AJCP / Original Article
A Comparative Evaluation of Capillary Electrophoresis,
Cation-Exchange High-Performance Liquid
Chromatography, and Matrix-Assisted Laser Desorption/
Ionization Time-of-Flight Mass Spectrometry for the
Screening of Hemoglobin Variants
Miao Xu, MM,1 Yajun Wang, MD, PhD,2 and Anping Xu, MM3,
1
Department of Laboratory Medicine, Weifang People’s Hospital, Weifang, China; 2Public Health Laboratory Centre, Kowloon, Hong Kong; and
3
Department of Laboratory Medicine, Peking University Shenzhen Hospital, Shenzhen, China.
Key Words: Hemoglobin variant; Capillary electrophoresis; Cation-exchange high-performance liquid chromatography; MALDI-TOF mass
spectrometry
Am J Clin Pathol 2021;XX:1–10
DOI: 10.1093/AJCP/AQAA260
ABSTRACT
Objectives: The present study was conducted to evaluate
the usefulness of capillary electrophoresis (CE), cation-
exchange high-performance liquid chromatography
(HPLC), and matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry (MS)
for the screening of hemoglobin (Hb) variants prevalent in
southern China.
Methods: A total of 102 types of Hb variants in 1,083
variant carriers were identified over a 5-year period. These
variants were analyzed by a CE method (Capillarys 3
TERA), a cation-exchange HPLC analyzer (Variant II
Turbo 2.0), and a MALDI-TOF MS system (QuanTOF).
Results: The presence of 85 (83.3%, 85/102), 84 (82.4%,
84/102), and 62 (60.8%, 62/102) Hb variants was
detected by Capillarys 3 TERA, Variant II Turbo 2.0,
and QuanTOF, respectively. Of the three methods, only
Capillarys 3 TERA recognized all 10 of the most frequent
Hb variants in southern China. There were six, two, and
three Hb variants that can only be detected by Capillarys 3
TERA, Variant II Turbo 2.0, and QuanTOF, respectively.
The detection limit of mass difference for QuanTOF was
approximately 11 to 20 Da.
Conclusions: MALDI-TOF MS is suitable for use as an
auxiliary method rather than a stand-alone method for the
screening of Hb variants prevalent in southern China.
© American Society for Clinical Pathology, 2021. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com
Key Points
• We presented a comparative evaluation of capillary
electrophoresis, cation-exchange high-performance liquid
chromatography, and matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF MS)
for the screening of hemoglobin (Hb) variants encountered in
southern China.
• Different Hb variants are found in different populations, and
none of the separation techniques presented, even when used in
combination, were able to detect all Hb variants.
• MALDI-TOF MS is suitable for use as an auxiliary method rather
than a stand-alone method for the screening of variants in China.
The detection limit of mass difference for QuanTOF was about 11
to 20 Da.
Hemoglobin (Hb) variants are common inherited dis-
orders characterized by structural abnormalities in the Hb
molecule, which are typically attributable to amino acid sub-
stitutions in globin chains. More than 1,300 Hb variants,
mainly caused by HBB and HBA gene mutations, have so
far been described in the database of human Hb variants
and thalassemias.1,2 They are found around the world, and
different frequencies of specific variants exist in each region.3
The most popular methods for the screening of Hb
variants are based on cation-exchange high-performance
liquid chromatography (HPLC) and capillary electro-
phoresis (CE) techniques, both of which can quantify
Hb A, Hb F, and Hb A2 along with Hb variant screening
in a highly reproducible system.4,5 In addition to these
two common methods, matrix-assisted laser desorption/
Am J Clin Pathol 2021;XX:1-10 1
DOI: 10.1093/ajcp/aqaa260
Xu et al / Three Methods for the Screening of Hb Variants

A Captions Z15 Z14 Z13 Z12 Z11 Z10 Z(A) Z8 Z(F) Z(D) –Z(S) Z(E) Z(A2) Z(C) Z1 B
Hb A Hb A0

