Clinica Chimica Acta 531 (2022) 145–151

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/cca

Analytical interference of 33 different hemoglobin variants on HbA1c

measurements comparing high-performance liquid chromatography with
whole blood enzymatic assay: A multi-center study
Bozena Zechmeister a, Tanja Erden b, Berit Kreutzig c, Matthias Weber d, Philippe Joly e, f, g,
Jürgen Erdmann h, Christine Brockmann-Hönig i, Andreas Fischer a, j, k, Abass Eidizadeh a, *
a
Institute for Clinical Chemistry/Interdisciplinary UMG Laboratory, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany
b
Tosoh Bioscience, Diagnostic Business Unit-Europe, Germany
c
Amedes MVZ wagnerstibbe f. Laboratoriumsmed. med. Mikrobiologie und Immunologie, Werner-von-Siemens-Straße 10, 37077 Göttingen, Germany
d
Bioscientia MVZ Labor Karlsruhe, Am Rüppurrer Schloss 1, 76199 Karlsruhe, Germany
e
Laboratoire Interuniversitaire de Biologie de la Motricité (LIBM) EA7424, Team « Vascular Biology and Red Blood Cell », Université Claude Bernard Lyon 1, France
f
Laboratoire d’Excellence du Globule Rouge (Labex GR-Ex), PRES Sorbonne, Paris, France
g
Laboratoire de Biochimie et de Biologie Moléculaire, UF de Biochimie des Pathologies Erythrocytaires, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon,
Lyon, France
h
Laborgemeinschaft Rottweil G.b.R., Klinikstr. 3, 78052 Villingen-Schwenningen, Germany
i
Aesculabor Hamburg GmbH, Haferweg 36, 22769 Hamburg, Germany
j
Vascular Signaling and Cancer, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
k
DZHK (German Centre for Cardiovascular Research), Partner Site Goettingen, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Background and aims: The concentration of glycated hemoglobin (HbA1c) is an essential diagnostic and thera­
HbA1c peutic biomarker in diabetes mellitus. However, it is known that Hb structural variants and synthesis disorders,
Hemoglobinopathy can affect the HbA1c measurement in different assays. Although the analytical interference of various hemo­
Hb variants
globinopathies on the chromatographic measurement of HbA1c using HPLC has been well studied, data on the
HPLC
Enzymatic assay
interference on the enzymatic assay are few.
Tosoh Materials and methods: In this multi-center study, a large number (n = 104) of 33 different hemoglobin variants
Abbott were collected over a period of one year and compared between an HPLC (Tosoh G8 and G11) and an enzymatic
Alinity assay (Abbott Alinity c).
Results: A good comparability between ion-exchange HPLC and the Alinity assay for most Hb variants was found.
However, we were able to determine for the first time that certain Hb variants (Hb Okayama, HbAE, Hb Lepore)
can lead to clinically relevant discordant results. HbF (>5%) can already cause a relevant aberration.
Conclusions: Overall, using the Abbott HbA1c assay in the presence of certain hemoglobin variants can induce
clinically relevant interference that can affect diagnosis and therapy monitoring decisions, mainly because the
enzymatic assay cannot provide any information about Hb variants.

1. Introduction
Type 2 diabetes mellitus is a global health threat. The number of
patients suffering from type 2 diabetes mellitus has quadrupled in the
last 40 years and is around 463 million people worldwide [1]. The
prevalence increases with age and was found to be over 30% in Germany
over the age of 80 [2]. A reliable diagnosis is crucial for an adequate
treatment to avoid in particular cardiovascular complications of
diabetes mellitus [3]. For this purpose, the measurement of hemoglobin
A1c (HbA1c) is an essential part of diabetes mellitus diagnostics and
therapy monitoring [4].
The tetrameric protein hemoglobin of healthy people can be divided
into three groups: HbA (approx. 97%), HbA2 (2.5%) and the fetal he­
moglobin HbF (<2%). Around 6% of HbA is glycated, with HbA1c being
the main component [5]. HbA1c is formed by a non-enzymatically
catalyzed covalent chemical binding of glucose to the N-terminal

* Corresponding author.
E-mail address: abass.eidizadeh@med.uni-goettingen.de (A. Eidizadeh).

