hemoglobinopathies: routine lab work-up and beyond - … · 2013-09-04 · nhs sickle cell &...
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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)
Hemoglobinopathies in the lab
• Indications for lab testing – Screening
• Pre-conceptual • Antenatal • Neonatal • Pre-operative/pre-anaesthesia
– Opportunistic testing – Monitoring of known disease
~ risk group vs universal
BCSH 2010 guidelines (BJH, 2010; 149: 35-49)
Hemoglobinopathies in the lab
• Indications for lab testing – Screening
• 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
– Opportunistic testing – Monitoring of known disease
BCSH 2010 guidelines (BJH, 2010; 149: 35-49)
Hemoglobinopathies in the lab
• Indications for lab testing – Screening
• 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
– Opportunistic testing – Monitoring of known disease
BCSH 2010 guidelines (BJH, 2010; 149: 35-49)
Hemoglobinopathies in the lab
• Indications for lab testing – Screening
• 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)
Hemoglobinopathies in the lab
• Indications for lab testing – Screening
• Pre-conceptual • Antenatal • Neonatal • Pre-operative/pre-anaesthesia
– Opportunistic testing – Monitoring of known disease
B. Gulbis. Training course on hemoglobin disorders 2011
Hemoglobinopathies in the lab
• Indications for lab testing – Screening
• Pre-conceptual • Antenatal • Neonatal • Pre-operative/pre-anaesthesia
– 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
– Opportunistic testing – Monitoring of known disease
BCSH 2010 guidelines (BJH, 2010; 149: 35-49)
Hemoglobinopathies in the lab
• 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
BCSH 2010 guidelines (BJH, 2010; 149: 35-49)
Hemoglobinopathies in the lab
• Indications for lab testing – Screening – Opportunistic testing
BCSH 2010 guidelines (BJH, 2010; 149: 35-49)
Hemoglobinopathies in the lab
• 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
BCSH 2010 guidelines (BJH, 2010; 149: 35-49)
Hemoglobinopathies in the lab
• Techniques
1. Routine setting = hematological/biochemical testing
2. Expert setting = molecular/DNA analysis
3. research setting = protein/(tandem)MS analysis
Hemoglobinopathies in the lab
• 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?)
Hemoglobinopathies in the lab
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)
Hemoglobinopathies in the lab 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:
“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
EMQ
N 2
002
Best
Pra
ctic
e Gu
idel
ines
• 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) – …
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)
• Morphology (desirable):
Hemoglobinopathies in the lab Routine setting
HbC crystal Sickle cell β-thalassemie major - target cells - teardrop cells - aniso/poikilo: +++
• Hb analysis techniques:
What do we want to know? – Variant Hb?
• HbS, HbC, HbE, HbD-Punjab, Hb-O-arab – % of HbF? – % of HbA2?
Hemoglobinopathies in the lab Routine setting
Hb analysis Interpretation
HbA2 < 2,5% α-thalassemia
HbA2 > 3,5% β-thalassemia
HbF > 1% δβ-thalassemia or HPFH
• Hb analysis techniques:
Hemoglobinopathies in the lab
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
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 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 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 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 Routine setting
• Hb analysis techniques
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
Hemoglobinopathies in the lab Routine setting
BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines
• Hb analysis techniques
IEF
Hemoglobinopathies in the lab
F. Cotton Enerca training days
Routine setting
• Hb analysis techniques
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
Hemoglobinopathies in the lab Routine setting
BCSH 2010 guidelines (BJH, 2010; 149: 35-49) EMQN 2002 Best Practice Guidelines
• 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 Routine setting
• Hb analysis techniques
CZE
Hemoglobinopathies in the lab Routine setting
• 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
Hemoglobinopathies in the lab Routine setting
Bain 2006, Haemoglobinopathy Diagnosis 2nd edition
• Functional tests:
Sickle solubility or “Itano” test – Sodium dithionite lowers oxygen tension – High phosphate buffer solution – formation of HbS crystals that refract light
Hemoglobinopathies in the lab Routine setting
HbS > 10 – 20%
Bain 2006, Haemoglobinopathy Diagnosis 2nd edition
• Functional tests:
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
Hemoglobinopathies in the lab Routine setting
Bain 2006, Haemoglobinopathy Diagnosis 2nd edition
• Functional tests:
Heinz body formation unstable variants (not specific)
DCIP HbE carrier screening osmotic fragility test thalassemia screening oxygen dissociation curve altered oxygen affinity variant
Hemoglobinopathies in the lab Routine setting
Bain 2006, Haemoglobinopathy Diagnosis 2nd edition
• 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
Hemoglobinopathies in the lab Routine setting
Bain 2006, Haemoglobinopathy Diagnosis 2nd edition
• 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
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations α2, α1: highly homologous unequal crossing over
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations
Hemoglobinopathies in the lab Expert setting
α+-thalassemia (African, Mediterranian)
α0-thalassemia (Asian)
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations gap-PCR (known) MLPA (known & unknown)
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations gap-PCR (known)
Hemoglobinopathies in the lab Expert setting
Molecular Genetics and Metabolism, 2010; 100: 51-56
• Techniques ~ DNA defects:
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
Hemoglobinopathies in the lab Expert setting
α+-thalassemia
α0-thalassemia ~ 80%
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations multiplex gap-PCR 7 most common deletions
Hemoglobinopathies in the lab Expert setting
Clark et al. Clin Lab Haem 2004; 26: 159-176
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations MLPA (known & unknown)
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations MLPA (known & unknown)
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations αT
α2 > α1
αT more severe α+
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations αT
- Hb Constant Spring (Asian)
- multiple αT2 (Mediterranian)
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
α-thalassemia: deletions > point mutations Direct sequencing
RE-PCR (HbCS) (ASO, ARMS)
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
β-thalassemia: deletions < point mutations known mutations
RE-PCR, ASO, ARMS unknown mutations Direct sequencing
Hemoglobinopathies in the lab Expert setting
Global migration
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
β-thalassemia: deletions < point mutations Hb Lepore
δβ deletion HPFH 1/2/3
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
β-thalassemia: deletions < point mutations gap-PCR MLPA
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
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
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• Techniques ~ DNA defects:
Hb variants: typically point mutations (single AA substitutions)
> 1000 Hb variants other: direct sequencing of α- or β-chain (γ- or δ-chain)
Hemoglobinopathies in the lab Expert setting
Harteveld et al. Clin Biocem 2009; 42: 1767-1779
• 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
Hemoglobinopathies in the lab Research setting
• Rationale for MS?
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
Hemoglobinopathies in the lab Research setting
• Rationale for MS?
detection of charge difference – Kleinert et al. 2008:
Hemoglobinopathies in the lab Research setting
56% undetected
• Rationale for MS?
detection of charge difference – Kleinert et al. 2008:
Hemoglobinopathies in the lab Research setting
32% undetected
71% undetected
• Rationale for MS?
detection of charge difference – Kleinert et al. 2008:
Hemoglobinopathies in the lab Research setting
32% undetected
92% detectable by MS
Hemoglobinopathies in the lab
• 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
Geen cumul
Hemoglobinopathies in the lab
• 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
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