Hb A2 Hb A1c Hb A2
Other Hb A

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

Hemoglobin Electrophoresis A1c Hemoglobin Electrophoresis

Normal
Name % Values (%) Fractions % mmol/mol Calculated %
Hb A 97.5 96.8-97.8 Hb A1c — 35 5.3
Hb A2 2.5 2.2-3.2 Other Hb A 2.3
Hb A0 90.8
Hb A2 2.3
C
Peak NGSP Area Retention Peak
Name (%) (%) Time (min) Area
A1a — 1.2 0.178 20,595
A1b — 1.0 0.244 16,797
F — 1.9 0.284 31,895
LA1c — 1.8 0.413 31,466
A1c 6.5* — 0.519 91,084
P3 — 3.6 0.805 62,244
P4 — 1.4 0.878 23,198
A0 — 83.8 1.020 143,517
*Value outside of Total Area: 1,708,795
expected range.

20.0

17.5

15.0

12.5
% Hb A1c

10.0
0.52

7.5 A1c
0.81
0.28
5.0 0.88
0.24 0.41
0.18
2.5 1.02

0.0

0.00 0.25 0.50 0.75 1.00 1.25

❚Figure 1❚ A sample without hemoglobin variants analyzed by the hemoglobin procedure of Capillarys 3 TERA (A), HbA1c procedure of
Capillarys 3 TERA (B), Variant II Turbo 2.0 (C), and QuanTOF (D). NGSP, National Glycohemoglobin Standardization Program.

ionization time-of-flight (MALDI-TOF) mass spec- of these techniques alone is unable to recognize all Hb
trometry (MS) has been proposed to distinguish Hb variants because each method has specific resolution
variants by their mass differences.6,7 However, any one and recognition capabilities for a given Hb variant.8

2 Am J Clin Pathol 2021;XX:1-10 © American Society for Clinical Pathology

DOI: 10.1093/ajcp/aqaa260
 AJCP / Original Article

D 0.0044
15,868
R: 772
0.0042

0.0040 15,127
R: 784
0.0038

0.0036

0.0034

0.0032

0.0030

0.0028

0.0026
Intensity (volts)

0.0024

0.0022

0.0020

0.0018

0.0016

0.0014 α-chain β-chain

0.0012

0.0010

0.0008
16,075
15,334 R: 487
0.0006 R: 498
15,948 16,031
0.0004 15,289 15,498 R: 8,005 R: 1,189
R: 2,613 15,743 16,173 16,485
R: 925 16,280 R: 587
R: 663 R: 733
0.0002 R: 1,115
0.0000
14,700 14,800 14,900 15,000 15,100 15,200 15,300 15,400 15,500 15,600 15,700 15,800 15,900 16,000 16,100 16,200 16,300 16,400 16,500 16,600 16,700 16,800 16,900

m/z

❚Figure 1❚ (cont)

Many commercial automatic devices based on HPLC (E), Z (S), Z (D), Z (F), Z (8), Z (A), Z10, Z11, Z12, Z13,
or CE have been developed for the analysis of Hb frac- Z14, and Z15 (software version: Phoresis 9.1.5; ❚Figure
tions.9 Several of them can switch between the Hb proce- 1A❚). The HbA1c procedure yields an HbA1c fraction and
dure and the glycated Hb (HbA1c) procedure, providing produces an atypical electropherogram in the presence of
not only HbA1c values but also information of Hb vari- Hb variants or additional peaks ❚Figure 1B❚.
ants.10,11 In this study, we evaluated CE, cation-exchange
HPLC, and MALDI-TOF MS for the screening of Hb Cation-Exchange HPLC
variants in southern China. We analyzed 1,083 samples The Variant II Turbo 2.0 (Bio-Rad), an HbA1c ana-
with Hb variants containing 102 types of gene mutations lyzer (HbA1c program, 1.5 minutes) based on the cation-
using Capillarys 3 TERA (Sebia), Variant II Turbo 2.0 exchange HPLC technique, produces a chromatogram
(Bio-Rad), and QuanTOF (Intelligene Biosystems). showing the proportion of each Hb fraction present in
a set of windows and the retention time of each Hb frac-
tion. As shown in the chromatogram (software version:
Materials and Methods Clinical Data Management System 5.1; ❚Figure 1C❚), in
Analyzers addition to the A0 and A1c windows, A1a, A1b, F, LA1c,
Capillary Electrophoresis P3, P4, variant window, and C windows are observed, and
Similar to the CAPILLARYS 2 Flex Piercing each one has a retention time range.
system,12 Capillarys 3 TERA (Sebia) separates and quan-
tifies normal Hb fractions and Hb variants through elec- MALDI-TOF MS
trophoresis in an alkaline buffer. This system can switch QuanTOF (Intelligene Biosystems), a recently de-
between a Hb procedure and an HbA1c procedure, both veloped MALDI-TOF MS system, has been evaluated
of which allow identification of Hb A, Hb F, Hb A2, and for Hb and HbA1c analysis.13 This system distinguishes
Hb variants. The Hb procedure gives an x-axis number, intact globin chains based on their mass differences. As
from 0 to 300, which represents a relative migration posi- shown in ❚Figure 1D❚, the spectrum of normal Hb shows
tion classified into 15 different zones: Z1, Z (C), Z (A2), Z the known masses for MH+1 of the α-chain (15,127 Da)

