Clinical Chemistry 46, No. 10, 2000
Effect of Hemoglobin Variants on Routine Glycohemoglobin Measurements Assessed by a Mass Spectrometric Method, Toyofumi Nakanishi,1 Ayako Miyazaki,1 Ken
Iguchi,2 and Akira Shimizu1,2* (1 Department of Clinical
Pathology and 2 Central Clinical Laboratory, Osaka Medical College, Osaka 569-8686, Japan; * address correspondence to this author at: Department of Clinical Pathology,
Osaka Medical College, 2-7, Daigakumachi, Takatsuki,
Osaka 569-8686, Japan; fax 81-726-84-6548, e-mail shimizu
@poh.osaka-med.ac.jp)
Comparative analyses of glycohemoglobin (HbA1c) in
samples containing hemoglobin (Hb) variants have
shown that different test systems may give discrepant
results (1–3 ). HPLC methods for such samples generally
underestimate the true HbA1c value, although a few
variants give a positive error for HbA1c. Immunoassays
may also underestimate the values if there is a change in
an epitope that contains glucose and N-terminal amino
acids of the Hb ␤ chains. Therefore, more information
needs to be collected on the effects of various Hb variants
on specific HbA1c test systems (1 ). The reference method
proposed by Kobold et al. (4 ) for measuring HbA1c is
based on electrospray ionization mass spectrometry (ESI/
MS) determination of the N-terminal residues of the Hb ␤
chains, which are released by enzymatic cleavage of the
intact Hb molecule with endoproteinase Glu-C. This
method gives accurate results for the percentage of glycated Hb at the N terminus of the ␤ chains even for
samples containing Hb variants. In the present study, we
compared the HbA1c values measured in samples with
various Hb variants by two commercial systems (HPLC
and immunoassay) and the ESI/MS method.
A total of 81 samples, from nondiabetic and diabetic
subjects, were analyzed, of which 45 were homozygous
for HbA and 36 were heterozygous for various Hb variants (Table 1). The structures of most Hb variants were
determined by MS and by DNA analysis, primarily to
elucidate the cause of the unexpected values of HbA1c
measured by HPLC; one case (HbM Boston) was examined for cyanosis-like symptoms (5 ). For high-resolution
HPLC to measure the content of the variants, we used
cation-exchange column chromatography with Polycat A
packing and a slow (90 min) gradient buffer change (6 ).
Because HbM was not separated by the HPLC, its content
was determined by conventional column chromatography (5 ).
For immunoassay, we used the DCA2000 method from
Bayer Diagnostics, which measures the intact glycated ␤
chain of hemoglobin without proteolytic digestion. Some
samples were also assayed by another immunoassay,
Unimate (Roche Diagnostics), to investigate possible alterations of antigenicity of the glycated N terminus of the
abnormal ␤ chain. In the Unimate assay, globin is digested by pepsin, and the released glycated peptide is
measured by latex agglutination immunoassay. Both immunoassays were performed according to the manufacturers’ instructions. For HPLC, a Hi-AUTOA1c HA-8150
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HbA1c analyzer (Kyoto Daiichi) was used to measure
HbA1c, according to the manufacturer’s instructions.
For ESI/MS, we followed the procedure described by
Kobold et al. (4 ) with modifications (7, 8 ). To calculate the
peak ratio of glycated and nonglycated ␤-N-terminal
hexapeptide, we monitored both singly and doubly protonated ions, whereas Kobold et al. (4 ) used only doubly
protonated ions. We used the expression 0.5 ⫻ the peak
area of doubly protonated ions ⫹ 1 ⫻ the peak area of
singly protonated ions for each peptide to calculate the
ratio of peak intensity of both peptides (7, 8 ), which
improved the reproducibility of the ratio of peptides
compared with the ratio calculated with only the doubly
protonated ions. In addition, for the calibrator, we mixed
synthetic peptides of nonglycated and glycated hexapeptide, VHLTPE and 1-deoxyfructosyl-VHLTPE (7, 8 ), instead of mixing HbA1c and HbA0, both purified by
cation-exchange chromatography and affinity chromatography (9 ). We also used immobilized Glu-C (7, 8 ), (Poroszyme; PE Biosystems), instead of the solution form of
the enzyme (4 ), the cost of immobilized enzymes per
experiment being much less than that of soluble enzymes.
The MS system was a TSQ 7000 triple-stage quadrupole
mass spectrometer with a conventional electrospray ion
source (Finnigan MAT). The intraassay CV of the ESI/MS
method in our hands was 1.3% for control samples with a
high percentage of HbA1c (10.3%; n ⫽ 10) and 1.8% for
those with a low percentage of HbA1c (4.9%; n ⫽ 10). The
interassay CV was 2.7% for high-HbA1c samples (10.2%;
n ⫽ 10) and 2.2% for low-HbA1c samples (5.0%; n ⫽ 10).
