SUPPORTING INFORMATION
EMQN Best Practice Guidelines for molecular and haematology methods for
carrier identification and prenatal diagnosis of the haemoglobinopathies
Joanne Traeger-Synodinos1, Cornelis L Harteveld2, John M. Old3, Mary Petrou4, Renzo
Galanello5, Piero Giordano2, Michael Angastioniotis6, Barbara De la Salle7, Shirley
Henderson3, Alison May8, on behalf of contributors to the EMQN haemoglobinopathies
best practice meeting.
1. Department of Medical Genetics, University of Athens, Choremeio Research
Laboratory, St Sophia's Children's Hospital, Athens 11527, Greece.
2. Department of Human and Clinical Genetics, Leiden University Medical Center,
Einthovenweg 20, 2333ZC Leiden, The Netherlands
3. National Haemoglobinopathy Reference Laboratory, Molecular Haematology, L4,
John Radcliffe Hospital, Oxford, OX3 9DU, UK.
4. Haemoglobinopathy Genetics Centre, University College London Hospitals NHS
Foundation Trust and Institute of Women’s Health, University College London, 8696 Chenies Mews, London WC1 E6HX, UK.
5. Ospedale Regionale Microitemie, Via Jenner (sn), 09121 Cagliari, Italy
6. Thalassaemia International Federation, PO Box 28807, 2083 Nicosia, Cyprus.
7. UK NEQAS, General Haematology, PO Box 14, WD18 0FJ, UK.
8. Department of Haematology, Cardiff University Medical School, University Hospital
of Wales, Heath Park, Cardiff, UK.
1
FIRST-LINE HAEMATOLOGICAL METHODS
Haemoglobin (Hb) pattern analysis
The methods for a presumptive identification of abnormal haemoglobins include

Haemoglobin electrophoresis at pH 8.6 using cellulose acetate membrane. This
method will reliably detect the common haemoglobin variants, ie Hb’s S, C, DPunjab, E, O-Arab and the Lepore Hbs. Hb H and Hb Barts may also be detected if
suitable run times are used. Many other variants are also detectable, eg Hb’s J’s,
N’s, Q’s, other Ds and Gs and Hasharon.

Haemoglobin electrophoresis at pH 6.0 using acid agarose or citrate agar gel. This
method is useful in selected cases for distinguishing Hb’s C, E, and O-Arab from
each other, also Hb S and Hb D-Punjab from each other. (Of note is that the
migration patterns are different for acid agarose or acid citrate agar gels).

Isoelectric focusing (IEF). IEF is a sensitive method, giving good separation of
haemoglobin variants but requires considerably more expertise for interpretation
than electrophoresis since adduct fractions also separate.

High Performance Liquid Chromatography (HPLC) is a recommended method for
simultaneous automatic detection and quantitation of haemoglobin fractions. Since
systems are automated, operation of analysers is simple. However, interpretation of
the chromatograms requires expertise. Also, attention must be paid to quality
control, especially for measurement of Hb A2. (16;17;53).

Capillary electrophoresis (CE) is a high through-put, automated alternative to
alkaline electrophoresis, providing separation and quantitation of normal and
abnormal Hb fractions.(16-18)
2
Recommendations for application and interpretation of haemoglobin pattern
analysis

When a haemoglobin variant is detected further separation tests are recommended
to confirm its identity. However, second or even third line separation procedures do
not always ensure identification for all variants. In these cases molecular (DNA)
analysis is the only method that ensures identification.

On most HPLC systems, derivatives of Hb S, and other similarly-eluting
haemoglobin variants, may co-elute with Hb A2 resulting in an overestimation of
the Hb A2 level (3.5-4.5%). In such cases the peak value should not be reported as a
percentage of Hb A2.

Measuring Hb A2 levels in the presence of Hb S and at least 50% Hb A (if no recent
transfusion history) is of no diagnostic value, since the presence of more than 50%
Hb A logically excludes co-existing β-thalassaemia. For the same reason Hb A2
estimation in the presence of  50% Hb A and Hb C, D, E, O, or any other variant is of no diagnostic value.