© American Society for Clinical Pathology Am J Clin Pathol 2021;XX:1-10 3

DOI: 10.1093/ajcp/aqaa260
Xu et al / Three Methods for the Screening of Hb Variants

and β-chain (15,868 Da), as well as the corresponding ❚Table 1❚

glycated α-chain (15,289 Da) and β-chain (16,031 Da). Numbers of Hemoglobin Variants Detected by the Possible
Combination of Three Methods
Samples were prepared according to the following
procedures: whole-blood samples collected in EDTA tubes Detectable by Detectable by Detectable by
Capillarys 3 TERA Variant II Turbo 2.0 QuanTOF No.
were washed three times with 0.9% saline solution, and
then erythrocytes were diluted 1:500 in deionized water and Yes Yes Yes 41
Yes Yes No 30
mixed 1:9 with 10 mg/mL sinapinic acid. Then, 3 μL of this
Yes No No 6
mixture was spotted onto a stainless steel MALDI target Yes No Yes 8
plate prior to MS acquisition. The acquisition parameters No Yes Yes 11
of QuanTOF are as follows: mass spectra were generated No No Yes 3
No No No 1
in positive ion mode, with source voltage 19 kV, laser fre- No Yes No 2
quency 5 kHz, laser energy 8 μJ, scanning speed 2 mm/s,
mass range 5,000 to 30,000 m/z, and 10-row scan per spot.
❚Table 2❚
Hemoglobin Variants Detected by Capillarys 3 TERA
Specimens
From January 2015 through May 2020, all speci- Migration
Position (x-
mens with suspected Hb variants were collected during Zone Axis Number) Hb Variants Detected in Each Zone
the measurements of HbA1c by Variant II Turbo 2.0 or
Z1 260-300 None
Capillarys 3 TERA. These samples were confirmed by Z(C) 250-259 Hb C
Sanger sequencing and separated into aliquots prior to Z(A2) 234-249 Hb Chad, Hb E-Saskatoon
storage at −80°C. In this study, each of the samples with Z(E) 221-233 HbE, Hb Savaria
Z(S) 210-220 Hb Ottawa, Hb S, Hb Handsworth, Hb
Hb variants (n = 1,083), which contained 102 types of Arya, Hb G-Makassar, Hb Lavagna
Hb variants, including 52 HBB and 50 HBA mutations, Z(D) 194-209 Hb G-Coushatta, Hb G-Honolulu, Hb
was further analyzed using QuanTOF and the Hb pro- G-Taipei, Hb G-Siriraj, Hb D–Los An-
geles, Hb G-Waimanalo, Hb Pyrgos,
cedure of Capillarys 3 TERA. In addition, because only
Hb Queens, Hb Etobicoke, Hb