In addition to the synthetic peptide calibrators in our
calculation method, we measured reference materials
kindly donated by the IFCC Working Group, which had
been prepared by mixing purified HbA1c and purified
HbA0 (9 ). Values for six samples measured in an average
of five analyses each by the modified ESI/MS method
[where y is the measured ratio of HbA1c/HbA0, and x (in
parentheses) is the target value of the working group]
were as follows: 0.180 (0.198), 0.134 (0.154), 0.101 (0.112),
0.065 (0.072), 0.034 (0.034), and 0.0 (0.0). The measured
values correlated well with the IFCC target values, but the
slope of the regression line was shifted from 1: y ⫽ 0.886
(⫾ 0.017)x ⫹ 0.002 (⫾ 0.002); r ⫽ 0.999; Sy兩x ⫽ 0.003.
Possible causes of this shift in slope include different
efficiencies of enzyme cleavage of the glycated and the
nonglycated ␤ chains, impurities in the reference materials, or errors in mixing reference materials. This is an
important issue to resolve, but the method we propose is
sufficient for assessing conventional methods of measuring HbA1c in samples containing abnormal Hb. We emphasize that the ratios of peak intensity of control samples
were highly reproducible in every assay on different days
(CV ⫽ 1⬃2%). Therefore, calibration was not necessary
each time.
As shown in Fig. 1, all three methods produced similar
results for samples without variants. The correlation between the values obtained by HPLC and by MS (r ⫽ 0.993)
was slightly better than that between immunoassay
(DCA2000) and MS (r ⫽ 0.975). Absolute values obtained
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Technical Briefs
by both HPLC and immunoassay were considerably
higher than those obtained by MS because of differences
in standardization. We therefore used the differences in
values among assays for normal Hbs to adjust the values
obtained for samples with variants to evaluate the discrepancies between assays.
In most samples containing variant Hb, HPLC divides
glycated Hb into two fractions, glycated HbA and gly-
cated variant Hb, which leads to underestimation of
HbA1c in the samples containing variant Hb. For variants
of the N terminus of the ␤ chain, MS includes neither
glycated nor nonglycated variant peptides in the measurement and thus reports an accurate ratio of glycated to
nonglycated HbA. For Hb Okayama (␤2His3 Gln) and
Hb Niigata (␤1Val3 Leu, with complete retention of the
initiator Met and ⬃20% acetylation of the N-terminal
Table 1. Amino acid substitution, content, and HbA1c values of samples with variant Hb measured by HPLC, DCA2000,
Unimate, and ESI/MS.a
HbA1c, %
Sample no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Variant name
Le Lamentin
M Boston
J Meerut
Niigata
Case
no.
1
Substitution
␣20His3Gln
␣58His3Tyr
␣120His3Gln
␤1Val3Leu,
AcetylMet-Leu
2
Okayama
Hoshida
Hokusetsu
Hamadan
J Lome
G Szuhu
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
Agenogi
Yoshizuka
Peterborough
Masuda
Riyadh
Takamatsu
Camden
Sagami
1
2
3
4
5
␤2His3Gln
␤43Glu3Gln
␤52Asp3Gln
␤56Gly3Arg
␤59Lys3Asn
␤80Asn3Lys
␤90Glu3Lys
␤108Asn3Asp
␤111Val3Phe
␤114Leu3Met,
119Gly3Asp
␤120Lys3Asn
␤120Lys3Gln
␤130Gln3Glu
␤139Asn3Thr
Content of
variant, %
HA-8150
32.0
20.0
27.7
43.2
3.7
4.5
4.7
13.8
4.8
5.5
5.2
3.5
4.8
5.4
5.1
NDb
4.6
4.2
4.0
3.7
43.7
28.2
49.5
45.2
41.9
53.1
51.2
50.2
ND
ND
ND
48.6
51.3
48.5
43.7
42.6
39.6
44.1
43.1
42.3
41.5
46.9
23.8
39.0
13.2
21.9
2.5
6.2
2.8
2.3
2.5
2.5
2.8
1.0
1.5
2.8
2.9
2.3
2.8
3.2
2.4
2.4
2.4
2.4
2.6
3.0
2.6
2.9
3.3
5.5
5.2
11.0
5.0
4.7
4.9
4.6
5.2
4.8
4.9
4.8
5.3
4.8
4.8
4.8
4.2
4.7
4.8
4.5
4.9
5.0
3.8
4.8
ND
5.7
5.1
11.0
ND
4.9
4.9
ND
ND
ND
ND
4.6
5.1
4.6
ND
ND
ND
ND
5.0
4.5
5.2
4.9
4.0
4.8
3.8
5.1
4.1
9.1
3.9
3.5
3.1
2.7
4.7
4.1
4.2
4.1
5.2
2.9
4.2
3.9
2.8
3.3
3.6
3.2
3.4
3.9
4.1
4.1
49.7
48.6
50.3
50.8
49.6
49.5
51.7
69.2
2.7
2.6
4.3
6.8
3.2
2.5
1.3
1.1
5.0
5.1
6.6
11.5
4.5
4.5
3.8
1.1
5.2
ND
ND
11.8
ND
4.5
ND
4.6
4.4
4.9
6.1
10.2
4.2
3.7
2.9
3.2
DCA2000
Unimate
ESI/MS
a
Unimate values were included where available. The content of variant components, determined by Polycat A column chromatography, was expressed as a
percentage of the sum of the variant and normal Hb. Glycated and other minor components were not inlcuded in the calculation. For Hb Niigata, the ratios of component
with N-terminal acetyl-Met-Leu, with Met-Leu, and with normal N-terminal Val were 9.6%:33.6%:56.8% (sample 4) and 9.8%:33.9%:56.3% (sample 5). The sum of the
components with acetyl-Met-Leu and Met-Leu is shown. Sample 36 was from a subject who was compound heterozygous for ␤⫹-thalassemia [31(A3 G)] and a variant,
Hb Sagami (10 ).