When a high percentage of Hb A2 is found in the presence of Hb S, but in the
absence of Hb A and in the presence of microcytosis, this may indicate either Hb
S/βo-thalassaemia or homozygous Hb S and -thalassaemia. In such cases family
and/or molecular analysis are recommended. ,

There are rare sickling variants (currently 11 known) with an additional amino acid
substitution to that of Hb S that mostly do not migrate or elute like Hb S (Table
2supp) and are only detectable at the protein level if a sickle solubility test is used
for all variant haemoglobins of any significant quantity (>20% total).
3

A sickle solubility test (see Supporting Information) can be used to confirm the
presence of Hb S if suspected (54).

When Hb H disease or other unstable haemoglobin variants are suspected, always
analyse fresh blood samples.

It is important to inspect carefully the chromatogram or electropherogram before
the results are authorized to check for baseline, peak resolution and overlapping
peaks.

An alkaline denaturation test may confirm the presence of significantly (>5%)
increased Hb F

DCIP (2.6 dichlorophenolindophenol)) test (see Second-line haematology tests) can
be used to screen for Hb E (28).

When the presence of a high affinity but electrophoretically silent Hb variant is
suspected, oxygen dissociation analysis may be useful, but definitive identification
is only achieved by molecular (DNA) analysis.
Quantification of Hb A2
Methods include (16;20;21):

Electrophoresis and elution, which is accurate but time-consuming.

Microchromatography, which is accurate but time-consuming.

HPLC which is accurate and high-throughput.

Capillary electrophoresis which is accurate and high throughput.