one method was performed for the former screening and Cocody, IVS-II-81 C→T, Hb Gavello,
collection of these Hb variants, the alternative lacking Hb Yaizu, Hb D–Ouled Rabah, Hb
HbA1c analysis by Variant II Turbo 2.0 or Capillarys 3 Montgomery, Hb Strumica, Hb
Matsue-Oki, Hb G-Philadelphia, Hb
TERA (HbA1c procedure) has been supplemented. This Shenzhen, Hb Tamano
study was approved by the ethics review committee of Z(F) 172-193 Hb G–San José, Hb Ta-Li, Hb G-Georgia,
the hospital where the samples originated, and signed in- Hb Deer Lodge, Hb Howick, Hb
Galliera II, Hb Roanne, Hb Bunbury,
formed consent was obtained from each participant. Hb Zhaoqing
Z8 156-171 Hb Port Phillip, Hb Manitoba I, Hb
Hinsdale
Z (A) 143-155 Hb Broomhill, Hb Melusine, Hb
Results Molfetta, Hb Ethiopia, Hb Hubei, Hb
Fort Dodge, Hb San Bruno, Hb La
Among the 102 Hb variants, 85 (83.3%, 85/102), Coruna, Hb Taipei-Tien, Hb Liaoning,
Hb Sichuan
84 (82.4%, 84/102), and 62 (60.8%, 62/102) were recog-
Z10 131-142 Hb Nouakchott, Hb Camden, Hb Hope
nized by Capillarys 3 TERA, Variant II Turbo 2.0, and Z11 112-130 Hb New York, Hb Marseille, Hb
QuanTOF, respectively. The numbers of Hb variants de- Dongguan
tected by the random combination of three methods are Z12 75-111 Hb Q-Thailand, Hb J-Bangkok, Hb
Ube-2, Hb J-Wenchang-Wuming,
shown in ❚Table 1❚. Hb I-Interlaken, Hb J-Toronto, Hb
There were no Hb variants detected in zone 1 (Z1) of the J-Sicilia, Hb J-Taichung, Hb Rambam,
electropherogram from Capillarys 3 TERA ❚Table 2❚. Half of Hb Coombe Park, Hb Shenyang, Hb
Jilin, Hb Heilongjiang, Hb Albany-
the Hb variants (42/84) were located in the variant window Suma
(retention time, 1.085-1.175 minutes) of the chromatogram Z13 64-74 Hb J-Lome, Hb Beijing, Hb
from Variant II Turbo 2.0 ❚Table 3❚. QuanTOF showed a mass J-Kaohsiung, Hb Zambia, Hb
J-Tashikuergan
difference of more than 50 Da in 28 of 62 Hb variants de-
Z14 51-63 Hb Hunan
tected compared with the corresponding normal globin chain Z15 0-50 HbI, Hb Shaare Zedek, Hb
❚Table 4❚. Based on visual inspection of MS spectra, QuanTOF Guangxi, Hb N-Timone, Hb
could not distinguish variant chains with a mass difference of Pôrto Alegre

