Hematopathology / Hb Variant Migration by CE Relative to HbA2
Expression of Hemoglobin Variant Migration by Capillary
Electrophoresis Relative to Hemoglobin A2 Improves
Precision
David F. Keren, MD,1,2 Renee Shalhoub, MT(ASCP),2 Ronald Gulbranson, MT(ASCP),2
and Deborah Hedstrom, MT(ASCP)2
Key Words: Hemoglobinopathy; Capillary electrophoresis; Hemoglobin S; Hemoglobin C; Hemoglobin A2; Hemoglobin G; Hemoglobin D
DOI: 10.1309/AJCPOF8V0JJOPSVF
Abstract
We report the precision of the mean migration
position of hemoglobin (Hb)S, HbC, HbG
(Philadelphia), and HbD (Los Angeles) in 193 samples
of whole blood assayed by capillary electrophoresis
(CE) and high-performance liquid chromatography
(HPLC). By expressing the migration of Hb variants
by CE relative to that of HbA2 in the same sample,
there was a significant improvement in the coefficient
of variation for each variant studied. The potential
usefulness of expressing Hb variants relative to that
of HbA2 was evaluated by comparing the separation
of 2 closely migrating Hbs. When expressed by their
initial migrations on CE, 25 of the 43 cases of HbG
and HbD overlapped. However, when the migrations
of these variants were expressed relative to the HbA2
in the same sample, the 24 cases of HbG separated
completely from the 19 cases of HbD. These findings
suggest that expressing Hb variants relative to an
internal standard, such as HbA2, may be of value for
establishing a library of variant Hbs evaluated by CE.
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DOI: 10.1309/AJCPOF8V0JJOPSVF
Identification of structural hemoglobin (Hb) variants
and thalassemias traditionally has relied on alkaline and acid
gel electrophoresis and, more recently, high-performance
liquid chromatography (HPLC).1-8 These methods detect
structural variants by electrophoretic migration or elution patterns. Precision of measurement of HbA2 is needed to detect
β-thalassemia and can be helpful in cases of α-thalassemia
and iron deficiencies.7,8
Capillary electrophoresis (CE) has been shown to be a
reliable alternative to HPLC and is one that provides a more
user-friendly interpretive format.9-17 All 3 methods readily
separate the most common variants such as HbS, HbC, and
HbE. However, some variants, such as HbG and HbD, are
more difficult to separate from each other because they have
identical migration on gels, overlapping elution times by
HPLC, and similar migration by CE. While HbG and HbD
are readily distinguished owing to their being α and β variants, respectively, the ability to separate such closely eluting
or migrating proteins can be used as a test of the precision of
each method.
To improve precision by HPLC, the Primus Ultra2 system (Trinity Biotech, Kansas City, MO) elution times are
expressed relative to the elution time of a standard (HbF,
HbA, HbS, or HbC) that was run through the same column
the same day. This approach presents data for the variant as an
arithmetic ratio to the standard with the nearest elution time.
Use of relative ratios by HPLC improves separation of closely
migrating Hbs.6,9
In this report, we describe our findings for the precision
of the migration position for common Hb variants evaluated
by CE. We have demonstrated that expressing the migration
© American Society for Clinical Pathology
Hematopathology / Original Article
position relative to HbA2 improves the ability of CE to
distinguish between 2 closely migrating variant Hbs (HbG
and HbD).
We recorded the positions of the peak of HbA2 and
the variant Hb in each sample. This approach allowed us to
express the actual migration of each variant and the migration
of each variant relative to that of HbA2.
Materials and Methods
Gel Electrophoresis
Selected variants were analyzed on alkaline and acid agarose gel electrophoresis using the Sebia Hydrasys 2 method
(Sebia) according to manufacturer’s guidelines. Each sample
was run with controls for HbA, HbS, HbF, and HbC.
Specimens
This was a retrospective study on 193 variant-containing
clinical samples of whole blood collected in EDTA that were
submitted for routine evaluation of hemoglobinopathies. They
were stored at 2°C to 8°C and processed by HPLC and CE
within 3 days following venipuncture. All samples used in the
study needed to contain HbA. We have included all cases that
contained 1 or more of the following Hbs: HbS, HbC, HbG,
or HbD. Each sample was examined by HPLC and CE. Our
study was conducted in accordance with a protocol (R-06703) approved by the institutional review board of St Joseph
Mercy Hospital, Ann Arbor, MI.
High-Performance Liquid Chromatography
HPLC was used to establish independently the initial
identification of the Hb variant. It was performed using the
Primus Ultra2 Resolution method, a cation exchange column.