b
ND, not determined.
Clinical Chemistry 46, No. 10, 2000
1691
Fig. 1. HbA1c values obtained by DCA2000, HiAUTOA1c, and MS methods.
F, results for homozygous HbA; E, samples heterozygous
for Hb variant and HbA. Numbers inside the open circles
correspond to those shown in Table 1. (A), comparison
between the percentages obtained by MS and DCA2000.
Deming regression analysis of results for homozygous
HbA samples: slope ⫽ 0.959 ⫾ 0.033; intercept ⫽
⫺0.551% ⫾ 0.246%; r ⫽ 0.975; Sy兩x ⫽ 0.510; n ⫽ 45.
(B), comparison between the percentages obtained by MS
and the Hi-AUTOA1c HPLC method. For homozygous HbA
samples, slope ⫽ 0.914 ⫾ 0.017; intercept ⫽ ⫺0.744%
⫾ 0.138%; r ⫽ 0.993; Sy兩x ⫽ 0.306; n ⫽ 45. For
superimposed plots, see Table 1.
Met), the HbA1c value obtained by HPLC was much
higher than that obtained by MS because the variant
components comigrated with the HbA1c fraction of HPLC.
Some variants also gave values by immunoassay
(DCA2000) considerably different from those obtained by
ESI/MS. After we adjusted the values obtained by MS to
correlate with the immunoassay values by using the
regression line obtained for normal Hb samples, the MS
values shifted upward. For example, the value for Hb
Niigata case 1 was 3.5% by immunoassay and initially
3.7% by MS, but 4.4% after adjustment. For Hb Niigata
case 2, the immunoassay value was 3.3%, whereas the MS
value was 3.8% (adjusted value, 4.5%). The relatively
lower values for Hb Niigata by immunoassay than by
ESI/MS may indicate low reactivity of the antibody
against the glycated N-terminal portion with variant
sequence Met-Leu-His. Unexpectedly, the sample with
Hb Okayama (␤2His3 Gln) did not produce a large
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Technical Briefs
discrepancy between values obtained by DCA2000 HbA1c
(5.5%) and those obtained by ESI/MS analysis (5.1%;
adjusted value, 5.9%). This sample also yielded nearly
equal values by Unimate (5.6% and 5.8%). Perhaps the
antibody used for these assays reacts to the same degree
with 1-deoxyfructosyl-Val-His-Leu and 1-deoxyfructosylVal-Gln-Leu, although the second amino acids of both
peptides are different. In Hb Sagami (␤139Asn3 Thr), the
value (1.1%) obtained by repeated immunoassay
(DCA2000) was much lower than that obtained by
ESI/MS (3.2%; adjusted value, 3.9%). The value obtained
with Unimate, an immunoassay with peptides released by
pepsin digestion, was 4.6%, which may be a more reasonable value. It is possible that the reactivity of the antibody
against glycated epitope in intact globin was substantially
lower in Hb Sagami. As we reported previously, the
oxygen affinity of this variant is 20% lower than that of
normal Hb (10 ), and the conformation of this variant may
differ greatly from that of normal Hb. Under mild conditions like the buffer for the immunoassay, the glycated
epitope on the variant Hb in the intact molecule may be
buried in the molecule.
Almost 800 kinds of variant Hbs have been reported.