Hb electrophoresis with automatic densitometry, which is not recommended.
Quantification of Hb F
Methods for measuring Hb F levels include:
4
 Alkali denaturation - The modified method of Betke (55)
has excellent
reproducibility, at low values of Hb F (<10%), giving worthwhile results in virtually
all clinical situations. However, it is technically exacting and is not often used.
 HPLC (17) and CE are much more accurate although some devices may overestimate
Hb F due to overlap with the first glycated fraction.
NOTE: some variant haemoglobins, or adducts of chromatographically silent
variants may co-elute with Hb F and cause false elevations, so confirmatory tests
may be required. The use of standards and internal controls are recommended (16).
Iron (Fe) status
There are several parameters that can be measured to evaluate the the iron status of an
individual, see below:
a) Zinc protoporphyrin (ZnPP). For this method the sample can be analysed from the
same tube as the blood count. Analysis is simple and fast, although it requires a
specific instrument. Zinc protoporphyrin is elevated in iron deficiency, but may be
falsely high in lead intoxication or if the bilirubin levels are raised. Some studies
have shown increased levels in thalassaemia carriers with normal iron levels and
varying degrees of discrimination between uncomplicated heterozygous thalassaemia
and iron deficiency have been reported.
b) Serum ferritin measurements are most commonly used for indicating iron deficiency,
but may be falsely elevated during infection, inflammation, liver disease or
neoplasia.
5
c) Transferrin saturation measures serum iron compared to the total iron binding
capacity (Fe/TIBC). However, serum iron may be falsely low in infection and
inflammation despite normal iron stores..
SECOND-LINE HAEMATOLOGICAL METHODS
There are a number of additional methods that may be useful for investigating unusual
samples that do not have a definite diagnosis with conventional methods.
Red Cell Morphology (RCM)
Thalassaemia carriers will present with classic target cells, microcytes, fragmentocytes,
basophilic stippling and anisopoikylocytosis.
Reticulocytes and F-cells can be counted using flow cytometry (25) or on a slide as a
smear (14).
Single tube osmotic fragility test (OF).
This easy and cheap method is largely used in emerging countries doing population
screening but can be very useful also in the modern laboratory. (25) .
Inclusion body test for alpha thalassaemia
Carriers of alpha thalassaemia with genotypes or combined genotypes causing loss of
about half the usual alpha globin production have a very small number of red blood
cells (often<1/5000) that contain sufficient HbH to precipitate on incubation with
certain oxidative dyes and be visualized on smears under the microscope. This test has
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to be carried out on freshly-taken blood and 150 fields may have to be scrutinized. It
has high specificity, giving a rapid confirmation of alpha thalassaemia for some making
it a valued and useful test. It is not sensitive enough to be used either for all types of
alpha thalassaemia or to exclude heterozygous alpha zero thalassaemia. Furthermore, it
cannot distinguish between the various genotypes.
Globin chain synthesis
This was one of the first methods used to support differential diagnosis of globin gene
disorders. However it is quite a cumbersome, time consuming method involving the use
of radio-isotopes. Nevertheless, it may provide useful information for diagnosing in
selected atypical cases. Once radioactively labelled the haem-free denatured and
solubilised globin chains can be separated for quantification by a number of methods:
a) CMC chromatography method. - Very accurate but time consuming method for
evaluating relative rate of globin chains synthesised in reticulocytes.
b) HPLC – Potentially a less time consuming method, but it needs careful
standardization to be accurate and reliable.
c) IEF- rapid and convenient, with potential to process multiple samples
simultaneously (27) .
Globin chain separation
Can be undertaken either by HPLC or IEF, and is useful for indicating which globin
chain is affected, thus giving evidence for the nature and potential significance of the
variant(s) present.
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Functional tests for Hb S
If there is an abnormal fraction that runs in the position of Hb S, then the variant can be
confirmed without molecular analysis, either using one of the commercially available
solubility tests or the sickling test.
Note: the solubility test may produce false positive results in the presence of elevated
Hb F or with unstable haemoglobin variants that usually do not migrate or elute on the
same position as Hb S. On the other hand there are 11 rare sickling variants with an
additional amino acid substitution to that of Hb S that mostly do not migrate or elute
like Hb S (Table 2Supp).
Mass spectrometry
Specialized method based on analysing tryptic digests of whole blood. Although there
may be only a small shift in mass for some common variants, it is very effective for the
