Bird, Robert

Brown, Nigel

Dimeski, Goce

University of Queensland eSpace

Clin Chem Lab Med 2012;50(8):1479–1482 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/cclm-2011-0772
 Letter to the Editor 
 Measurement of glycated hemoglobin in a patient 
with homozygous hemoglobin E 
 Goce  Dimeski 1,2, *,  Robert  Bird 3 and  Nigel  Brown 1 
 
1
  Pathology Queensland ,  Chemical Pathology, Princess 
Alexandra Hospital, Brisbane ,  Australia 
 
2
  School of Medicine ,  Southside Clinical School, The 
University of Queensland, Brisbane ,  Australia 
 
3
  Pathology Queensland ,  Haematology, Princess Alexandra 
Hospital, Brisbane ,  Australia 
 Keywords:  HbA 1c ;  HbE;  HPLC;  interference. 
 A recently diagnosed diabetic patient with homozygous HbE 
presented for HbA 1c estimation. The sample was fi rst analyzed 
using ion-exchange chromatography on a Bio-Rad Variant 
V-II (Bio-Rad, Hercules, CA, USA) using the V2_A 1c _NU 
program and it did not produce a HbA 1c result but showed 
an A
o
 area peak of 84.4 % . The sample was then analyzed 
on a second ion-exchange chromatography system, Bio-
Rad D-10 analyzer using software version 3.60, and it also 
failed to provide a HbA 1c result. The D-10 variant-window 
peak area was 92.8 % , and the A
o
 area peak of 1.9 % . The HbF 
fractions by the two different analyses were 3.3 % and 3.5 % , 
respectively. The results are shown in Figure  1 . The sample 
was then analyzed on a boronate affi nity method, In2it ana-
lyzer (Bio-Rad) and the result was 9.7 % . An aliquot of the 
sample was also analyzed on a COBAS Integra 800 (Roche 
Diagnostics, Germany) immunoassay method and the result 
was 6.9 % . This was the fi rst HbA 1c request received on this 
patient. There were three non-fasting glucose results for this 
patient, one with the HbA 1c request which was 11.8 mmol/L, 
and the two prior over a 2 week period were 14.4 mmol/L 
and 8.2 mmol/L. The 9.7 % HbA 1c result is equivalent to an 
approximately average glucose concentration of 12.8 mmol/L 
whereas the 6.9 % HbA 1c result equates to 8.4 mmol/L. The 
measured glucose results do not represent a large enough time 
period to provide more accurate approximation of the aver-
age glucose result. A fourth sample collected 2 months later 
had a glucose of 9.0 mmol/L. We measured the fructosamine 
( # 11930010, Roche Diagnostics) on a Beckman DxC800 
(Beckman Coulter, Brea, CA, USA) and the result was 349 
 µ mol/L (RI: 190 – 285). This suggests the HbA 1c In2It result 
refl ects the glycemic status more accurately than the Integra 
immunoassay result. 
 The patient had been confi rmed 6 days prior to the HbA 1c 
request as HbE homozygous with HbA 2 of 3.6 % , which is 
lower than usually seen in this condition (expected  > 4.0 % ) 
 (1) . The total HbA, HbA 2 and HbE area using the VII 
 β -thalassemia short program was 93.6 % . The requesting doc-
tor was not aware how such a variant may affect HbA 1c esti-
mations and the laboratory was not informed of the condition. 
HbE homozygote patients may present with mild hemolysis, 
normal or low hemoglobin and MCV and the blood fi lm may 
show target cells  (2) . The hematology results for this patient 
were in line with published literature: total Hb 110 (RI: 
115 – 160 g/L), RBC 5.5 (RI: 3.8 – 5.2 × 10 12 /L), MCV 56 (RI: 
80 – 100 fL) and MCH 20.0 (RI: 27.5 – 33.0 pg) (Sysmex 
XE-5000, Roche, Australia), and the blood fi lm showed 
target cells. 
 HbE is primarily found in people originating from South-
East Asia, with a prevalence of 30 % – 40 % in some parts of 
Thailand, Cambodia and Laos. This varies across the remain-
ing South Asian nations, but falls off to much lower levels 
across Southern Europe, the Middle East, and West Africa 
