Background
A project for the standardization of HbA2 was launched by the IFCC back in 2004.

Materials and methods
In this work we report on the state-of-the-art of the project on standardization of HbA2. Data obtained from various EQAS studies, and from previous experimental evaluations, are presented.

Results
We have proven that biases between various commercial methods are still currently significant. We have also shown that calibration by commutable control materials may halve the inter-method variability.

Conclusions
The foundation of the reference system for HbA2, together with a brief preliminary presentation of the proposed primary reference measurement procedure based on ID-MS are outlined

A. Mosca

R. Paleari

AIR Universita degli studi di Milano

eJIFCC2018Vol29No4pp298-302
Page 298
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Standardization of the HbA
2
 assay
Renata Paleari, Andrea Mosca
Department of Physiopathology and Transplantation, Center for Metrological Traceability  
in Laboratory Medicine (CIRME), University of Milano, Italy
A R T I C L E  I N F O A B S T R A C T
Background
A project for the standardization of HbA
2
 was launched 
by the IFCC back in 2004.
Materials and methods
In this work we report on the state-of-the-art of the 
project on standardization of HbA
2
. Data obtained 
from various EQAS studies, and from previous experi-
mental evaluations, are presented. 
Results
We have proven that biases between various com-
mercial methods are still currently significant. We 
have also shown that calibration by commutable con-
trol materials may halve the inter-method variability.
Conclusions
The foundation of the reference system for HbA
2
, to-
gether with a brief preliminary presentation of the 
proposed primary reference measurement proce-
dure based on ID-MS are outlined.
Corresponding author:
Prof. Andrea Mosca
Dip. Fisiopatologia mct, L.I.T.A.
Via Fratelli Cervi 93
20090 Segrate, (MI)
Italy
Phone: +39 02 5033 0422
Email: andrea.mosca@unimi.it 
Key words:
HbA
2
, β-thalassemia, 
standardization, traceability
   