characterisation of Hb variants especially if used in conjunction with other methods
(29). However this method may identify the specific amino-acid change but does not
necessarily confirm the DNA sequence variation or, in the case of duplicated genes such
as - or -, the gene involved
DCIP (2.6 dichlorophenolindophenol) test
The DCIP test is also, available in a kit form, can be used for Hb E carrier screening
whenever more complex and expensive tests are not readily available (28).
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Heinz body formation
This is not a very specific, but useful for detecting the presence of some unstable
variants
Oxygen dissociation curve
The measurement of the oxygen dissociation curve may be useful for confirming the
presence of Hb variants with altered oxygen affinity. However, the abnormal fraction
should ideally be purified first, the measurement is complex and few laboratories are
equipped to do it. Pragmatic alternatives are the p50 saturation measurement in the
presence of erythrocytosis and elevated PCV for variants with increased O2 affinity or
some degree of cyanosis in carriers of variants with reduced O2 affinity.
9
Table 1supp. Traditional and HGVS nomenclature of globin gene variants
referred to in these guidelines
Traditional nomenclature
HGVS nomenclature
Variants involving HBB gene 1
Hb Lepore Hollandia
NG_000007.3:g.63290_70702del
Hb Lepore-Baltimore
NG_000007.3:g.63564_70978del
Hb Lepore-Boston-Washington
NG_000007.3:g.63632_71046del
Hb C
c.19G>A
Hb C-Harlem
c.[20A>T;220G>A]
Hb C-Ndjamena
c.[20A>T;112T>G]
Hb C-Ziguinchor
c.[20A>T;176C>G]
Hb DPunjab
c.364G>C
Hb E
c.79G>A
Hb Hope
c.410G>A
Hb I-Toulouse
c.199A>G
Hb Jamaica Plain
c.[20A>T;205C>T]
Hb North Shore.
c.404T>A
Hb OArab
c.364G>A
Hb O-Tibesti
c.[34G>A;364G>A]
Hb Quebec-Chori
c.263C>T
Hb S
c.20A>T
Hb S-Antilles
c.[20A>T;70G>A]
Hb S-Clichy
c.[20A>T ;26A>C]
Hb S-Cameroon
c.[20A>T;271G>A]
Hb S-Oman
c.[20A>T;364G>A]
Hb S-Providence
c.[20A>T;249G>T or 249G>C]
Hb Shelby
c.394C>A
Hb S-South End
c.[20A>T;399A>C]
Hb S-Travis
c.[20A>T;428C>T]
10
 -101 (CT)
c.-151C>T
 -92 (CT)
c.-142C>T
 +33 (CG)
c.-18C>G
IVS2-844 (CG)
c.316-7C>G
 +1480 (CG)
c.*6C>G
Cap+1 (AC)
c.-50A>C
 IVS1-6 (TC)
c.92+6T>C
Variaants involving HBA1 or HBA2 genes 2
--FIL
NG_000006.1:g.11684_43534del31851
--MEDI
NG_000006.1:g.24664_41064del16401
--SEA
NG_000006.1:g.26264_45564del19301
--THAI
NG_000006.1:g.10664_44164del33501
-()20.5
NG_000006.1:g.15164_37864del22701
-3.7 (Type I)
NG_000006.1:g.34164_37967del3804
-3.7 (Type II)
Not defined
-3.7 (Type III)
Not defined
-4.2
Not defined
Hb Adana
c.179G>A (HBA2 or HBA1)
Hb Agrinio
c.89T>C
Hb Constant Spring
c.427T>C
Hb Hasharon
c.142G>C
Hb Taybee
c.118_120delACC (HBA1)
2 gene variant at the initiation codon
c.2T>C
(e.g. ATGACG
IVS1 donor site (-GAGGT- 5 base pair
c.95+2_95+6delTGAGG
deletion)
2 gene polyadenylation signal
c.*93_*94delAA
11
AATAAA->AATA- 2 gene polyadenylation signal
c.*92A>G
AATAAA->AATGAA
2 gene polyadenylation signal
c.*94A>G
AATAAA->AATAAG
1
NM_000518.4<http://www.ncbi.nlm.nih.gov/nuccore/NM_000518.4> HBB;
http://www.ncbi.nlm.nih.gov/nuccore/NG_000007.3
2
http://www.ncbi.nlm.nih.gov/nuccore/NG_000006.1 NM_000517.4
<http://www.ncbi.nlm.nih.gov/nuccore/NM_000518.4> HBA2; NM_000558.3 HBA1.
All variants are on the HBA2 gene unless stated otherwise between brackets.
12
Table 2supp. The sickling variants with two amino acid substitutions
Haemoglobin
Amino acid substitution
HPLC
Hb S-South End
beta 6(A3) GluVal & beta 132(H10)
Runs with Hb A
LysAsn
Hb S-Antilles
beta 6(A3) GluVal & beta 23(B5) ValIle
Separate peak
Hb C-Ziguinchor
beta 6(A3) GluVal & beta 58(E2) ProArg
Unknown
Hb C-Harlem
beta 6(A3) GluVal & beta 73(E17) AspAsn Runs with Hb A2
Hb S-Providence
beta 6(A3) GluVal & beta 82(EF6) LysAsn
Hb S-Travis
beta 6(A3) GluVal & beta 142(H20) AlaVal Separate peak
Hb C-Ndjamena
beta 6(A3) GluVal & beta 37(C3) TrpGly
Separate peak
Hb S-Oman
beta 6(A3) GluVal & beta 121(GH4)
Separate peak
Separate peak
GluLys
Hb S-Clichy
Beta6(A3) GluVal & beta 8(A5)LysThr]
Separate peak
Hb S-Cameroon
beta 6(A3) GluVal & beta 90(F6) GluLys
Separate peak
Hb Jamaica Plain
beta 6(A3) GluVal & beta 68(E12) LeuPhe
Runs with Hb S
13
Table 3supp. Known causes underlying Hb A2 levels outside the normal range
Increase (excluding -thalssaemia variants)
Reduction
Genetic
Genetic
KLF1 variants
-thalassaemia
Triplicated  gene
-chain variants
Some unstable variants
-chain variants
Hb variants eluting with or close Hb A2
Hb Lepore1
-thalassaemia2
 and  thalassaemia; some mild 
thalassaemia variants
Acquired
Acquired
Hyperthyroidism
Severe iron deficiency anaemia
Megaloblastic anaemia
Sideroblastic anaemia
Aplastic crisis in HS
Lead poisoning
Antiretroviral drugs
Leukaemia, aplastic anaemia
Pseudoanthoma elasticum
Note 1: a) In Hb Lepore carriers, the expected parameters are: 2.0-2.5% Hb A2, 1-3%
Hb F, 3-5% Hb Lepore-Boston-Washington. However, falsely high Hb A2 levels may
be observed (up to 10-15%) when using HPLC, since Hb Lepore co-elutes with Hb A2.
Note 2: Co-inheritance of -thalassaemia, particular Hb H disease may reduce Hb A2
levels.
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