4 Am J Clin Pathol 2021;XX:1-10 © American Society for Clinical Pathology

DOI: 10.1093/ajcp/aqaa260

❚Table 3❚
Hemoglobin Variants Detected by Variant II Turbo 2.0
Retention Hb Variants Detected in Each
Analyte Window Time, mi) Window
A1a 0.120-0.200 None
A1b 0.200-0.250 None
F 0.250-0.330 Hb Shaare Zedek
Between F 0.330-0.365 Hb Marseille, Hb N-Timone, Hb
and LA1c Graz
LA1c 0.365-0.465 None
A1c 0.460-0.590 HbI, Hb Pyrgos, Hb Hope,
Hb Coombe Park, Hb
J-Tashikuergan
Between LA1c 0.590-0.670 Hb Zambia, Hb Jilin, Hb Hunan
and P3
P3 0.670-0.890 Hb Ube-2, Hb J-Wenchang-
Wuming, Hb Beijing, Hb
I-Interlaken, Hb Taipei-Tien,
Hb Beziers, Hb Camden, Hb
Fuchu-I, Hb Shenyang, Hb
Heilongjiang, Hb Albany-Suma
P4 0.810-0.960 Hb J-Bangkok, Hb J-Lome, Hb
J-Kaohsiung, Hb J-Toronto,
Hb Wiangpapao, Hb J-Sicilia,
Hb Guangxi, Hb Rambam
A0 0.955-1.085 Hb Inglewood, Hb North Man-
chester, Hb Fort Dodge, Hb
Deer Lodge, Hb Shizuoka, Hb
Taradale, Hb Dongguan, Hb
Hengyang
Variant window 1.085-1.175 Hb E, Hb Q-Thailand, HbG-
Coushatta, Hb G-Honolulu,
Hb G-Taipei, Hb Ottawa, Hb
G-Siriraj, Hb D–Los An-
geles, Hb G-Waimanalo,
Hb Port Phillip, Hb Queens,
Hb Manitoba I, Hb G–San
José, Hb Ta-Li, Hb Ethi-
opia, Hb M-Milwaukee-I, Hb
Handsworth, Hb Hubei, Hb
Loves Park, Hb Etobicoke, Hb
San Bruno, Hb E-Saskatoon,
Hb J–Altgeld Gardens,
Hb Galliera II, Hb Savaria,
Hb Cocody, Hb Arya, Hb
Hinsdale, Hb Bunbury, Hb
Gavello, Hb Yaizu, Hb D–
Ouled Rabah, Hb G-Makassar,
Hb Lavagna, Hb Montgomery,
Hb Strumica, Hb Matsue-Oki,
Hb G-Philadelphia, Hb Shen-
zhen, Hb Tamano, Hb S, Hb
Zhaoqing
C 1.175-1.245 Hb C, Hb Chad
within 10 Da (n = 22). Still, it could distinguish 8 of 22 variants
with a mass difference of 11 to 20 Da and readily distinguished
those with a mass difference of more than 20 Da (Table 4).
These data suggested that the detection limit of mass differ-
ence for QuanTOF was approximately 11 to 20 Da.
Six (Hb Melusine, Hb La Coruna, Hb G-Georgia,
Hb Roanne, Hb Sichuan, Hb Pôrto Alegre), two (Hb
Wiangpapao and Hb Beziers), and three (Hb Phnom
© American Society for Clinical Pathology
AJCP / Original Article
❚Table 4❚
Hemoglobin Variants Detected by QuanTOF
Absolute Mass
Difference, Da Detectable Hb Variants Detected
<1 No None
1-10 No None
11-20 Partly Hb Molfetta, Hb J-Sicilia, Hb La
Coruna, Hb Deer Lodge, Hb Nouak-
chott, Hb Tamano, Hb Dongguan,
Hb Strumica, Hb Port Phillip, Hb
J-Lome
21-30 Yes Hb New York, Hb Q-Thailand,
Hb J-Kaohsiung, Hb S, Hb
M-Milwaukee-I, Hb J–Altgeld Gar-
dens, Hb Shizuoka, Hb Taipei-Tien,
Hb Graz, Hb Taradale, Hb Broomhill,
Hb Galliera II, Hb Liuzhou
31-40 Yes Hb Inglewood, Hb North Manchester,
Hb Loves Park, Hb Marseille, Hb Ty
Gard, Hb South Florida, Hb Fuchu-I
41-50 Yes Hb J-Taichung, Hb Montgomery, Hb
Liaoning, Hb Hunan
>50 Yes Hb J-Bangko, Hb G-Coushatta, Hb
G-Taipei, Hb Pyrgos, Hb G–San
José, Hb Ta-Li, Hb Fort Dodge,
Hb Howick, Hb Hope, IVS-II-81
C→T, Hb Rambam, Hb Gavello, Hb
G-Makassar, Hb Lavagna, Hb Shen-
zhen, Hb Hengyang, Hb Ottawa,
Hb Phnom Penh, Hb Queens, Hb
I-Interlaken, Hb J-Toronto, Hb Mani-
toba I, Hb Ethiopia, Hb Handsworth,
Hb Hubei, Hb Etobicoke, Hb Shen-
yang, Hb J-Tashikuergan
Penh, Hb Ty Gard, Hb Liuzhou) Hb variants could only
be detected by Capillarys 3 TERA, Variant II Turbo 2.0,