 (1) . However, with global migration HbE is likely to pres-
ent more frequently in developed nations. Worldwide, HbE 
is the second most common hemoglobin variant, and has a 
single amino acid substitution, lysine for glutamic at position 
26 in the  β chain  (3) . It is estimated 30 million South-East 
Asians are carriers of HbE and one million are HbE homozy-
gous  (1) . 
 The four analyzers we evaluated gave different results 
due to different methodologies used to detect the glycated 
hemoglobin. HbA accounts for approximately 60 % of the 
glycated hemoglobins in a normal patient. HbA is glycated 
at the N-terminal valine residues of the  β chain  (4) . The two 
ion-exchange HPLC methods correctly showed that there was 
no HbA 1c , as the patient has no HbA. We recognize that ion-
exchange HPLC methods can be powerful tools in detecting 
and identifying the presence of some Hb variants. Incidental 
discovery of an index case can identify a family with a vari-
ant, prompting medical advice. 
 The Integra 800 immunoassay method, like many other 
immunoassay methods utilize an antibody that recognizes 
the glycated N-terminal four amino acids of the  β chain  (5) . 
 *Corresponding author: Goce Dimeski, Chemical Pathology, 
Princess Alexandra Hospital, Ipswich Road, Woolloongabba 
Brisbane, Queensland 4102, Australia
Phone:  + 61 7 31762290, Fax:  + 61 7 31767070, 
E-mail:  goce_dimeski@health.qld.gov.au
 Received October 23, 2011; accepted February 9, 2012; 
previously published online March 3, 2012 
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1480  Dimeski et al.: Measurement of glycated hemoglobin in homozygous HbE
Since these amino acids are the same for HbA and for HbE, 
one would expect that the Integra result would be accurate. 
The Integra method has been shown not to be affected by 
heterozygous HbE variants  (3) . However, our other patient 
data suggests that the immunoassay underestimated the 
patient ’ s glycemic status, perhaps due to a decreased affi nity 
of the antibody to HbE. Therefore, we recommend that each 
immunoassay be examined for accuracy with homozygous 
HbE samples. Boronate affi nity measures all glycated hemo-
globins, and it has been reported to be unaffected by variant 
0.150
A1c Concentration=%
20.0
17.5
15.0
12.5
10.0A
1c
%
7.5
5.0
0.
19
0.
30
0.
54
0.
70
A
1c
 -
0.
92
1.
51
1.
61
1.
73
-2.5
0.0
0.0 0.5 1.0 2.0 3.01.5
Time, min
2.5
A1c Concentration=8.5%
Total area:
Peak name
A1a ---
---
---
---
---
---
---
---
0.3
1.1
0.2
3.1
0.5
5.3
6.5
83.1
0.11
0.24
0.40
0.55
1.55
1.64
1.75
1.85
4818
19,477
3605
55,383
9179
95,367
116,355
1,498,590
1,802,773
A1b
Unknown
F
P3
P4
Ao
E,D
Peak
area
Calibrated
area%
Retention
time, min
Area%
0.125
0.100
V
ol
ts
0.075
0.050
0.
11
0.
24
0.
40 0.
55
1.
55
1.
64
1.
75
1.
850.025
0.000
0.0 0.5 1.0 2.0 3.01.5
Time, min
2.5
Total area:
Peak name
A1a ---
---
---
---
8.5
---
---
---
0.8
1.9
0.4
2.3
---
3.6
1.9
83.0
0.19
0.30
0.54
0.70
0.92
1.51
1.61
1.73
27,641
64,932
12,360
75,856
204,696
120,055
63,403
2,783,172
3,352,115
A1b
F
LA1C
A1c
P3
P4
Ao
Peak
area
Calibrated
area%
Retention
time, min
Area%
A
B
Figure 1
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Dimeski et al.: Measurement of glycated hemoglobin in homozygous HbE  1481
hemoglobin forms, although it cannot detect the presence of 
variants  (6) . Based on published data and the lack of interfer-
ence from Hb variants on affi nity chromatography systems, it 
seems likely that the In2it result provides the best refl ection 
of the patient ’ s glycemic status. Availability of more glucose 
and fructosamine data for this patient would have provided 
a better guide as to whether this is actually true. To our 
knowledge there is no data comparing the In2it method to 
a more sophisticated affi nity chromatography method with 
