalassemia

HbA2 > 3,5% β-thalassemia

HbF > 1% δβ-thalassemia or HPFH

Routine setting

HbA2 measured on Bio-Rad Variant HPLC in 100 normal non iron depleted or α-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

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

Acid agarose/citrate agar electrophoresis pH 6.0

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

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

CE-HPLC

IEF – Identification of:

• HbA, HbF, HbS, HbA2, HbC, HbD, HbE, HbO-arab – Advantages

• Semi-automated (relatively long run-times) • Identification of more variants than CAE • Small sample volume (or eluate from dried blood spot)

– Disadvantages • Quantification of HbA2 not validated (CV > 20%) • Separation in post-translational derivatives: difficult interpretation

F. Cotton Enerca training days

Routine setting

CZE – Identification + quantification of:

• HbA, HbF, HbS, HbA2, HbE, HbC, HbD, HbG + others – Advantages

• Quantification of HbA2 and HbF • HbE separation from HbA2 • Reliable HbA2% when HbS present (PTM fractions under main peak) • Easy-to-use system, high through-put automation, multi-use • Detection of minor variants • Reliable detection of HbH and Hb Bart’s • No interference of bilirubin

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

• Functional tests:

Sickle cell or “Emmel” test – Drop of blood mixed with 2% Na-metabisulphite – Between glass slide and cover slip – Sealed with molten paraffin wax – Microscopic evaluation (24hrs)

Not very suitable in routine lab

Bain 2006, Haemoglobinopathy Diagnosis 2nd edition

Sickle solubility or “Itano” test – Sodium dithionite lowers oxygen tension – High phosphate buffer solution – formation of HbS crystals that refract light

HbS > 10 – 20%

heat test or isopropanol test – Use

• Detection of unstable hemoglobins • Heat test more sensitive than isopropanol test

– Pitfalls • Aged samples of high HbF% false positive results • Positive and negative controls necessary

Heinz body formation unstable variants (not specific)

DCIP HbE carrier screening osmotic fragility test thalassemia screening oxygen dissociation curve altered oxygen affinity variant

• Other:

microcolumn chromatography HbA2 alkali denaturation HbF Kleihauer Betke HbF cells (HPFH vs δβ-thal) Immunoassay HbS, HbC, HbE, HbA (> 10%) Flowcytometry HbF cells globin chain electrophoresis α- or β-chain abnormality spectrometry (absorbance) methemoglobins

• Molecular/DNA analysis Southern blotting

PCR-based techniques (among others) – Allele Specific Oligonucleotide hybridization (ASO) – Amplification Refractory Mutation System (ARMS) – Restriction-endonuclease PCR (RE-PCR) – gap-PCR – Multiplex Ligation-dependent Probe amplification (MLPA) – Denaturing gradient gel electrophoresis (DGGE) – Single-strand confirmation polymorphism (SSCP)

Hemoglobinopathies in the lab Expert setting

Harteveld et al. Clin Biocem 2009; 42: 1767-1779 EMQN 2002 Best Practice Guidelines

• Techniques ~ DNA defects:

α-thalassemia β-thalassemia Hb-variants

Harteveld et al. Clin Biocem 2009; 42: 1767-1779

α-thalassemia: deletions > point mutations α2, α1: highly homologous unequal crossing over

α-thalassemia: deletions > point mutations

α+-thalassemia (African, Mediterranian)

α0-thalassemia (Asian)

α-thalassemia: deletions > point mutations gap-PCR (known) MLPA (known & unknown)

α-thalassemia: deletions > point mutations gap-PCR (known)

Molecular Genetics and Metabolism, 2010; 100: 51-56

α-thalassemia: deletions > point mutations multiplex gap-PCR 7 most common deletions - α3.7 African, Mediterranian, (Southeast) Asian - α4.2 Southeast Asian and Pacific - - SEA Southeast Asian - - MEDI Mediterranian - (α)20.5 Mediterranian - - FIL Fillipino - - THAI Thai

α+-thalassemia

α0-thalassemia ~ 80%

α-thalassemia: deletions > point mutations multiplex gap-PCR 7 most common deletions

Clark et al. Clin Lab Haem 2004; 26: 159-176

α-thalassemia: deletions > point mutations MLPA (known & unknown)

α-thalassemia: deletions > point mutations αT

α2 > α1

αT more severe α+

α-thalassemia: deletions > point mutations αT

- Hb Constant Spring (Asian)

- multiple αT2 (Mediterranian)

α-thalassemia: deletions > point mutations Direct sequencing

RE-PCR (HbCS) (ASO, ARMS)

β-thalassemia: deletions < point mutations known mutations

RE-PCR, ASO, ARMS unknown mutations Direct sequencing

Global migration

β-thalassemia: deletions < point mutations Hb Lepore

δβ deletion HPFH 1/2/3

β-thalassemia: deletions < point mutations gap-PCR MLPA

Hb variants: typically point mutations (single AA substitutions)

> 1000 Hb variants clinically important: - Hb S ASO – ARMS – direct sequencing – RE-PCR - Hb C ASO – ARMS – direct sequencing - Hb E ASO – ARMS – direct sequencing – RE-PCR - Hb D-Punjab ASO – ARMS – direct sequencing – RE-PCR - Hb O-arab ASO – ARMS – direct sequencing – RE-PCR

Hb variants: typically point mutations (single AA substitutions)

> 1000 Hb variants other: direct sequencing of α- or β-chain (γ- or δ-chain)

• Other potential techniques?

DNA analysis: RT-PCR, QMPSF, micro-array…

Mass spectrometry (MS)

Hemoglobinopathies in the lab Research setting

• Rationale for MS?

detection of charge difference Gel electrophoresis, IEF, CZE,… CE-HPLC (affinity stationary poly asp phase) detection of mass to charge ratios of ionized molecules

detection of charge difference – Kleinert et al. 2008:

• Known variants with ∆ charge overrepresented in HbVar database • Only 5 of 20 AA contain basic or acidic side chain • Estimation of potential variants (known + unknown) ~ DNA • Calculation of pI and differentiation charged vs neutral variants

56% undetected

32% undetected

71% undetected

32% undetected

92% detectable by MS

• RIZIV - INAMI – Identificeren en doseren van HbF door alkaliresistentie B200 – Fotometrische dosage HbA2 B400

– Elektroforese (alkalische pH) opsporen Hbpathie B500 – Chromatografie opsporen Hbpathie B500 – Isoëlektrische focalisatie opsporen Hbpathie B500

– Identificatie abnormaal Hb door 2e elektroforese B400

– Identificatie HbS en HbD door oplosbaarheidstest B100

– Aantonen instabiele Hb door hitte/isopropanol denaturatie B100 http://www.riziv.fgov.be/care/nl/nomenclature/

Geen cumul

• RIZIV - INAMI – Opzoeken van genetische anomalieën volgens de methoden van

hybridisatie van DNA-fragmenten B 8000

Indien via een CME, kost voor de patiënt +/- 10 euro

http://www.riziv.fgov.be/care/nl/nomenclature/

Questions?

The end

hemoglobinopathies: routine lab work-up and beyond - … · 2013-09-04 · nhs sickle cell &...

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