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Annals o f Clinical & Laboratory Science, vol. 30, no.

2, 2000 191

Chromatographic Measurements of Hemoglobin A2 in Blood


Samples that Contain Sickle Hemoglobin

1 2 3
Masih Shokrani, Falicia Terrell, Ernest A. Turner, and Maria del Pilar Aguinaga
Comprehensive Sickle Cell Center, Meharry Medical College, Nashville, Tennessee; 1Department of
Microbiology, Immunology & Genetics, Department of Pediatrics, 3 Department of Obstetrics & Gynecology

Abstract. In the sickle cell syndromes, Hb A2 measurements aid in the differential diagnosis of sickle cell anemia
from sickle-beta-thalassemia. The purpose of this study is to assess the Hb A2levels in samples containing sickle
hemoglobin (Hb S) by the use of an automated high performance liquid chromatography system (HPLC-
Variant (3-thalassemia Short Program). The blood samples analyzed were from individuals of African descent
living in the state ofTennessee who had either sickle cell trait (Hb AS), sickle cell disease (Hb SS), or sickle cell-
hemoglobin C disease (Hb SC). Interestingly, the Hb A2 levels determined by HPLC were found elevated in
samples containing Hb S. The Hb A^ mean in Hb AS samples (n=l46) is 4.09% (SD + 0.42, range 2.20 to
5.20%); in Hb SS samples (n=33) it is 3.90% (SD 1.08, range 0.60 to 5-90%); and in Hb SC samples (n=27)
it is 4.46% (SD 0.70, range 2.30 to 5.91%). The H b A2 mean by HPLC in normal individuals (Hb AA,
n=70) is 2.57% (SD 0.25, range 2.1 to 3.0%), and the Hb A 2 range in (3-thalassemia carriers is 4 to 9%. Our
results show that the Hb A2 levels in Hb S-containing samples partially overlap with those expected from (3-
thalassemia carriers. The hemoglobinopathy laboratory should be aware of this apparent elevation in Hb A2
levels determined by HPLC in individuals carrying Hb S. Other factors, such as family history and clinical
symptoms, should be taken into account before a diagnosis of sickle cell trait, sickle-beta-thalassemia, or sickle
cell anemia is made.

Keywords: Hemoglobin A2, hemoglobin S, high-performance liquid chromatography, thalassemia diagnosis

Introduction (3-thalassemia when determined by capillary IEF. The


purpose of this report is to assess Hb A 2 levels by HPLC
The quantitation of H b A2 is im portant for the in liquid blood samples containing Hb S. We have
accurate diagnosis of hemoglobin disorders such as found that Hb A2 levels quantitated by HPLC are
the (3-thalassemias, or hem oglobin variants in elevated in samples from non-thalassemic individuals
combination with (3-thalassemia (eg, sickle-beta- that carry Hb S. We have not observed this increase
thalassemias). Hb A2 is known to be elevated in (3- in Hb A2 levels in samples that contain only Hb AA.
thalassemia trait [1], sickle-beta-thalassemia [2], and HPLC reference ranges for Hb A2 in sickle cell trait,
sickle cell disease with a-thalassemia [3]. In sickle sickle cell anemia, and Hb SC disease should be
cell trait, Hb A 2 determined by microchromatography established in order to prevent an erroneous association
has been found slightly elevated [4], and significantly of these disorders with (3-thalassemia.
elevated when determined by capillary isoelectric
focusing electrophoresis (IEF) [5]. However, no Methods
overlap in H b A2 levels was seen between Hb S -
containing samples and samples from patients with Blood specimens were collected in heparinized
capillaries or in ethylene diaminetetraacetare (EDTA)
Address correspondence to Maria del Pilar Aguinaga, Ph.D., microvettes (Sarstedt, Inc., Newton, NC). The criteria
Comprehensive Sickle Cell Center, Meharry Medical College, for sample inclusion were: (1) presence of Hb S, (2)
1005 Dr. D . B. Todd Jr. Bvld., Nashville, T N 37208; tel 615 327
6528; fax 615 327 5511; e-mail maguinaga@mmc.edu.
sample age < 7 days after collection, (3) symmetrical

0091-7370/00/0200-0191 $1.00; 2000 by the Association of Clinical Scientists, Inc.