and QuanTOF, respectively ❚Figure 2❚, ❚Figure 3❚, and
❚Figure 4❚. The theoretical molecular weight was con-
sistent with the measured mass difference between the
variant and normal chains by QuanTOF (Figure 4). None
of these methods could detect Hb Westmead.
❚Table 5❚ shows the 10 most frequent Hb variants in
southern China, which represents 81.8% (886/1,083) of all
α- and β-chain variant carriers. Capillarys 3 TERA could
recognize all of them. However, Variant II Turbo 2.0 failed
to detect Hb New York and Hb Broomhill, and QuanTOF
could not distinguish Hb E, Hb G-Honolulu, and Hb Ube-
2. The CE, cation-exchange HPLC, and MALDI-TOF
MS findings for the remaining 92 Hb variants are listed in
Supplementary Table 1 (all supplemental materials can be
found at American Journal of Clinical Pathology online).
Discussion
In this study, we presented a comprehensive evalu-
ation for the recognition of Hb variants encountered
in southern China by three methods using various
Am J Clin Pathol 2021;XX:1-10 5
DOI: 10.1093/ajcp/aqaa260
Xu et al / Three Methods for the Screening of Hb Variants
A Hb A0
(A1c)
Other Hb
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
C Hb A0
Hb F or variant
Hb A1c
Other Hb
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
E Hb A0
Hb F or variant
Hb A1c
Other Hb
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
❚Figure 2❚ Hemoglobin variants that can only be detected by Capillarys 3 TERA: Hb Melusine (A), Hb La Coruna (B), Hb
Roanne (C), Hb Sichuan (D), Hb G-Georgia (E), and Hb Pôrto Alegre (F). Arrows indicate the presence of hemoglobin variants.
principles. Most Hb variants in this study can easily be
detected by CE or cation-exchange HPLC techniques,
and 96.1% (98/102, Table 1) of Hb variants can be de-
tected when these two techniques are combined, sug-
gesting that both CE and cation-exchange HPLC are
appropriate and complementary methods for screening
Hb variants. However, our data point out that QuanTOF
is not suitable to be used as a stand-alone method for
the screening of Hb variants because it could only detect
60.8% of total Hb variants.
As shown in our study, Hb E is the most frequent
variant in southern China, followed by Hb New York,
6 Am J Clin Pathol 2021;XX:1-10
DOI: 10.1093/ajcp/aqaa260
B Hb A0
5
Hb A2 Hb A1c 2 Hb A2
6
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
D Hb A0
Hb A2 Hb A2
(A1c) Other Hb
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
F Hb A0
1 2
Other Hb Hb A2
Hb A2 3
Hb F or variant
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
Hb Q-Thailand, Hb J-Bangkok, Hb G-Coushatta, Hb
G-Honolulu, and Hb G-Taipei. In comparison, the other
common Hb variants worldwide (Hb S, Hb C, and Hb D)
are rare. The CE method can detect all top 10 most fre-
quent variants in southern China. Nevertheless, Variant II
Turbo 2.0 failed to detect two of them, which could result in
the risk of missing such cases when screening for Hb vari-
ants. In addition, it has been shown that the β-thalassemia
short program of the Bio-Rad Variant Classic HPLC in-
strument also cannot detect Hb New York.14 As such, we
consider that CE is more competent for first-line screening
in clinical laboratories in southern China.
© American Society for Clinical Pathology
 AJCP / Original Article