https://doi.org/10.1016/j.cca.2022.03.028
Received 30 March 2022; Accepted 30 March 2022
Available online 1 April 2022
0009-8981/© 2022 Elsevier B.V. All rights reserved.
B. Zechmeister et al.
valine of the β-chain of HbA1. HbA1c levels reflect the mean blood
glucose concentration over 2–3 months and correlate with cardiovas­
cular complications [4].
Various methods are available for measuring HbA1c. Most of them
show a high level of standardization and are certified by the National
Glycohemoglobin Standardization Program (NGSP) [6]. The hemoglo­
bin fractions can be separated either methodically by their charge dif­
ference (cation-exchange matrices and capillary electrophoresis) or by
structural differences (affinity chromatography, immuno- and enzy­
matic assay). The immunoassay or ion-exchange high-performance
liquid chromatography (HPLC) are the most common methods in use
[7]. Cation-exchange HPLC is a well established and widely used method
for HbA1c measurement, which enables reliable detection of the HbA1c
fraction and can identify possible hemoglobin variants, even if they may
interfere with the HbA1c [8–9]. Although enzymatic-photometric
methods are relatively new, studies have shown good precision and
accuracy [10–11]. The principle bases on a proteolytic cleavage of the N-
terminal fructosyl dipeptide from lysed whole blood and subsequent
spectrophotometric measurement of the activity of a fructosyl dipeptide
oxidase [12]. Hemoglobinopathies are the most common monogenetic
diseases worldwide and affect about 5% of the total population,
although the prevalence can be much higher regionally [13–14]. Two
main groups can be distinguished: hemoglobin synthesis disorders
(thalassemia syndromes) and hemoglobin structural variants [13]. A
significant interference of hemoglobin variants with the HbA1c mea­
surement was demonstrated in numerous studies with specific hemo­
globin variants for the HPLC methods [14–15]. It is worth noting that
although some hemoglobinopathies can affect the HbA1c measurement
through hemolysis, reduced erythrocyte lifespan, technical interference
independent from the used method or alterations in glycation rate
caused by changes of the chemical surrounding [16–17]. HPLC is a very
reliable method of detecting them and thus providing valid HbA1c
measurements [8]. On the other hand, there is hardly any data on the
possible influence of hemoglobinopathies on the enzymatic measure­
ment method, except for very common Hb variants, which were also
taken into account when evaluating the assay [12]. However, the
automated enzymatic analysis only gives the result about the HbA1c
concentration and no information about hemoglobin variants or possible
interferences.
The aim of this study was to compare the influence of many different
hemoglobin variants on the HbA1c measurement between an HPLC and
an enzymatic method. For this purpose, 104 blood samples were
collected from patients from the routine diagnostics of six participating
centers in a multi-center study over a period of one year. More than 33
different hemoglobinopathies were measured using the Tosoh G8 HPLC
method and the latest generation of Tosoh analyzers, the Tosoh G11, in
variant and β-thalassemia mode. The results were compared with the
enzymatic HbA1c determination using the Abbott HbA1c enzymatic
assay on the Alinity c, the latest analyzer generation from Abbott.
2. Material and methods
2.1. Study and experimental design
In this multi-center study, EDTA whole blood samples (n = 104) with
33 different hemoglobin variants were collected from six centers over a
period of one year (2019–2020). The six centers were: (1) Institute for
Clinical Chemistry/Interdisciplinary UMG Laboratory, University Med­
ical Center Goettingen (2) Amedes MVZ wagnerstibbe f. Laborator­
iumsmed. med. Mikrobiologie und Immunologie Göttingen (3)
Bioscientia MVZ Labor Karlsruhe (4) Laborgemeinschaft Rottweil G.b.R.
Villingen-Schwenningen (5) Amedes Aesculabor Hamburg (6) Labo­
ratoire de Biochemie et de Biologie Moléculaire Lyon, France. The