192 Annab o f Clinical & Laboratory Science

Hb A 2 chromatogram peak (Fig. 1), and (4) subjects cation exchange HPLC. The resin of the column has
from 1 to 60 years of age. Samples from subjects being a negatively charged surface group that will bind the
treated with hydroxyurea, a medication prescribed for positively charged protein (Hb), while the more
treatment of sickle cell disease that increases Hb F negatively charged molecules will elute readily from
levels, were excluded from this study, as were samples the column. A higher cation concentration will be
diagnosed as sickle-beta-thalassemia (Fig. 1) or samples required to elute the positively charged hemoglobin
with asymmetrical Hb A2 peak (Fig. 2). Blood from the column. The ionic strength of the elution
specimens were received in the laboratory by postal buffer mixture is increased by raising the percent
service, courier, or walk-in. Red cell hemolysates were contribution of elution buffer #2. As the ionic strength
run by alkaline electrophoresis (pH 8.6) (Helena of the mixture increases, more strongly retained
Laboratories, Inc., Beaumont, TX) and by isoelectric hemoglobins elute from the column [8],
focusing electrophoresis (IEF) (Wallace Laboratories,
Inc., Akron, OH) before HPLC analysis. The Bio- Results
Rad Variant (3-thalassemia Short Program was used
for Hb quantitations. The Variant (Bio-Rad Labora For this study, 146 Hb AS samples, 33 H b SS samples,
tories, Inc., Hercules, CA) is a fully automated HPLC and 28 Hb SC samples were analyzed by HPLC.
system that separates a hemoglobin mixture into its Results showed that in Hb AS samples, the mean for
individual components by pumping liquids through a Hb A^ is 4.09% (SD 0.42, range 2.20 to 5.2%). In
column at high pressure. It is a fast and reproducible Hb SS samples, the mean for Hb A 2 is 3.90% (SD +
method that can be used to separate and determine 1.08, range 0.60 to 5.90%. In H b SC samples, the
area percentages for various hemoglobins, including mean for Hb A^ is 4.46% (SD 0.70, range 2.30 to
Hb F and Hb A r It can also provide qualitative 5.91%). The corresponding hemoglobin quantitations
determinations of abnormal hemoglobins. The (3- for Hb F, Hb A, Hb S, and Hb C are shown in Tables
thalassemia Short Program utilizes the principle of 1, 2, and 3 for the sample populations mentioned

Elution Time (min.)


E lu tio n T im e (m in.)

Fig. 1. Bio-Rad HPLC chromatogram showing a typi Fig. 2. Bio-Rad HPLC chromatogram showing an
cal Hb A2 peak (symmetrical peak). This patient was atypical Hb A2 peak (asymmetrical peak, having a
diagnosed as sickle-beta-thalassemia. shoulder). This patient was diagnosed as sickle cell
trait.
HPLC analysis ofH b A 2 in blood that contains H b S 193

above. The reference range for Hb A2in normal adults It has been postulated that, in the presence of
is 2.1 to 3.0%, with a mean of 2.57% [8] using the P-chain hemoglobin variants, the association of the
Bio-Rad Variant system. OC-globin chain with the (3-globin chain variant is
impaired, which fosters the association of CX-chains with
Discussion 8-chains, increasing the Hb A^ levels [10,11]. Other
factors that play a role in this increase are the
The sensitivity of HPLC has significantly contributed accumulation of Hb S adducts and sample aging. The
to the accurate quantitation of hemoglobin variants. Hb S adducts include glycohemoglobin [11] and other
In this report we have established reference ranges for post-translational modification products of Hb S. The
H b A2 quantified by HPLC (Bio-Rad Variant (3- effect of sample aging is observed in the accumulation
thalassemia Short Program) in samples containing of glycohemoglobin in blood samples over time [7].
Hb S. We found that blood samples containing H b S If Hb Scontaining blood samples are stored for 2
usually have increased Hb A2levels when compared to weeks, Hb A2 levels are increased, possibly due to
previously published HPLC reference ranges [8,9]. The accumulation of glycohemoglobin [7,12]. We did not
HPLC manufacturer states that Hb S byproducts could determine glycohemoglobin in our samples. Since the
coelute with Hb A2 [8]. We have not observed increased blood samples used for this study were relatively fresh
H b A2 values by HPLC in normal Hb AA samples. (1 week) and we still observed a marked increase in
Hb A2, we postulate that other factors may be involved
in the increase of Hb A2 in Hb S-containing samples
Table 1: Hemoglobin percentages in Hb AS samples when determined by HPLC.
(sickle cell trait), based on HPLC analysis. Concurrently elevated levels of Hb A2 and Hb F
Hb N Mean SD (%) Range (%) offer a practical way to diagnose carriers o f the
p-thalassemia gene or other Hb disorders that lead to
A 146 53.802.93 (47.70 - 63.60) an increase in this hemoglobin. The HPLC manu
S 146 34.663.71 (21.2 0 - 42.10) facturer indicates that a range for Hb A^ of 4 to 9% is
a2 146 4.090.42 (2.20 - 5.20) typical of heterozygous P-thalassemia [8]. O ur results
F 98 0.980.67 (0.20 - 3.90) show that in samples containing Hb S, the Hb A^ values
are elevated above the normal reference ranges given
by the manufacturer [8] and also above those that we
previously reported in a healthy African American
Table 2: Hemoglobin percentages in Hb SS samples population (Hb A^ range = 0.05 to 3.4%, mean 1.2%)
(sickle cell disease), based on HPLC analysis. [9]. W ith the system used in the present study, Hb A2
Hb N Mean SD (%) Rang; (%) levels in samples containing Hb S partially overlap with
those expected for P-thalassemia carriers. When Hb
S 33 86.743.62 (80.40 -- 94.70)
A2 quantitations in Hb Scontaining samples are
A, 33 3.901.08 (0.60 -- 5.90)
reported, it should be recognized that the apparently
F 33 6.802.95 (1 .3 0 - 11.40)
elevated Hb A2 values may well be within the normal
range for those samples. The elevated Hb A2 values
should be correlated with the clinical picture and family
Table 3: Hemoglobin percentages in Hb SC samples
studies to help establish the diagnosis.
(hemoglobin SC disease), based on HPLC analysis.