A 20.0 B 20.0

17.5 17.5

15.0 15.0

12.5 12.5 0.77

% Hb A1c

% Hb A1c
10.0 10.0
0.48
0.39
7.5 A1c 7.5 0.47

0.26 A1c
0.24
5.0 0.23 0.77 5.0
0.16 0.32 0.37
0.85
0.16 0.89
2.5 2.5 0.11
0.951.00 0.99

0.0 0.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 0.00 0.25 0.50 0.75 1.00 1.25 1.50
Time (min) Time (min)

❚Figure 3❚ Hemoglobin variants that can only be detected by Variant II Turbo 2.0: Hb Wiangpapao (A) and Hb Beziers (B).
Arrows indicate the presence of hemoglobin variants.

Theoretically, each Hb variant has a characteristic
electrophoretic band on an electrophoretogram or an
elution peak on a chromatogram. Although the electro-
phoretic migration or HPLC position of a particular Hb
variant will have some variability, the excellent repeata-
bility shows that these techniques can serve as screening
tools for Hb variants. Moreover, the relative positions of
Hb variants, such as those that migrate very close to each
other, should be analogous regardless of the system. Thus,
although this study was carried out on only three devices,
the relative electrophoretic migration and HPLC posi-
tions of Hb variants should be similar on other devices
based on the same techniques as well.15 For example, the
migration data from Capillarys 3 TERA should be iden-
tical to the other Sebia CE systems, including Capillarys 2
Flex Piercing, Minicap, Capillarys 3 OCTA, and so on. As
a limitation of this study, we did not use the β-thalassemia
short program of the Bio-Rad Variant Classic HPLC in-
strument for the screening of Hb variants, which may re-
sult in a different detection rate of Hb variants from that
using Variant II Turbo 2.0.
The absence of some variant peaks on cation-
exchange HPLC or CE analysis chromatograms could
be explained by the overlap of peaks between regular
Hb fractions (eg, Hb A and HbA2) and Hb variants
with no or minor changes in electrical charge.16 Four
Hb variants in the present study—Hb Phnom Penh,
Hb Ty Gard, Hb Liuzhou, and Hb Westmead—could
not be initially identified by either HPLC or CE. They
were noticed for the following reasons: a marked dis-
cordance was observed between the results of HbA1c by
Variant II Turbo 2.0 and fasting plasma glucose for the
individual with Hb Phnom Penh, chromatograms of
Hb Ty Gard and Hb Liuzhou from Variant II Turbo 2.0
showed no sign of Hb variant but a slightly abnormal
HbA1c peak, and the Hb Westmead carrier also har-
bored Hb E identified by both cation-exchange HPLC
and CE. For these reasons, Sanger sequencing was per-
formed for these samples.
Although MALDI-TOF MS has been evaluated and
proposed for Hb analysis,7,17 a critical drawback of this
method that should be known is that its insufficient reso-
lution prevents the detection of Hb variants with small
mass differences in globin chains.18 In the present study,
two intact globin chains could not be separated unless their
masses differed from one another by more than approxi-
mately 11 to 20 Da, which represents the detection limit of
mass difference for QuanTOF. Furthermore, the resolution
of MALDI-TOF MS alone was insufficient to distinguish
some of the most common variants worldwide, such as Hb
C, Hb D, or Hb E. Despite that, it is an indispensable supple-
mentary tool because it complements traditional techniques
and allows the recognition of chromatographically or elec-
trophoretically silent Hb variants, such as Hb Phnom Penh,
Hb Ty Gard, and Hb Liuzhou, in this study. There should
be more Hb variants like this in a real condition because the

© American Society for Clinical Pathology Am J Clin Pathol 2021;XX:1-10 7

DOI: 10.1093/ajcp/aqaa260
Xu et al / Three Methods for the Screening of Hb Variants

A 15,127
0.0022
0.0021 15,867
0.0020
0.0019
0.0018
0.0017
0.0016
0.0015
Intensity (volts)

0.0014
0.0013
0.0012
0.0011
0.0010
0.0009
0.0008
0.0007
0.0006
0.0005
15,239 15,336 16,078
0.0004
0.0003
16,029
0.0002
0.0001
0.0000
14,800 14,900 15,000 15,100 15,200 15,300 15,400 15,500 15,600 15,700 15,800 15,900 16,000 16,100 16,200 16,300 16,400 16,500 16,600 16,700 16,800

m/z

B 0.0080
0.0078 15,127
0.0076
15,868
0.0074
0.0072
0.0070
0.0068
0.0066
0.0064
0.0062
0.0060
0.0058
0.0056
0.0054
0.0052
0.0050
Intensity (volts)