reference HbF samples (n = 10) were obtained in different concentra­
tions from the European Reference Laboratory for Glycohemoglobin
(Winterswijk, Netherlands). The blood samples were selected because
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Clinica Chimica Acta 531 (2022) 145–151
conspicuous chromatograms indicative of a hemoglobin variant were
visible using ion-exchange HPLC in the respective centers. In centers (3)
and (6), molecular genetic and mass spectrometric analysis were carried
out to precisely determine the hemoglobinopathy. Aliquots of the
respective blood samples were sent on dry ice and stored in the center
(1) at − 70 ◦ C. After thawing, all samples were measured directly and
immediately on all three analysis systems (Tosoh G8, G11 and Alinity c)
in the center (1). There was no refreezing. The study was conducted
according to the World Medical Association Declaration of Helsinki and
approved by the Ethics committee of the University Medical Center
Göttingen (approval no. 27/11/19An).
2.2. HbA1c and Hb variant measurement
The collected EDTA whole blood samples were measured using ion-
exchange HPLC on the Tosoh G8 and Tosoh G11 both in variant and in
β-thalassemia mode. In the β-thalassemia mode, the Hb fractions can be
analyzed more precisely for the purpose of determining the hemoglobin
variant thanks to a longer chromatographic runtime. All samples were
also measured using the enzymatic HbA1c method on the Abbott Alinity
c with the Abbott Alinity c hemoglobin A1c Reagent Kit. In the enzy­
matic method, the erythrocytes are first lysed and the hemoglobin is
then converted into methemoglobin to get the total hemoglobin. The
HbA1c is detected by proteolytically cleavage the glycated N-terminal
dipeptide of the β-chain. This is then specifically oxidized with the
fructosyl peptide oxidase, the resulting H2O2 reacts with a chromogen,
which is measured spectrophotometrically. All HbA1c measurement
results were expressed in relation to total-Hb in both the IFCC (The In­
ternational Federation of Clinical Chemistry and Laboratory Medicine)
unit (mmol/mol) and converted to the NGSP unit (%).
2.3. Statistics
Data analysis and statistical evaluation was performed with Micro­
soft Excel and GraphPad Prism. The results are presented as means ±
standard deviation. Mean deviation with the indication of minimum and
maximum value or as deviation in % are given. Whiskers boxplots show
median, 25th and 75th percentiles, as well as maximum and minimum
values. Statistical significance was calculated with the Friedman t-test
with a significance level of 5% (*p < 0.05; **p < 0.02).
3. Results
3.1. Comparison of Hb variants between HPLC and enzymatic
determination methods
33 hemoglobin variants were measured on Tosoh G8 and G11 in
variant and β-thalassemia mode and compared to the Alinity c. Mean
deviation between the three analyzers were calculated (Tables 1 and 2).
Most common Hb variants were summarized in Table 1. A clinically
significant difference was assumed by a deviation about 5%. As shown,
there is no clinically significant difference between methods when
measuring the concentration of HbA1c in most samples with more
common haemoglobinopathies, like HbAS, HbAD and HbAC (Table 1).
No clinically significant differences were found between Tosoh G8 and
G11 for all Hb variants, indicating a good comparability (Tables 1 and
2). Clinically significant difference between HPLC and enzymatic assay
could be determined for the more frequent hemoglobinopathies: Hb
Okayama, HbAE, Hb Lepore and by increased HbA2 and HbF fraction
(Table 1). Invalid HbA1c results were found in Hb variants such as Hb
Okayama, Hb SS and Hb South Florida. In these samples, it was not
possible to identify the HbA1c concentration; the expected value was
either falsely high measured (Hb Okayama) or not detected. This could
be reliably detected in the HPLC method and designated as invalid.
In the enzymatic method these variants were often output with an
error message (like for HbSS and Hb South Florida). With Hb Okayama,
B. Zechmeister et al. Clinica Chimica Acta 531 (2022) 145–151

