DIAGNOSIS OF THE

THALASSAEMIA SYNDROMES:

MEASUREMENT OF HAEMOGLOBIN A2

Barbara Wild
UK National External Quality Assessment Scheme
London
 Globin biosynthesis
Hb F!
100! Hb A!


80!

%!


60!

40!

20! +



0!
3
6
Birth
3
6

The importance of Hb A2 measurement

Hb A2 is measured as a proportion of the total

haemoglobins present, not as an absolute amount

Hb A2 measurement is used as a marker for beta

thalassaemia trait. Carrier detection is important because:

Beta thalassaemia carriers are asymptomatic but

homozygous beta thalassaemia is a life-threatening
disorder
The importance of Hb A2 measurement
Accurate and reliable measurement of Hb A2 is essential
for the diagnosis of beta thalassaemia trait
Small difference (if any) between normal & abnormal levels

Antenatal women should be screened for beta

thalassaemia trait
Carriers: recommend partner testing
prediction of genetic risk

Failure to detect condition may result in newborn with a

medically significant condition
Screening for beta thalassaemia trait

Full blood count with red cell indices:

RBC, Mean Cell Volume and Mean Cell
Haemoglobin
Hb A2 %
Hb F %
Screen for haemoglobin variants
Iron status - ferritin, zinc protoporphyrin

Family history
 Measurement of Hb A2
Automated methods
High Performance Liquid Chromatography
Capillary electrophoresis
Mass spectrometry

Manual methods
Hb electrophoresis with elution
Microcolumn chromatography

Interpretation
Normal: 2.2-3.5% (usually<3.3%)
Beta thalassaemia trait: >3.5%
4
 High performance liquid chromatography
General principle
Utilises a weak cation-exchange column
Hb molecules adsorb onto the column saurated with
low ionic strength buffer
Buffer with increased ionic strength used to elute
haemoglobins from column
Haemoglobins will elute when ionic strength of
eluting solution exceeds that of the haemoglobins

Retention time of a particular haemoglobin is

characteristic and reproducible, but not unique
HPLC analysis - normal adult
Beta thalassaemia trait
Sickle cell trait
Hb S+thalassaemia
 chain variant

Consider total Hb A2
and
review red cell indices

Note:
also check for carry-over
Hb Lepore trait
Hb Kenya trait
Hb Fort Worth trait
Capillary electrophoresis
Utilises a thin capillary of silica, diameter approx 50-75m
Inner surface of the capillary has a negative charge
High voltage applied (10-30kv) capillary generates
endo-osmotic flow (EOF) towards cathode
Hbs separated because of different charges-fractions
move towards the cathode because of EOF

Electropherograms of peaks of a particular haemoglobin

is characteristic and reproducible, but not unique
19
Capillary electrophoresis
 High throughput haemoglobin variant
mutation analysis and protein biomarker
quantitation using dried blood spots

Neil Dalton, Charles Turner & Yvonne Daniel

The use of Mass Spectrometry for screening and
identification of the haemoglobinopathies

MS technique based on mass differences in globin chains

Initially used for identification of variants detected on

screening

Being developed as potential approach for

haemoglobinopathy screening
ESI-MS: normal whole blood

Original
spectrum

Deconvoluted
spectra
Electrospray ionisation mass spectrometry
Hb Johnstown
15126.6
Patient FP

15867.4

+14.1

High throughput haemoglobin variant mutation analysis and protein
biomarker quantitation using dried blood spots

Wild-type T1 VHLTPEEK
MW 951.5

Doubly charged peptide, m/z 476.8

Product ion (y4), m/z 502.3

Sickle T1 VHLTPVEK
MW 921.5

Doubly charged peptide, m/z 461.8

Product ion (y4), m/z 472.5
Wild-type T1 isolation
Sickle T1 isolation
 26

High throughput haemoglobin variant mutation analysis and

protein biomarker quantitation using dried blood spots
Protein/peptide quantitation: Antenatal screening for -
thalassaemia trait

HbA2 is about 2% of total haemoglobin

4% in -thalassaemia trait

HbA is 22








HbA2 is 22

Could the / ratio be used

as a biomarker for -thalassaemia trait?
What are the differences in the peptides?
 T1 T2 T3

Beta Val-His-Leu-Thr-Pro-Glu-Glu-Lys Ser-Ala-Val-Thr-Ala-Leu-Trp-Gly- Val-Asn-Val-Asp-Glu-Val-Gly-Gly-

Lys Glu-Ala-Leu-Gly-Arg
Delta Val-His-Leu-Thr-Pro-Glu-Glu-Lys Thr-Ala-Val-Asn-Ala-Leu-Trp-Gly- Val-Asn-Val-Asp-Ala-Val-Gly-Gly-
Lys Glu-Ala-Leu-Gly-Arg
T4 T5 T6