eJIFCC2018Vol29No4pp298-302
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Renata Paleari, Andrea Mosca
Standardization of the HbA2 assay
WHY TO STANDARDIZE 
The promotion of the standardization of routine 
laboratory methods is one of the mission state-
ment of the International Federation of Clinical 
Chemistry (IFCC) general policy. 
In the specific case of the methods used for the 
determination of hemoglobin A
2 
(HbA
2
), their 
standardization is a real need, not only an aca-
demic specific exercise. Indeed, there are sev-
eral important motivations to meet this:
a) most of the routine laboratory methods are 
poorly aligned;
b) important decision limits have been pro-
posed as markers for the diagnosis of thal-
assemia syndromes;
c) the correct diagnosis of carriers in couples 
at risk is the basic instrument to reduce the 
burden of thalassemia syndromes;
d) β-thalassemia is one of the few examples 
among genetic disorders in which identifi-
cation of carriers is performed, mostly, by 
simple hematological and biochemical tests.
The evidence that the laboratory methods are 
not aligned may come from the information 
provided by the external quality assessment 
schemes (EQAS) or by specific investigations. We 
have previously reported that the overall inter-
laboratory CVs evaluated from a pilot exercise 
in Italy with 48 Italian laboratories were in the 
order of 6 to 8% and that the fraction of labo-
ratories reporting unacceptable results ranged 
from 17 to 32% [1]. We have also shown that 
data collected in Italy from a mandatory exercise 
performed in Tuscany and surrounding regions 
reported CV values between methods similar to 
those found in another EQAS exercise provided 
by a manufacturer [2].
Method
Between-run 
reproducibility
Bias versus consensus mean
r
Slope Intercept
Bio-Rad D10 1.7 0.978 to 1.018 0.07 to 0.24 0.9981
Bio-Rad Variant II β-thalassemia 0.9 0.866 to 0.905 0.20 to 0.38 0.9977
Bio-Rad Variant II dual kit 1.7 1.026 to 1.047 -0.23 to -0.14 0.9995
Menarini HA8160 2.3 0.790 to 0.826 0.56 to 0.72 0.9976
Trinity Premier High Resolution 6.6 1.012 to 1.087 -0.08 to 0.24 0.9941
Trinity Premier Quick Scan 1.4 1.115 to 1.156 -0.42 to -0.24 0.9985
Sebia Capillarys 2 FP 1.9 0.898 to 0.946 -0.19 to 0.02 0.9969
Tosoh G8 1.9 1.150 to 1.180 -0.63 to -0.50 0.9992
Table 1 Performance characteristics  
of  different methods for HbA
2
 testing
Data were derived from reference 3. Reproducibility is expressed as CV %. 
For slope and intercept, the 95% confidence intervals are reported.
eJIFCC2018Vol29No4pp298-302
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Renata Paleari, Andrea Mosca
Standardization of the HbA2 assay
Besides that, we have recently evaluated the 
performance of current high-performance meth-
ods for HbA
2
 by measuring 40 blood samples in 
double over two separate days by the HPLC and 
one capillary electrophoresis system [3]. We 
have found a mean imprecision ranging from 
0.9 to 6.6 %, and a significant bias between the 
methods which were however quite well corre-
lated (r between 0.9941 and 0.9995). In Table 1 
the main data concerning the performance of the 
evaluated methods are summarized. In the same 
work we have tested the commutability of var-
ious control materials and we have shown that 
it is possible to reduce the variability between 
the methods by using a couple of commutable 
home-made control materials as calibrators. 
With regard to the overall variability, we have 
seen that after calibration the CVs were reduced 
from 6.8 to 3.4 %, from 6.6 to 4.6 %, and from 6.7 
to 3.0 % for HbA
2
 to concentrations lower than 
3.0 %, between 3.1 and 4.5 % and above 4.6 %, 
respectively.
HOW TO BUILD A ROBUST 
STANDARDIZATION SYSTEM
In 2004, the IFCC approved a project for the de-
velopment of a complete reference system for 
HbA
2
, as shown in Fig. 1. At the top of the trace-
ability chain, pure recombinant hemoglobins 
(HbA and HbA
2
) are used to calibrate the refer-
ence measurement procedure. This reference 
measurement procedure is based on peptide 
mapping and isotopic dilution mass spectrometry 
Figure 1 IFCC reference system and traceability chain 
for HbA
2 
measurement
Reference materials
(recombinant HbA0 and HbA2)
Reference Measurement
Procedure
(ID-LC-MS/MS)Certified Reference Material
 