Table 1
Summary of hemoglobin (Hb) variants compared between Tosoh G8, G11 and Alinity c. Mean values (in IFCC and NGSP units) and mean deviations in HbA1c
concentrations between HPLC (Tosoh G8 and G11) to enzymatic assay (Alinity c) from common Hb-variants are given (min. n = 3 per Hb-variant). NA: not applicable.
†
In one sample, the Alinity c gave a normal, valid HbA1c value (4.4%).
Mean (±SD) Mean Deviation (min; max)

Hb Variant G8 G11 Alinity G8 to Alinity G11 to Alinity G8 to G11

% mmol/ % mmol/ % mmol/

mol mol mol

HbA1c Hb Okayama (n ¼ 6) 51.8 539.4 48.0 501.0 5.7 39.0 88.7% (87.7; 87.7% (86.2; 7.3% (2.8;
invalid (1.9) (22.8) (1.9) (20.9) (0.6) (6.9) 89;6) 88.7) 10.9)
HbSS (n ¼ 4) NA NA NA NA NA NA NA NA NA
Hb South Florida (n ¼ 3) NA NA NA NA NA† NA† NA NA NA
HbA1c Undefined β-Variants 5.0 31.6 (9.9) 5.2 33.0 5.3 34.7 − 3.4% (− 42.4; − 2.2% (− 67.7; − 1.3% (− 19.5;
valid (n ¼ 12) (0.9) (1.3) (14.1) (1.4) (15.2) 15.0) 20.9) 16.0)
HbF (n ¼ 11) 6.5 47.6 6.4 46.9 5.9 40.8 11.5% (5.5; 10.5% (3.9; 1.4% (0; 3.3)
(1.1) (12.5) (1.1) (12.6) (0.8) (8.8) 22.9) 21.7)
HbAD (n ¼ 8) 5.7 38.6 5.6 22.9 (9.0) 5.7 39.0 − 0.7% (− 31.5; 0.8% (− 35.1; − 1.8% (− 4.4;
(1.3) (14.1) (1.4) (0.8) (9.2) 6.9) 8.5) 2.6)
HbAC (n ¼ 8) 8.1 64.5 8.0 64.8 7.7 61.0 3.7% (− 2.4; 9.6) 3.4% (0; 8.5) 0.3% (− 2; 4)
(2.5) (27.0) (2.3) (25.3) (2.3) (25.0)
HbJ (n ¼ 5) 7.1 54.0 6.4 46.4 (7.4) 6.6 48.4 4.3% (− 16.9; − 4.8% (− 21.0; 4.3% (− 16.9;
(1.1) (12.2) (0.6) (0.4) (4.5) 28.4) 13.4) 28.4)
HbAE (n ¼ 5) 7.9 63.3 7.3 56.1 8.2 66.8 − 10.8% (− 26.2; − 10.7% (− 21.4; − 9.0% (− 19.1;
(2.7) (29.6) (2.0) (22.9) (2.0) (21.5) 4.3) − 3.6) 0.9)
HbSC (n ¼ 4) 3.5 26.3 4.2 28.3 6.9 51.4 2.0% (− 1.2; 5.2) 2.0% (-1.2; 5.2) 0
(3.5) (28.0) (3.1) (26.1) (1.4) (16.1)
Hb Lepore (n ¼ 4) 4.6 27.0 (4.9) 4.8 28.8 (7.1) 5.5 37.0 − 13.4% (–32.0; − 7.2% (− 15.8; − 3.5% (− 14.0;
(0.4) (0.7) (0.8) (8.4) − 2.2) 1.5) 2.5)
α-Thalassemia (n ¼ 4) 11.0 96.5 10.9 96.3 6.4 46.5 3.8% (1.4; 6.3) 2.3% (0; 4.7) 0.9% (− 0.6;
(4.3) (47.6) (4.4) (47.5) (0.4) (4.1) 1.6)
HbAS (n ¼ 4) 6.0 41.6 6.0 42.6 5.5 36.2 5.6% (− 13.3; 6.2% (− 15; 34) − 1.1% (− 5.3;
(1.2) (12.9) (1.2) (13.9) (0.7) (7.4) 31.5) 2.9)
Increased HbA2 (n ¼ 3) 9.8 83.7 10.3 88.7 5.1 31.9 18.0% (1.9; 23.5% (3.5; − 6.0% (− 16.5;
(4.8) (52.6) (4.6) (50.8) (0.1) (0.9) 34.2) 43.5) 0)

Table 2
Summary of rare hemoglobin (Hb) variants compared between Tosoh G8, G11 and Alinity c. Deviations in HbA1c concentrations are given from rare Hb-variants
between Abbott enzymatic assay (Alinity) and HPLC Tosoh (G8 and G11). NA: not applicable.
Hb variante Alinity G8 G11 Deviation (%)