Beta Leu-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg Phe-Phe-Glu-Ser-Phe-Gly-Asp-Leu- Val-Lys

Ser-Thr-Pro-Asp-Ala-Val-Met-Gly-
Asn-Pro-Lys
Delta Leu-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg Phe-Phe-Glu-Ser-Phe-Gly-Asp-Leu- Val-Lys
Ser-Ser-Pro-Asp-Ala-Val-Met-Gly-
Asn-Pro-Lys
T7 T8 T9

Beta Ala-His-Gly-Lys Lys Val-Leu-Gly-Ala-Phe-Ser-Asp-Gly-

Leu-Ala-His-Leu-Asp-Asp-Leu-Lys
Delta Ala-His-Gly-Lys Lys Val-Leu-Gly-Ala-Phe-Ser-Asp-Gly-
Leu-Ala-His-Leu-Asp-Asp-Leu-Lys
T10 T11 T12

Beta Gly-Thr-Phe-Ala-Thr-Leu-Ser-Glu-Leu-His-Cys-Asp-Lys Leu-His-Val-Asp-Pro-Glu-Asn-Phe- Leu-Leu-Gly-Asn-Val-Leu-Val-Cys-

Arg Val-Leu-Ala-His-His-Phe-Gly-Lys
Delta Gly-Thr-Phe-Ser-Thr-Leu-Ser-Glu-Leu-His-Cys-Asp-Lys Leu-His-Val-Asp-Pro-Glu-Asn-Phe- Leu-Leu-Gly-Asn-Val-Leu-Val-Cys-
Arg Val-Leu-Ala-Arg
T13 T14 T15

Beta Glu-Phe-Thr-Pro-Pro-Val-Gln-Ala-Ala-Tyr-Gln-Lys Val-Val-Ala-Gly-Val-Ala-Asn-Ala-Leu- Tyr-His

Ala-His-Lys
Delta Asn-Phe-Gly-Lys Glu-Phe-Thr-Pro-Gln-Met-Gln-Ala- Val-Val-Ala-Gly-Val-Ala-Asn-Ala-Leu-
Ala-Tyr-Gln-Lys Ala-His-Lys
T16

Delta Tyr-His
Measurement of : globin peptide ratio
Samples subjected to tryptic digestion
Multiple Reaction Monitoring undertaken for
T2, T3 and T14 peptides
T2, T3 and T13 peptides
: peptide ratios calculated

Study validated the quantitative : globin peptide ratio as a

surrogate marker of Hb A2

Developed within concept of National Screening

Programme needs
Daniel et al 2007
Interpretation of Hb A2 levels
Hb A2 percentage is increased in:

Beta thalassaemia trait

Presence of an unstable haemoglobin
Hyperthyroidism
Some cases of congenital dyserythropoietic
anaemia, type I
HIV infection

Sickle cell trait or anaemia

 HPLC analysis sickle cell trait

Normal FBC



Hb S% : 35-45



Hb A2 may be raised

 Hb Yokohama trait

RB AS RB AC Dad SF Dad FA

FA Dad SF Dad AC RB AS RB

RB Dad AFSE RB Dad AA

Interpretation of Hb A2 values

Haemoglobin A2 percentage is decreased in

thalassaemia
Delta/beta thalassaemia
thalassaemia trait or haemoglobin H disease

Severe iron deficiency

National Sickle & Thalassaemia
Screening Programme
Established to provide a linked screening
programme for antenatal women and newborn
Universal screening
Established laboratory standards
Standardised reporting formats
Standardised methodology (newborn)

Decision algorithm (antenatal)

NSC&TSP: High Prevalence Screening FBC and HPLC

Hb Variant

HbS, HbC, HbD,

No variant
HbE, Hb OArab Other variant
Hb Lepore

refer to
Test partner Consultant
Haematologist*

MCH >= 27
MCH<27

HbA2 > 4.0 HbA2 =< 4.0

HbA2 >= 3.5 HbF > 5% orHbF > 5% HbF =< 5%
HbA2 < 3.5
beta thal trait

refer to
Consultant No further
Test partner MCH < 25 MCH >= 25 Test partner
Haematologist* action

Consider Iron deficiency

ethnic group alpha thal

High risk of low risk of No further

alpha zero alpha zero action***
thalassaemia** thalassaemia**

Test partner No further action

 From : Haemoglobinopathy diagnosis, BJ Bain

Silent thalassaemia trait (normal MCV, MCH, and Hb A2 %)

Almost silent thalassaemia trait (reduced MCV, MCH, normal Hb A2 %)

Indices typical of thalassaemia trait but Hb A2 % normal

 36

Risk assessment:
UK National screening programme

The following conditions will be missed:

Silent or near silent beta thalassaemia carrier
Possible beta thalassaemia carrier obscured by severe
iron deficiency
Alpha zero thalassaemia occurring outside of the
defined at-risk family origins
Dominant haemoglobinopathies where the woman has
no haemoglobinopathy
Any significant variant not detected by HPLC
Normal Hb A2 thalassaemia in Europe

Aim: To determine the extent of the problem

associated with normal Hb A2 thalassaemia
mutations

Subjects: 226 patients from Tunisia, Greece, Cyprus

and UK

Criteria for selection: Hb A2 values of 3.3-3.8%

Methods: Samples analysed by ARMS-PCR &

sequencing

Old et al , Ithanet project, International Thalassaemia

Normal Hb A2 thalassaemia in Europe

22 cases were outside of the average A2 and MCH

groups
Of these:
All of the IVS1-6 patients had a reduced MCH
10/13 of the CAP+1 patients had a reduced MCH

An additional 35 patients with Hb A2 values >3.5% gave

normal gene sequencing results
Hb A2 values of a standard -thalassaemia mutation
(IVSI-5 GC): 4.5% - 6.5%
7
6
5
4
No 3
2
1
0
5
5

5
3.

3.

4.

4.

4.

5.

5.

5.

6.

6.
% A2
Hb A2 values of an atypical -thalassaemia mutation
(CAP+1 AC)

16
14
12
10

No 8 CAP+1
6
4
2
0
1

5
3.

3.

3.

3.

3.

4.

4.

4.
% Hb A2
Average values

mutation cases Hb A2 MCH MCV

+1480 (CG) 18 2.9 28.2 89
-101 (CT) 42 3.8 29.0 89
CAP+1 (AC) 75 3.7 25.4 79
IVSI-6 (TC) 34 4.2 22.7 72
Poly A (AG) 10 3.9 24.7 76
Poly A (TC) 5 4.0 22.4 73
Poly A (-AT) 2 3.8 22.7 72
Poly A (-AA) 8 4.0 23.6 73
Patients with a raised Hb A2 and no -thalassaemia

25 patients had a normal -globin gene sequence

average values: Hb A2 MCH MCV
3.8 28.8 87

3 had MCH below 27 pg with normal -genotype

Possible causes: mutation in LCR or enhancer sequence

21 had a MCH above 27pg: Are these patients normal?

Possible known causes:
HIV drug treatment,
Hyperthyroidism
Is it the tail end of the range for normal individuals?
UKNEQAS:
UK National External Quality Assessment Scheme

Participants are required to give analytical results and an

interpretation

With increase in technologies:

Results of Hb A2 measurement related to
methodology used

Identified differences in values obtained from

different technologies and/or kits
Normal sample:Hb A2 2.6%
Beta thal trait sample: Hb A2 4.8%
Borderline sample: Hb A2 3.7%
Performance scoring for Hb A2:
Considerations for UKNEQAS

Use of different normal ranges

variation even within same instrument group

Use of a universal cut-off

Instrument bias impact on borderline values
 48

Measurement of Hb A2
ICSH recommendations ISLH Oct 2011
Previous ICSH recommendations written in 1978

Hb A2 is measured as a percentage of haemoglobin

present relative to any other haemoglobin present not an
absolute value

Therefore analytically important to measure the A2 and any

other fractions present separation, resolution and
integration crucial

In the presence of an Hb A2 variant, it is the total of the

normal and abnormal Hb A2 which is significant
 49

ICSH recommendations ISLH Oct 2011

Fraction separation by
Electrophoresis with elution or microcolumn chromatography
Quantification by spectrophotometry at 415nm

HPLC

Capillary Zone Electrophoresis

Capillary Isoelectric Focusing

DNA analysis is required for the characterization of

beta thalassaemia mutations
 50
50

ICSH recommendations ISLH Oct 2011

Measurement of the Hb A2 alone cannot absolutely confirm
or exclude the carrier state as the there may be little
difference between A2 in normals and some beta
thalassaemia carriers

Precision levels should be +/- 0.1% of the final answer (SD

0.05%)

Common beta thalassaemia trait Hb A2 = 4.0 - 6.0%

Beta thalassaemia trait overall usually Hb A2 = 3.5 - 7.0%
Normal subjects usually Hb A2 = 2.2-3.3%
Current developments
Instrument calibration-use of calibrant(s)

Target value for performance scoring:

all methods mean
method-specific mean current target
submethod-specific mean

Development of new Hb A2 reference material

Acknowledgements

For information on beta thalassaemia mutations in Europe:

Dr John Old
National Haemoglobinopathy Reference Laboratory, Oxford

For assessment of Hb A2 in UKNEQAS scheme:

Barbara Dela Salle, UKNEQAS
Hannah Batterbee, Royal Hallamshire Hospital / UKNEQAS