This method relates the retention time of unknown Hbs to
that of a calibrating standard containing 4 Hbs: HbF, HbA,
HbS, and HbC. Whole blood specimens collected in EDTA
were lysed with the hemolyzing reagent (provided by the
manufacturer) for injection into the HPLC column. Elution of
adsorbed Hbs used a gradient formed by 2 mobile phases of
Bis-Tris and 1 mmol of potassium cyanide with different pH
values and ionic strengths, as previously described.3,5,6
Capillary Electrophoresis
CE was performed using the Sebia Capillarys 2 system
(Sebia, Norcross, GA). Manufacturer guidelines were followed in performing the analysis, as previously described.6
The Sebia Capillarys 2 records Hb migration on the x-axis
from 0 to 300. On each sample, when present, HbA is standardized to migrate at position 150. When a sample lacks
HbA, the instrument uses the archived information from the
most recent samples passing through that column to estimate
the position of HbA in order to generate the migration point.
While this provides reliable quantitative data for the variant,
in the absence of HbA, the migration position is imprecise.
When HbA is not present in a sample, the manufacturer recommends that the sample be diluted 1:1 with a normal sample
to obtain a better migration position of the Hbs present in that
sample. In addition to determining the migration position, the
presence of HbA generates a grid that divides the migration
into 12 zones. The manufacturer provides a table delineating
the variants it has determined to be present within each zone.
Statistics
The Student paired t test was used to determine the
significance of difference of coefficients of variation (CVs)
between migrations measured directly compared with migrations expressed relative to that of HbA2 in the same sample.
Results
Use of Migration Position by CE and Elution Time
by HPLC to Evaluate Variant Hbs
By CE, the mean migration position (x-axis value) and
SD for HbA2, HbS, HbC, HbG, and HbD were quite reproducible with CVs that ranged from 0.79% to 1.11% ❚Table 1❚.
The 2 most closely migrating variants in this study were HbG
and HbD. Both migrated in zone 6, with mean migrations of
205 and 208, respectively ❚Figure 1❚. Despite the tight SDs,
migration data alone were not able to consistently separate
the 24 cases of HbG from the 19 cases of HbD ❚Figure 2A❚.
Indeed, 25 of the 43 samples of HbG and HbD overlapped.
The absolute elution time recorded by HPLC was used to
compare separation of HbG and HbD cases with that of CE.
Similar to CE, by HPLC 30 of the 43 cases overlapped when
the absolute elution time was measured ❚Figure 2B❚.
Variant to HbA2 Ratio by CE and Relative Elution Time
by HPLC to Evaluate Variant Hbs
To evaluate whether an internal standard would improve
precision, the migration positions by CE were calculated
as an arithmetic ratio relative to that of HbA2 in the same
❚Table 1❚
Precision of Variant Location by Migration Position
HbA2
(n = 193)
Mean*
245
1 SD
2.3
Coefficient of 0.95
variation (%)
HbS
(n = 96)
HbC
(n = 54)
HbG
(n = 24)
HbD
(n = 19)
215
2.3
1.06
254
2.8
1.11
205
1.6
0.79
208
2.0
0.96
Hb, hemoglobin.
* Mean migration units (range, 1-300).
© American Society for Clinical Pathology
Am J Clin Pathol 2012;137:660-664
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Keren et al / Hb Variant Migration by CE Relative to HbA2
A
B
15
14 13
12
11 10 9 8
HbA
7
6 5 4 3 2
1
15
14 13
12
11 10 9 8
HbA
7
6 5 4 3 2
HbD
1
HbG
HbA2
HbA2 HbG
2
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
❚Figure 1❚ Capillary electrophoresis pattern with zones above and migration numbers below the electrophoretogram. A,
Hemoglobin (Hb)G migrates in zone 6 and HbG2 in zone 1. Fractions are as follows: HbA, 64.9%; HbG, 33.0%; HbA2, 1.4%; and
HbG2, 0.7%. B, HbD migrates in zone 6. Fractions are as follows: HbA, 55.4%; HbD, 41.5%; and HbA2, 3.1%.
❚Table 2❚
Precision of Variant Location Relative to HbA2
Mean
1 SD
Coefficient of
variation (%)
HbS/HbA2
(n = 96)
HbC/HbA2
(n = 54)
HbG/HbA2 HbD/HbA2
(n = 24)
(n = 19)
0.877
0.007
0.80
1.036
0.003
0.34
0.840
0.005
0.59
0.856
0.004
0.47
Hb, hemoglobin.
A
sample. The results shown in ❚Table 2❚ indicate that precision
improved for all variants (P < .001), as shown by a decrease
in the CVs. When the ratio (variant/HbA2) was used for HbG
and HbD, there was complete separation of all 43 cases ❚Figure 3A❚.
The counterpart of expressing the CE migration point
relative to that of HbA2 is the expression of the HPLC
elution time relative to that of HbS recommended by the
manufacturer. This arithmetic ratio improved the separation
B
7
7
HbD
HbG
6
5
No. of Cases
No. of Cases
5
4
3
2
1
0
200
HbD
HbG
6
4
3
2
1
202
204
206
208
Migration Position
210
212
0
4.800
5.000
5.200
5.400
5.600
5.800
Elution Time (min)
❚Figure 2❚ A, Capillary electrophoresis migration position for 24 cases of hemoglobin (Hb)G and 19 cases of HbD. B, Highperformance liquid chromatography elution time for 24 cases of HbG and 19 cases of HbD.