% mmol/mol % mmol/mol % mmol/mol G8 to Alinity G11 to Alinity G8 to G11

Hb α-Variant 9.0 74.6 7.8 61.7 7.6 60.2 − 15.3 − 17.4 1.7
Hb Athens-Georgia 5.4 35.9 5.8 39.8 5.8 40.6 6.8 8.0 − 1.2
Hb D Iran 4,8 29,33 3,3 12,57 4,11 21,42 − 1,5 − 0,69 − 0,81
Hb D Ouled-Rabah 5,2 33,49 5,9 40,98 3,62 16,07 0,7 − 1,58 2,28
Hb Fort de France 5.2 33.6 5.4 35.5 5.5 36.7 3.7 5.6 − 2.0
Hb G-Copenhagen 6.2 43.7 4.3 23.5 4.2 22.4 − 44.1 − 47.6 2.3
Hb G-Szuhu 4.9 30.4 5.5 36.6 6.4 46.8 10.9 23.9 − 17.0
Hb G-Szuhu 4.8 29.0 5.0 31.1 5.2 34.3 4 9.2 − 5.8
Hb Hafnia NA NA NA NA NA NA NA NA NA
Hb Hamilton NA NA 5.2 33.0 5.1 32.9 NA NA 0.7
Hb Hamilton 14.0 129.4 5.7 39.0 5.6 38.6 − 145.6 − 146.4 0.3
Hb I-Philadelphia 5.6 37.5 6.0 42.0 6.3 45.9 6.6 11.8 − 5.8
Hb Korle-Bu 5.0 30.9 6.0 42.0 6.3 45.3 16.6 20.6 − 5.0
Hb Long Island-Marseille 4.6 26.9 NA NA NA NA NA NA NA
Hb N-Baltimore 7.7 60.5 5.4 35.5 5.4 36.3 − 42.5 − 40.5 − 1.4
Hb Nouakchott 5.5 36.8 6.0 42.0 5.5 36.6 8.3 0 8.3
Hb O-Arab 4.3 23.0 4.3 23.5 3.7 17.3 0 − 14.9 13.0
Hb O-Arab 5.1 32.5 5.3 34.4 4.7 28.4 3.7 − 7.3 10.3
HbSD NA NA NA NA NA NA NA NA NA
Hb Setif 5.7 39.2 NA NA 7.1 54.8 NA 20.5 NA
Hb Setif 5.4 35.9 5.8 39.8 6.1 43.9 6.8 12.4 − 6.3
Hb Tacoma 7.4 57.4 8.1 65.0 8.1 65.1 8.6 8.7 − 0.1
Hb Winnipeg 6.0 41.8 6.8 50.8 6.8 51.1 11.7 12.1 − 0.4

however, normal high HbA1c values were output on the Alinity without again without a flag.
a corresponding error message. Because Hb Okayama migrates with
stable HbA1c in the HPLC, this leads to a falsely high HbA1c value on the
3.2. Comparison of rare Hb variants between HPLC and enzymatic
HPLC, which can be clearly marked as invalid when looking at the
determination methods
chromatogram (Fig. 1A). Alinity reports normal HbA1c values, resulting
in significant inter-method variation (Fig. 1B). Alinity also incorrectly
Table 2 shows the deviation of different rare variants of hemoglobin
determined a normal HbA1c result in one Hb South Florida sample,
on the HbA1c concentration measurement between HPLC and