0.0048
0.0046
0.0044
0.0042
0.0040
0.0038
0.0036
0.0034 15,154
0.0032
0.0030
0.0028
0.0026
0.0024
0.0022
0.0020
0.0018 15,334
0.0016 16,075
0.0014
0.0012
0.0010 15,354
0.0008 15,365 15,499 15,934
0.0006 15,285 16,03116,174
0.0004 15,560 15,742 16,298 16,483
0.0002
0.0000
14,600 14,800 15,000 15,200 15,400 15,600 15,800 16,000 16,200 16,400 16,600 16,800 17,000

m/z

❚Figure 4❚ Hemoglobin variants that can only be detected by QuanTOF: Hb Phnom Penh (theoretical mass: 15,240 Da vs ac-
tual mass: 15,239 Da) (A), Hb Liuzhou (theoretical mass: 15,155 Da vs actual mass: 15,154 Da) (B), and Hb Ty Gard (theoretical
mass: 15,899 Da vs actual mass: 15,898 Da) (C). Arrows indicate the presence of hemoglobin variants.

initial screening methods in this study were cation-exchange
HPLC and CE but not MALDI-TOF MS. It is worth noting
that the assay sensitivity is highly dependent on the devel-
oped method, and different MALDI-TOF MS platforms
have different assay sensitivities. Besides, MALDI-TOF MS
combined with other technologies can significantly increase
sensitivity for the detection of Hb variants.19
In conclusion, the CE method is more suitable for
first-line screening of Hb variants in clinical laboratories
in southern China, while MALDI-TOF MS should be ap-
plied as an auxiliary method. In addition, our study again
demonstrated that none of the separation techniques pre-
sented, even when used in combination, were able to de-
tect all Hb variants.
Corresponding author: Anping Xu, MS; xuanping0528@aliyun.
com.

8 Am J Clin Pathol 2021;XX:1-10 © American Society for Clinical Pathology

DOI: 10.1093/ajcp/aqaa260
 AJCP / Original Article

C 0.0080 15,127
0.0076

0.0072

0.0068

0.0064

0.0060

0.0056

0.0052
Intensity (volts)

0.0048 15,868
0.0044

0.0040

0.0036

0.0032
15,898
0.0028

0.0024

0.0020

0.0016 15,334
0.0012 16,075
0.0008 16,094
15,228 15,498
15,743 16,027
0.0004 15,558 16,299 16,486
0.0000
14,600 14,700 14,800 14,900 15,000 15,100 15,200 15,300 15,400 15,500 15,600 15,700 15,800 15,900 16,000 16,100 16,200 16,300 16,400 16,500 16,600 16,700 16,800 16,900 17,000 17,100

m/z

❚Figure 4❚ (cont)

❚Table 5❚
Ten Most Frequent Hemoglobin Variants in Southern China
QuanTOF
Capillarys 3 Variant II Turbo 2.0
Hb Variant No. TERA (Zone) (Analyte Window) Detectable (Mass Difference) Mass of Variant Chain, Da
HbE 251 Z(E) Variant window No (<1.0 Da) 15,867
Hb New York 162 Z11 Undetectable Yes (30.0 Da) 15,898
Hb J-Bangkok 126 Z12 P4 Yes (58.0 Da) 15,926
Hb Q-Thailand 117 Z12 Variant window Yes (22.1 Da) 15,149
Hb G-Coushatta 59 Z(D) Variant window Yes (58.1 Da) 15,810
Hb G-Honolulu 53 Z(D) Variant window No (<1.0 Da) 15,126
Hb G-Taipei 48 Z(D) Variant window Yes (72.1 Da) 15,796
Hb Broomhill 33 Z(A) Undetectable Yes (26.0 Da) 15,101
Hb Ube-2 22 Z12 P3 No (<1.0 Da) 15,128
Hb Ottawa 15 Z(S) Variant window Yes (99.1 Da) 15,226

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