homozygous HbE samples. 
 In South-East Asian countries particularly where the 
homozygous HbE variant is prevalent the affi nity chromato-
graphy methods are the methods of choice if diabetic patients 
are to be managed appropriately. Additionally, in cases where 
hemolytic anemia is present, appropriate management to 
achieve good glycemic control will need to be supplemented 
by fructosamine estimates. 
 In summary, to provide reliable and useful glycated 
hemoglobin results when variants or chromatograms with 
potential variants are identifi ed on HPLC systems, results 
should be checked with an affi nity chromatography method 
that has been shown not to have interference from the 
particular variant. Affi nity chromatography systems as point 
of care systems are now readily available (and relatively 
inexpensive). Therefore, laboratories running HPLC systems 
should invest in such analyzer combinations, specifi cally 
in a point of care method that has been thoroughly evalu-
ated for interferences. Another possibility would be to send 
out the sample for analysis by an affi nity chromatography 
method to enable accurate results to be obtained to best 
guide patient care. Fructosamine also must be considered 
as an alternative method for monitoring glycemic control 
in such patients, especially if the erythrocyte lifespan may 
be altered. 
 Confl ict of interest statement 
 Authors ’ confl ict of interest disclosure: The authors stated that 
there are no confl icts of interest regarding the publication of this 
article. 
 Research funding: None declared. 
 Employment or leadership: None declared. 
 Honorarium: None declared. 
0.08C D
0.07
0.06
0.05
0.04
0.03
0.
14 0
.2
3
0.
33
0.
44
1.
44
0.
20
0.
28
0.
62
1.
36
A
1c
0.02
0:0
Peak table - ID: Peak table - ID:
Peak
Unknown
A1a
A1b
F
Ao
Variant-window
Total area:
Concentration: %
0.0---
R. time
0.14
0.23
0.33
0.44
1.44
1.56
1,886,646
Height
3224
5283
2069
8722
15,560
696,646
Area
7021
22,843
5396
65,176
36,251
1,749,978
Area%
0.4
1.2
0.3
3.5
1.9
92.8
Peak
A1a
A1b
LA1c/CHb-1
A1c
P3
Ao
Total area: 1,804,707
R.time
0.20
0.28
0.62
0.81
1.36
1.45
Height
3583
3866
3386
6224
20,270
433,336
Area
13,855
26,575
28,828
66,801
89,516
1,579,131
Area %
0.8
1.5
1.6
5.4
5.0
87.5
1:00 2:00 3:00
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0:0 1:00 2:00 3:00
Concentration: %
5.4A1c
 Figure 1  Chromatograms from Bio-Rad HPLC systems. 
 (A) Variant II V2_A 1c _NU program; (B – D) 10 software version 3.60 of a sample with homozygous HbE variant and normal patient samples. 
Figure 1 (continued)
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1482  Dimeski et al.: Measurement of glycated hemoglobin in homozygous HbE
 References 
 1.  Colah R, Gorakshakar A, Nadkarni A. Global burden, distribution 
and prevalence of b-thalasaemia and hemoglobin E disorders. 
Expert Rev Hematol 2010;3:103 – 17. 
 2.  Bain B. Haemoglobinopathy diagnosis. 1st ed. Melbourne, 
Australia: Blackwell Science, 2001:165. 
 3.  Little RR, Rohlfi ng CR, Hanson S, Connolly S, Higgins T, 
Weykamp C, et al. Effects of hemoglobin E and D traits on 
glycated hemoglobin (HbA1c) measurements by twenty-three 
methods. Clin Chem 2008;54:1277 – 82. 
 4.  Little RR, Roberts WL. A review of variant hemoglobins interfer-
ing with hemoglobin A1c measurement. J Diabetes Sci Technol 
2009;3:446 – 51. 
 5.  Fleming JK. Evaluation of HbA1c on the Roche COBAS Integra 
800 closed tube system. Clin Biochem 2007;40:822 – 7. 
 6.  Sacks DB. Hemoglobin variants and hemoglobin A1c analysis: 
problem solved ? Clin Chem 2003;8:1245 – 7. 
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Measurement of glycated hemoglobin in a patient with homozygous hemoglobin E

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