147
B. Zechmeister et al.
enzymatic assay. For these Hb variants, only single samples were
available. There are variants where the proper peak could not be
detected by HPLC like Hb Setif or Hb Long Island-Marseille and the
enzymatic method still could measure the concentration of HbA1c;
leading in false HbA1c report (Table 2). There also seems to be strong
interference with the enzymatic assay, particularly with Hb Hamilton,
Hb G-Copenhagen and Hb N-Baltimore, leading to discrepant results
between the methods. However, it should be noted that only individual
samples were measured here. Like Hb South Florida, Hb Long Island-
Marseille also shows a change in the β-chain N-terminus and should
lead to an invalid HbA1c result. Here the Alinity shows an HbA1c result
without an error message, in contrast to the HPLC method (Table 2).
3.3. The influence of high HbF levels on HbA1c determination methods
The results show the interference of elevated levels of HbF on the
HbA1c concentration when measured with the enzymatic assay and with
the HPLC method. There is a significant difference between the HbA1c
concentrations determined on the Alinity and Tosoh devices (p < 0.02).
Overlapping with the labile HbA1c in the HPLC method results in a
falsely low HbA1c concentration determination (Fig. 2A). This is also the
case when using the enzymatic method, since the HbF is included in the
total Hb determination. Fig. 2B shows that even at HbF concentrations
Fig. 2. The influence of HbF on the HbA1c determination methods. (A) Example of a HPLC chromatogram of Tosoh G8 with the strong peak of 38.8% HbF (F)
detected near to the stable HbA1c (SA1C), which overlay the labile HbA1c peak (LA1C + ) and influences the determination of HbA1c (black arrows). (B) Inter­
ference of HbF (%) (n = 10) on HbA1c measurement via the enzymatic method (Alinity) and HPLC method (G8 and G11) for different HbF concentrations.
Interference is presented with bias (%) for enzymatic assay to G8 (red line) and G11 (blue line).
148
Clinica Chimica Acta 531 (2022) 145–151
Fig. 1. Comparison of HbA1c results for Hb
Okayama between Tosoh G8/G11 and
Alinity. (A) Example of a HPLC chromato­
gram of Tosoh G8 with the strong additional
peak of Hb Okayama detected with the same
retention time as the stable HbA1c (SA1C),
leading to false high HbA1c determination
(black arrow). (B) Comparison of HbA1c
concentration in the samples with Hb
Okayama between Abbott Alinity enzymatic
assay and HPLC method on Tosoh G8/G11.
The results are presented as mean ± SD (n =
6) compared with the Friedman t-test. **p <
0.02.
below 10%, clinically relevant deviations between Alinity and Tosoh
appear which increase dramatically from 10%. The elevated level of
HbF, in our samples between 4.1 and 42%, clearly influences the proper
detection of the HbA1c as well as the enzymatic detection.
4. Discussion
Some authors estimate 400,000 people in Germany are predicted to
be gene carriers of hemoglobinopathies, whereas other data lead to
higher estimates for the prevalence [18]. Due to increasing immigration
from other countries with a higher occurrence of Hb variants, an in­
crease in the prevalence in medical laboratories is also expected [19].
HbA1c determination is relevant for the diagnosis, prognosis, and
therapy of diabetes mellitus and structural changes in hemoglobin can
affect common commercially available HbA1c assays [20]. It is therefore
important to know the possible interference of hemoglobin variants on
the HbA1c determination for the different methods in order to be able to
make reliable statements about the HbA1c value. A full validation is not
possible for all available HbA1c assays due to more than 1000 different
naturally occurring Hb variants [21], solely through point mutations,
but at least the most common and occasionally occurring known vari­
ants can be collected and evaluated [22].
In our study we tried to collect a wide range of different
B. Zechmeister et al.
hemoglobinopathies in order to analyze them using two HbA1c deter­
mination methods that are most often used in clinical diagnostics, ion-
exchange HPLC (Tosoh G8 and G11) and an automated enzymatic
assay (Abbott Alinity). In a multi-center study, we were able to collect 33
different hemoglobinopathies in 104 blood samples from patients from
six centers and compare them between the Tosoh G8, G11 and the
Alinity c in order to identify possible clinical relevance of HbA1c de­
viations caused by the hemoglobinopathies interferences.
In accordance with national guidelines for quality assurance in
medical laboratories in Germany, we assumed a difference between two
methods of more than 5% in the concentration of HbA1c as clinically
relevant. In most of the hemoglobinopathies we tested, there was no
clinically significant differences between the HPLC and enzymatic
methods. However, with specific Hb variants, clinically relevant HbA1c
false measurements can occur due to interference. In our study, we were
able to identify the following Hb variants with possible discrepant re­
sults: Hb Okayama, Hb South Florida, HbAE, Hb Lepore and HbF. All of
these variants can be reliably detected with the Tosoh devices and are
reported through error flags for the correct interpretation of the chro­
