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9
| 3 | 2008
Klinisk Biokemi i Norden
(Fortsætter side 10)
produce the same numerical values facilitating the
use of the same clinical information all over the
world.
The first methods
HbA1c was incidentally discovered as a third peak
in chromatography of hemoglobins. The peak was
later explained as a result of glycation of hemoglo-
bin and correlated to the glucose concentration (7).
Both ion exchange chromatography (e.g. Bio-Rad,
Tosoh, Mono S) and affinity chromatography (e.g.
Primus) have since then been utilized to separate the
peak of glycated hemoglobin from the non glycated
hemoglobins and measure the fraction of glycated
hemoglobin. Immunologic methods have also been
developed, with the poly- or monoclonal antibodies
raised against the glycated N-terminal amino acid
valine and the following 4-6 amino acids of beta
chains (Roche, Bayer).
Due to different analytical specificities of the chro-
matographic procedures, different standardizations
of HbA1c assays have been developed over time.
Thus, the HbA1c values we have seen until now,
have been traceable either to DCCT (The Diabetes
Control and Complications Trial) for which the
standardization of HbA1c later has been run by the
organization National Glycoprotein Standardization
Program (NGSP)(8), the Japanese Diabetes Society
(JDS/JSCC) standardization, or to the Swedish Mono
S procedure (9). During the process of further stan-
dardization these procedures have been termed
Designated Comparison Methods (DCM).
IFCC reference system
Given the great value of HbA1c measurement, the
IFCC in 1995 raised a working group for standardi-
zation of HbA1c with a mission to develop a reference
measurement system for HbA1c. As a first step of
standardization it was agreed to exactly define the
analyte as those hemoglobin beta chains where the N
terminal amino acid valine is glycated. Consequently
the measurand, that is the quantity to which the
measurement value is assigned, was defined as the
fraction between the amount of N-terminal glycated
beta chains and the total amount of beta chains. The
HbA1c-WG also prepared primary calibrators, pure
HbA1c (mono-glycated beta-chain and non-glycated
alpha-chain) and pure HbA0 (non-glycated beta-
chain and non-glycated alpha-chain) to calibrate the
reference methods (10). A reference method was
developed as follows: Hemoglobin is cleaved into
peptides by the proteolytic enzyme endoproteinase
Glu-C and the resulting glycated and non-glycated
N-terminal hexapeptides of the beta-chains are sepa-
rated from the crude mixture of peptides by reverse-
phase HPLC. The hexapeptides are quantified by
mass spectrometry or capillary electrophoresis at
UV detection. The reference method were unani-
mously accepted by the National Societies of Clinical
Chemistry following a ballot 2001 and published
as an “Approved IFCC reference method for deter-
mination of HbA1c in human blood” (11). The
complicated method is not aimed for routine use. A
linear relation and strong correlation was obtained
between the reference method and all DCM and rou-
tine methods. However, when comparing results with
the NGSP certified methods the HbA1c values were
20-35% lower (12).
The reference method, calibrated with the primary
reference materials, is now used by 14 selected labo-
ratories around the world within the IFCC network
to assign HbA1c values to whole blood secondary
reference materials. The secondary reference materi-
als are used by all manufactures to calibrate the field
methods. The manufacturers calibrators have assig-
Fig 2. Glycation sites of hemoglobin. Red colour represents
the N-terminal amino acids valine of beta and alpha chains.
Green colour indicates the amino acids lysine. Some of them
are glycated with increased glucose concentration. The cyan
coloured hexapeptide of beta chains (glycated and non-
glycated) is used for the determination of HbA1c in the IFCC
reference system.