The MS method proposed here may offer the best assessment method for newly developed HbA1c measurement
systems, especially for samples containing variant Hb.
This work was supported by 1997–2000 Grant-in-Aid
09557220 for Scientific Research (B) from the Ministry of
Education, Science, and Culture of Japan.
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study on a new hemoglobin variant, Hb MOsaka. Biochim Biophys Acta
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human hemoglobins. Simultaneous quantitation of foetal and glycated
hemoglobins. J Chromatogr 1988;434:95–110.
7. Nakanishi T, Shimizu A. Ratio of the ion peak intensity of glycated to
non-glycated hexapeptides from the N-terminal of hemoglobin ␤-chain measured by LC-ESI mass spectrometry. J Mass Spectrom Jpn 1999;47:389 –
91.
8. Nakanishi T, Shimizu A. Determination of ionization efficiency of glycated
and non-glycated peptides from the N-terminal of hemoglobin ␤-chain by
electrospray ionization mass spectrometry. J Chromatogr B 2000;in press.
9. Finke A, Kobold U, Hoelzel W, Weykamp C, Miedema K, Jeppsson J-O.
Preparation of a candidate primary reference material for the international
standardisation of HbA1c determinations. Clin Chem Lab Med 1998;36:
299 –308.
10. Miyazaki A, Nakanishi T, Kishikawa M, Makagawa T, Shimizu A, Mohammed
Mawjood AH, et al. Compound heterozygosity for ␤⫹-thalassemia
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Simplified Multiplex-PCR Diagnosis of Common
Southeast Asian Deletional Determinants of ␣-Thalassemia, Samuel S. Chong,1,2,3* Corinne D. Boehm,3 Garry R.
Cutting,3 and Douglas R. Higgs4 (1 Departments of Pediatrics, Obstetrics & Gynecology, and Laboratory Medicine,
National University of Singapore and National University
Hospital, Singapore 119074, Singapore; 2 Johns Hopkins
Singapore Pte. Ltd., Singapore 117610, Singapore; 3 Department of Pediatrics and McKusick-Nathans Institute of
Genetic Medicine, Johns Hopkins University School of
Medicine, Baltimore, MD 21287; 4 MRC Molecular
Haematology Unit, Institute of Molecular Medicine, Oxford OX3 9DU, United Kingdom; * address correspondence to this author at: Department of Pediatrics, National
University of Singapore, Level 4, Main Building, National
University Hospital, 5 Lower Kent Ridge Road, Singapore
119074, Singapore; fax 65-779-7486, e-mail paecs@
nus.edu.sg)
␣-Thalassemia double-gene deletions in cis are clinically
significant because homozygosity or compound heterozygosity for such deletions leads to fetal demise or death
shortly after birth. They account for the majority of cases
of hydrops fetalis among couples of Southeast Asian
origin.
We have sequenced the ⫺⫺THAI breakpoint junction in
a patient and included this allele in a single-tube multiplex-PCR test for detecting common Southeast Asian
␣-thalassemia determinants. The assay was tested on
genomic DNA samples and found to detect the ⫺␣3.7,
⫺␣4.2, ⫺⫺FIL, ⫺⫺SEA, and ⫺⫺THAI determinants of
␣-thalassemia.
␣-Thalassemia is common throughout the tropics and
subtropics and accounts for the majority of cases of
hydrops fetalis among couples of Southeast Asian origin
(1–3 ). Couples who are both carriers of a deletion that
removes both the adjacent ␣2- and ␣1-globin genes on one
of their chromosomes 16 (⫺⫺) are at 25% risk of having
fetuses with hemoglobin (Hb) Barts hydrops fetalis syndrome. Such pregnancies are accompanied by increased
risks of maternal complications [reviewed in Ref. (3 )].
The ⫺⫺SEA deletion is the most common double-gene
deletion in cis among Southeast Asians, whereas the
⫺⫺FIL and ⫺⫺THAI deletions account for a smaller percentage of the double-deletion alleles (3, 4 ). Unlike the
⫺⫺SEA deletion, the latter two deletions remove the entire
␣-globin gene cluster (5–7 ), and fetuses homozygous or
compound heterozygous for the ⫺⫺FIL and ⫺⫺THAI deletions are thought to undergo early fetal demise and
abort spontaneously. However, compound heterozygosity of ⫺⫺SEA with either ⫺⫺FIL or ⫺⫺THAI produces Hb
Barts hydrops fetalis syndrome, highlighting the importance of accurate genotype analysis. Although carriers of
single gene deletions most often do not present with any
clinical or hematological findings, these deletions are
nonetheless clinically important in the context of compound heterozygosity with the double-gene deletions
(⫺⫺/⫺␣), which produces Hb H disease.
We characterized the breakpoint junction sequence of