matograms. Due to the enzymatic determination, such variants cannot
be signalized by the Alinity, which leads to the release of possibly
invalide HbA1c results in laboratory practice.
Hb Okayama has a near N-terminal substitution (α2β2 2(NA2)His-
>Gln), which co-migrates with HbA1c in the chromatographic separa­
tion using ion-exchange HPLC and leads to an overlay, which suggests
falsely high HbA1c values [23–24]. Using the Tosoh, this variant is
easily recognizable (both in variant and β-thalassemia mode) and the
measurement results can be labeled accordingly. However, the Alinity
gives HbA1c results without an error feedback, all of which were normal
range in our case. The presence of Hb Okayama cannot be detected
enzymatically and the values would be reported regardless. To our
knowledge, however, there are no other studies that have examined the
validity of the enzymatic HbA1c results in the presence of Hb Okayama,
for example in correlation with the fructosamine value or the mean
blood glucose concentration. Here, for example, the HbA1c results of the
Alinity in an Hb Okayama sample would have to be compared with those
of a boronate affinity chromatography, which is unaffected by Hb var­
iants [7],in order to possibly really confirm that these values should be
properly validated.
The influence of HbF on both HPLC and enzymatic methods has
already been shown [25–26]. Elevated HbF (ααγγ) levels may affect
HbA1c assays [25]. Elevated HbF occurs in diseases such as thalassemia
and leukemia, as well as in hereditary persistent fetal HbF [27]. An
elevated HbF level is more than 2% of the total hemoglobin [28]. In
hereditary persistent fetal Hb, HbF levels can rise up to 30% in patients
who are asymptomatic [29]. Even if the manufacturer does not recom­
mend the output of an HbA1c value for an HbF of more than 20%, no
interference with the Tosoh method could be determined in studies up to
30% [25]. However, increased HbF already interfere with the enzymatic
method from 10% [12]. In our study, a clinically relevant discrepancy of
more than 5% between Tosoh and Alinity was found already under 10%.
HbF co-migrates at higher levels with the labile HbA1c in HPLC causing
a major bias. The HbF interference is also already declared by the
manufacturer Abbott for the enzymatic assay from a concentration of
5%. The manufacturer’s information suggests a falsely low HbA1c result
as a consequence.
The manufacturer Abbott denies a difference due to HbAC, HbAD,
HbAE, HbAS and high HbA2 values, even if the manufacturer states a
possible difference of up to 4.2% for HbAC. So far, common Hb variants
(HbAS, HbAE, HbAC and HbAD) have not shown any interference with
the enzymatic method [12]. The Abbott assay has already shown good
correlation with HPLC in routine samples without Hb variants in other
studies [12,30–31]. Especially for the heterozygous HbAS, HbAC, HbAE
and HbAD variants, various studies have been carried out on their
interference with common HbA1c assays, the results of which are
cumulatively documented on the NGSP website (https://www.ngsp.
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Clinica Chimica Acta 531 (2022) 145–151
org). With the Tosoh G11 and G8, these variants also do not cause any
interference, but can be detected in variant mode [32–33]. Also an in­
fluence of HbAC, HbAD Punjay, HbAE and HbAS on the enzymatic
Abbott method on the Architect analyzer could be ruled out [22].
On the Alinity, our study was only able to show a clinically relevant
difference to the Tosoh analyzers through heterozygous HbAS and
HbAE. A difference between the G8 and G11 could also be seen in the
presence of an HbAE variant. In contrast to previous studies, as
mentioned above, we were able to show an interference from the com­
mon variants HbAS and HbAE on the enzymatic method. An influence of
HbAE on the HPLC Tosoh G8 also seems to shown by our data.
Hb South Florida is a β-chain mutation that directly affects the N-
terminus (α2β2 1(NA1) Val ->Met), so HbA1c detection is no longer valid
[34–35]. Therefore, we could not show HbA1c results using HPLC in our
study. Values above the linearity range of the assay were given for two of
the three samples on the Alinity, so that these improbable values would
possibly be interpreted as implausible if the enzymatic method were
used alone. However, a false normal HbA1 value of 4.4% was reported
for one sample without any error flag. In this case, an incorrect result
would have been reported with no indication that an Hb variant was
present.
Hb Lepore is a structural abnormality in which the β-globin gene
fuses with the γ-globin gene. Highly increased HbF and HbA2 levels can
be seen in the HPLC [36]. Our study was able to determine clinically
relevant differences in Hb Lepore between the enzymatic method and
HPLC, so that we can assume an interference of Hb Lepore in the
enzymatic determination. As far as we know, this is the first study that
could show an influence of Hb Lepore on the Abbott enzymatic assay.
In addition to the exceptions described, the results of the Tosoh
Analyzers coincide in many cases with the Alinity. The enzymatic Abbott
method is also an NGSP-certified method with IFCC traceable calibra­
tors, so that good comparability with other routine methods, such as the