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Hematopathology / Original Article
A
B
12
14
HbD
HbG
12
No. of Cases
10
No. of Cases
HbD
HbG
10
8
6
4
8
6
4
2
2
0
0.81
0.82
0.83
0.84
0.85
0.86
0.87
Variant Migration Position/HbA2 Migration Position
0
0.88
0.90
0.92
0.94
0.96
0.98
Variant Elution Time/HbS Elution Time
❚Figure 3❚ A, Capillary electrophoresis variant migration relative to hemoglobin (Hb)A2 migration for 24 cases of HbG and 19
cases of HbD. B, High-performance liquid chromatography elution time relative to HbS elution time for 24 cases of HbG and 19
cases of HbD.
of the HbG and HbD cases by HPLC ❚Figure 3B❚. Only 2
cases still overlapped.
Discussion
HPLC is a reliable, rapid, precise procedure that has the
advantage of a well-characterized library of Hb variants.1-8,18
In people without the common variants HbS and HbE, HPLC
provides an accurate measure of HbA2. However, HPLC has
the disadvantage of providing a complex elution pattern. This
is due to several elution peaks from breakdown and posttranslational products of Hbs.6,13 Some of these products, such as
those from HbS, reside in the HbA peak, thereby decreasing
the percentage of HbS measured in heterozygotes.6,8 Other
HbS products comigrate and increase measured HbA2.8,13,18,19
In addition, on the Primus Ultra2 HPLC, common variants,
including HbE, HbG, and HbD, comigrate with HbA2, eliminating the ability to accurately measure HbA2 in their presence. By HPLC, the closely migrating Hbs such as HbG and
HbD can be distinguished from each other by virtue of the
unique presence of the product of the variant HbG α chain
with δ (HbG2). However, the elution times of HbG and HbD
themselves overlap in a few cases, even when expressed relative to that of HbS.17
CE has been used to evaluate hemoglobinopathies for
variant Hbs for 20 years.20-26 With early CE techniques, it
was demonstrated that CE could provide an accurate measurement of HbA2 and was superior to microcolumn techniques
for measuring HbA2 in patients who had HbS.22,26 Yet, inhouse methods and early commercial ventures were deemed
unlikely to compete with the rapid, accurate HPLC methods
available at that time.11
Developed in the past decade, the Sebia Capillarys has a
superior throughput compared with HPLC systems, provides
a more straightforward pattern for interpretation, and does not
have the need to account for glycated and breakdown products when measuring the most common variant Hbs.12,13,17,27
Previously, we reported that the percentage of HbS in heterozygotes is higher when measured by CE than by HPLC
because of the inability of the latter to account for fractions
of HbS that coelute with HbA2 and HbA.13 Furthermore,
HbA2 is not measurable by the Primus Ultra2 HPLC method
in the presence of the common variants HbG, HbD, or HbE,
while CE provides an accurate determination of HbA2 in these
situations.13,15,17
The present study sought to determine the precision of
CE in the identification of relatively common and 2 closely
migrating Hb variants. We found that the CVs of HbS, HbC,
HbD, and HbG hover around 1% from the direct migration
data. However, despite these tight CVs, we were not able to
separate HbG and HbD, finding that 25 of the 43 cases overlapped. These overlapping variants are used here to challenge
the precision of CE and HPLC in separating 2 closely migrating and relatively common variants.
The Primus Ultra2 HPLC technique expresses the elution
of Hbs relative to 1 of 4 standards (HbF, HbA, HbS, or HbC)
to improve precision over expressing the result as its absolute
elution time.3,5 On the current 43 cases of HbG and HbD, the
direct elution time was able to separate all but 2 of the cases.
To see if comparison with a standard would improve
precision on CE, we expressed the migration of relatively
© American Society for Clinical Pathology
Am J Clin Pathol 2012;137:660-664
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DOI: 10.1309/AJCPOF8V0JJOPSVF
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Keren et al / Hb Variant Migration by CE Relative to HbA2
common Hb variants relative to HbA2 as an internal standard. We found that this improved the precision of HbS,
HbC, HbD, and HbG as measured by their CVs. Furthermore, when this arithmetic ratio was used for the 2 closely
adjacent Hbs, it resulted in complete separation of all 43
cases of HbG and HbD.
These studies confirm the original observation by Louahabi et al12 demonstrating that precision of several common
Hb variants was improved by expressing the migration relative to that of HbA2 in the same specimen. The results of the
present study suggest that expression of other variants relative
to the migration of HbA2 may be helpful in creating a more
precise library of variants for CE. However, we note that
HbA2 migrates relatively near the variants studied. It may be
that variants with a more anodal migration may require a different internal standard to achieve a similar result.
From 1Department of Pathology, The University of Michigan, Ann
Arbor; and 2Warde Medical Laboratory, Ann Arbor.
Address reprint requests to Dr Keren: Dept of Pathology,
The University of Michigan Hospital and Health Systems, 5242
Medical Science 1, 1301 Catherine, SPC 5602, Ann Arbor, MI
48109-5602.
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© American Society for Clinical Pathology