Manufacturer’s internal
reference measurement 
procedureManufacturer’s 
working calibrator
Manufacturer’s standing
measurement procedure
Manufacturer’s 
product calibrator
Routine measurement procedure
Patient Sample
IFCC
WG-HbA2
Manufacturer
Individual
laboratory
HbA2 traceability chain
(lyophilized hemolysate)
eJIFCC2018Vol29No4pp298-302
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Renata Paleari, Andrea Mosca
Standardization of the HbA2 assay
(ID-MS), as described previously [2]. Specific 
tryptic fragments of the δ and α chains (i.e. δT2 
and αT5) are selected as signature peptides rep-
resenting either HbA2 or total hemoglobin, and 
quantified. The principle of the method together 
with the main performance characteristics are 
under publication. According to the traceability 
chain reported in Fig. 1, the reference measure-
ment procedure will then be used to assign the 
HbA
2
 value to certified reference materials which 
will be produced in collaboration with the Joint 
Research Centre (JRC). 
We have already prepared and characterized 
one pilot batch of this material consisting of a 
stabilized hemolysate in the lyophilized form [4]. 
The material was found to be quite stable with 
respect to HbA
2
 content for at least seven years 
when stored at -20°C, commutable with the 
majority of routine methods and to have a total 
hemoglobin concentration and methemoglobin 
(MetHb) content similar to that of fresh blood. 
Manufacturers will then use these certified ref-
erence materials to calibrate their methods and 
to assign values traceable to the primary refer-
ence measurement procedure to their calibra-
tors. Finally, the laboratorians using a routine 
method traceable to a reference measurement 
procedure will hopefully provide a more robust 
and accurate result.
TIME-SCALE FOR IMPLEMENTING 
THE REFERENCE SYSTEM
The first major step is to publish and validate 
the reference measurement procedure. The 
principle of this novel method is under publica-
tion at the moment of writing this report. The 
validation of the method according to the var-
ious ISO standards will require more work and 
the involvement of another laboratory because 
the minimum number of three laboratories is 
required in order to assign the values to any cer-
tified reference material. We estimate at least 
one more year to accomplish this second step. 
Almost in parallel we hope to be able to prepare 
the certified material to be used as calibrator in 
collaboration with the JRC. So, it is not unlikely 
that a couple of years will be necessary in order 
to have the manufacturers align their methods. 
CONCLUSION
Some are of the opinion that the novel molecular 
techniques such as next generation sequencing 
(NGS) may substitute the use of HbA
2
 in the field 
of thalassemia screening and diagnosis. We 
have performed a survey among various opin-
ion leaders and apparently this will not be the 
case, at least in the next coming years. Indeed, 
the following points were drawn:
1. Measuring a protein is not the same as se-
quencing the DNA. Measuring HbA
2
 is a func-
tional analysis at protein level, and it will nev-
er be replaced by DNA technology. 
2. NGS requires a lot of work for the interpre-
tation and, in many cases, the significance of 
novel SNPs or variants of unknown signifi-
cance is not known. In addition, it is really 
critical to know who is going to perform this 
interpretation.
3. There are still unresolved ethical problems 
in handling “incidental findings” with DNA 
technologies. 
4. NGS is very expensive and it is unlikely that 
it could be implemented in poor countries 
where the burden of thalassemia syndromes 
is greater than in some richer countries. 
5. On the contrary, measuring HbA
2
 is simple 
and fast and it has to be considered that in 
prenatal screening, as for couples at risk, it 
is desired to have the result in 1 or 2 days. 
As such it can be concluded that HbA
2
 assay still 
remains a gold standard for thalassemia screen-
ing and consequently its standardization is a ma-
jor and topical issue. 
eJIFCC2018Vol29No4pp298-302
Page 302
Renata Paleari, Andrea Mosca
Standardization of the HbA2 assay
Acknowledgements
We would like to acknowledge the following per-
sons who gave their opinion, as experts in the field, 
with regards to the need for a standardization 
of HbA
2
 measurements: Prof. Suthat Fucharoen 
(Mahidol University, Nakornpathom, Thailand), 
Dr. Cornelis L. Harteveld (Leiden University Medical 
Center, Leiden, the Netherlands), Dr. Serge Pissard 
(INSERM, Paris, France), Prof. Vip Viprakasit 
(Mahidol University, Bangkok, Thailand), Prof. 
Henri Wajcman (INSERM, Creteil, France). 

REFERENCES
1. Paleari R, Giambona A, Cannata M, Leto F, Maggio A, 
Mosca A. External quality assessment of hemoglobin A2 
measurement: data from an Italian pilot study with fresh 
whole blood samples and commercial HPLC systems. Clin 
Chem Lab Med 2007;45:88-92.
2. Paleari R, Caruso D, Kaiser P, Arsene CG, Schaeffer-Reiss 
C, Van Dorsselaer A, Bissé E, Ospina M, De Jesús VR, Wild 
B, Mosca A, on behalf of the IFCC Working Group on Stan-
dardisation of Hemoglobin A2 (WG-HbA2). Developing a 
reference system for the IFCC standardization of HbA2. 
Clin Chim Acta. 2017;467:21-6.
3. Paleari R, Ceriotti F, Harteveld CL, Strollo M, Bakker-
Verweij G, ter Huurne J, Bisoen S, Mosca A. Calibration 
by commutable control materials is able to reduce inter-
method differences of current high-performance meth-
ods for HbA2. Clin Chim Acta 2018;477:60-5.
4. Paleari R, Muñoz A, Mosca A, on behalf of the IFCC 
Working Group on Standardization of HbA2 (WG-
HbA2). Towards the development of a certified refer-
ence material for hemoglobin A2. Clin Chem Lab Med 
2010;48:1611-8.


Standardization of the HbA2 assay

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Standardization of the HbA2 assay

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