Hb N Mean SD (%) Range(%) Conclusions


S 28 46.461.27 (43.70 - 49.20)
C 28 44.251.74 (40.40 - 46.90) The HPLC Hb A2 levels in subjects older than 1 year
27 4.46+0.70 (2.3 0 -5 .9 1 ) and younger than 60 years of age having sickle cell
A,
F 27 2.17+1.51 (0.5 0 -6 .1 0 ) trait (Hb AS) or sickle cell disease (Hb SS and Hb SC)
have been assessed. These results confirm the elevation
194 Annals o f Clinical & Laboratory Science

of Hb A2 levels in individuals with Hb AS, Hb SS, 3. Balias SK, Gay RN, Chehab FE Is H b A2 elevated in
and Hb SC, as determined by the BioRad Variant adults with sickle-alpha-thalassemia (beta(S)/beta(S);
HPLC System. We conclude that in Hb AS, levels of -alpha/-alpha)? Hemoglobin 1997;21:405-450.
H b A2lower than 5.2% are normal. In Hb SS and Hb 4. Huisman THJ, Shroeder WA, Brodie AN, Mayson SM,
JakwayJ. Microchromatography of hemoglobins, III:
SC samples, levels of Hb A2lower than 5.9% may also
a simplified procedure for the determination of Hb Aj-
be normal if there is no other sign of P-thalassemia.
J Lab Clin Med 1975;86:700-702.
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cell disease patient, one should also consider family Hempe JM. Hemoglobin A2 levels in healthy persons,
history, clinical symptoms, drug treatment, and sample sickle cell disease, sickle cell trait, and P-thalassemia by
aging before the diagnosis o f an associated Pi- capillary isoelectric focusing. Am J Clin Path 1997;
thalassemia disorder is made. 107:88-91.
6. Ou CN, Buffone GJ, Reimer GL, Alpert AJ. High-
Acknowledgments perform ance liquid chrom atography o f hum an
hemoglobins on a new cation exchange. J Chromatog
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7. Kutlar A, Kutlar F, Wilson JB, Headlee M G, Huisman
reading of the manuscript, Ms. Mai Williamson for
TH J. Quantitation of hemoglobin components by
technical assistance, Ms. Deborah Majors for statistical
high-performance cation-exchange liquid chrom
analyses, and Ms. Tasha White and Ms. Carolyn Tansey atography: its use in the diagnosis and in the assessment
for typing the manuscript. Supported by grants from of distribution of hemoglobin variants. Am J Hematol
the Tennessee Department of Health and Environment 1984;17:39-53.
(GR-6 10510-600), and the National Heart, Lung and 8. BioRad. Variant Beta-Thalassemia Short Program
Blood Institute (P60 HL-38737 and K14 HL-03141). Procedure Manual. BioRad Laboratories, Inc., Hercules,
CA, 1994.
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