Tosoh, should be expected, at least for hemoglobinopathy-free routine
samples [6]. Routine HbA1c measurements can be considered as a
screening tool of hemoglobin variants when using the Tosoh [37]. It is
worth noting that there are Hb variants that cannot lead to valid HbA1c
values with any method, such as the presence of homozygous HbSS or
HbCC or in the case of HbSC disease, which leads to a change in the
erythrocyte lifespan [38].
4.1. Limitations of the study
As a limitation of our data, we state that although we compared the
two methods with each other, we did not use a third method that would
have been independent of hemoglobinopathies as a comparison. In
future studies, boronate affinity chromatography, as a largely
interference-free method for the overall determination of glycated Hb,
would be useful as a confirmation test for such comparisons. Inciden­
tally, although boronate affinity chromatography is not influenced by
hemoglobin variants, a study was able to show the interference of HbF
[25]. Another weakness in our study is the small number of samples per
Hb variant. Although we had a relatively solid number of samples of
more than five for some variants (HbF, HbD, HbAC, Hb Okayama etc.),
other were only measured as a single sample, so that no statistical
statement can be made. Moreover, not all of the termed hemoglobin­
opathies have been confirmed by genetically or mass spectrometric in­
vestigations. Most of the samples we use have been described as
hemoglobin variants by abnormal chromatograms in routine diagnostics
and the variant has been determined by interpreting the chromatograms
in the β-thalassemia mode of the Tosoh devices. However, our procedure
corresponds more closely to the conditions that prevail in routine di­
agnostics in the medical laboratory.
4.2. Conclusion
To our knowledge, this is the first study that was able to show the
B. Zechmeister et al.
influence of more than 33 hemoglobinopathies, especially very rare Hb
variants, on the enzymatic determination of HbA1c compared to con­
ventional HPLC methods. Also, our study was the first using the Abbott
enzymatic assay on Abbott’s new generation analyzer, the Alinity. We
were able to demonstrate that Tosoh’s G8 and G11 analyzers are reliably
able to detect hemoglobin variants, which means that only valid HbA1c
values can be released. Both analyzers are also very well comparable and
concordant with each other. The Abbott enzymatic assay is robust
against many hemoglobinopathies, especially the common ones, and is
comparable to the HPLC method, but there are exceptions that could
lead to invalide HbA1c values (e.g. in the presence of Hb Okayama, Hb
South Florida, Hb Lepore and high HbF values). We were furthermore
able to show a clinically relevant influence of the more common vari­
ants, HbAE and HbAS, on the Alinity, although this has not yet been
demonstrated. In particular, the enzymatic assay cannot provide any
information about the presence of an Hb variant which should be
considered when using this method for HbA1c alone. With a low prev­
alence of hemoglobin variants, the use of an enzymatic assay for HbA1c
measurement is definitely suitable, but with an increased prevalence, as
is to be expected in Western Europe in the future due to migration
movements, another method with the possibility of detecting Hb vari­
ants should be preferred to prevent the transmission of false HbA1c,
which would possibly delay or prevent the diagnosis and therapy of
diabetes mellitus.
Declarations
Institutional Review Board Statement and Informed Consent
Statement
The study was conducted according to the World Medical Associa­
tion Declaration of Helsinki and approved by the Ethics committee of the
University Medical Center Göttingen (approval no. 27/11/19An).
Informed consent was obtained from all subjects involved in the study.
No clinical trial registry was necessary.
Availability of data and materials
The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy
and ethical reasons.
CRediT authorship contribution statement
Bozena Zechmeister: Conceptualization, Methodology, Software,
Validation, Formal analysis, Investigation, Resources, Data curation,
Writing – original draft, Writing – review & editing, Visualization,
Project administration. Tanja Erden: Conceptualization, Methodology,
Software, Validation, Investigation, Resources, Writing – review &
editing. Berit Kreutzig: Resources, Writing – review & editing. Mat­
thias Weber: Validation, Resources, Writing – review & editing. Phil­
ippe Joly: Resources, Writing – review & editing. Jürgen Erdmann:
Resources, Writing – review & editing. Christine Brockmann-Hönig:
Resources, Writing – review & editing. Andreas Fischer: Software,
Validation, Writing – review & editing. Abass Eidizadeh: Conceptual­
ization, Methodology, Software, Validation, Formal analysis, Investi­
gation, Resources, Data curation, Writing – original draft, Writing –
review & editing, Visualization, Supervision, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We thank Tosoh Bioscience for providing reagents, calibrators,
control material and support as well as temporarily the Tosoh G11
analyzer for the study.
150
Clinica Chimica Acta 531 (2022) 145–151
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