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Articles |
1
Department of Clinical Chemistry, Hôpital Erasme, Université Libre de Bruxelles, 808 route de Lennik, B1070 Brussels, Belgium.
2
Analis SA, 14 rue Dewez, B5000 Namur, Belgium.
a Author for correspondence. Fax 32 2 555 6655; e-mail fcotton{at}ulb.ac.be.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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analytical methods
Capillary electrophoresis.
We tested the "Analis kit for Hb
A2 quantification and Hb Variants screening with the
Beckman P/ACETM" (Analis, Namur, Belgium). The reagents
for this assay are patented and proprietary (8). Assays
were performed following the manufacturer's instructions on a Beckman
P/ACE 5500 System (Beckman Inc.) equipped with a 25 µm (i.d.)
x 24 cm (effective length 17 cm) fused-silica capillary thermostated
at 26 °C. The capillary was first rinsed for 0.5 min under a
pressure of 138 kPa with the "initiator" solution (solution of a
polycation in a malic acid buffer, pH 4.7). This process coats the
capillary wall with the polycation because a large number of anchor
points form between the negative charges of the silanol groups of the
capillary glass and the positive charges of the polycation. This
rinse was followed by a 1.0-min rinse with the "buffer" solution
(solution of a sulfated polyanion in arginine buffer, pH 8.7), which
adds a second layer of coating because a large number of anchor points
form between the polycation and the polyanion. This double layer of
polymer provides a large number of negative sulfated charges facing the
inside of the capillary, creating a zeta potential higher than the
original charge from the silanol groups. This double layer produces a
strong, reproducible electroosmotic flow and a decrease in
protein adhesion to the capillary wall.
After hemolysis with a provided reagent [10 µL of whole blood + 50 µL of hemolyzer (arginine buffer, pH 8.7)], the sample was injected for 3 s by pressure (3.5 kPa). The buffer solution was then injected twice (1 s and 10 s under pressure) to clean the outside of the capillary and to push the sample inside. Separation of Hbs according to their mass-to-charge ratio was performed at 10.8 kV for 6.0 min in the same buffer solution. After separation, the coating was removed by first rinsing the capillary with the "conditioner" solution (0.2 mol/L NaOH) for 0.75 min under pressure (138 kPa) and for another 0.75 min under pressure and with current (200 µA) and then rinsing with water for 0.5 min. Detection was made at 415 nm using an on-line ultraviolet-visible light detector. Electropherograms were analyzed with the Beckman P/ACE Station software, Ver. 1.0. Results were expressed as the corrected area percentage for each Hb fraction. Expected values provided by the manufacturer, obtained from a population of 120 randomly selected ambulatory and hospitalized subjects, were 2.1–3.3% for Hb A2 and 0.1–1.1% for Hb F.
Automated HPLC.
Automated cation-exchange HPLC
(VariantTM Hemoglobin System Beta-Thalassemia Short
Program; Bio-Rad Laboratories) was used as the comparison method for Hb
A2 and Hb F, following the manufacturer's instructions
(3, 9). The expected values provided by
the manufacturer were 2.1–3.0% for Hb A2 and <1.0% for
Hb F.
Anion-exchange chromatography.
Microcolumn anion-exchange
chromatography (MAEC; Quik-SepTM Hemoglobin A2
Test System; Isolab) was used as the comparison method for Hb
A2 only. The method was applied following the
manufacturer's instructions (4). The expected values
provided by the manufacturer were 1.5–3.0% for Hb A2.
analytical performance
Imprecision.
Total imprecision for migration times and
concentrations of Hb fractions were determined by analyzing three
samples once a day for 10 days and by calculating the CVs.
Linearity.
The linearity of Hb A2 and Hb F
quantification was evaluated by assaying mixtures of different volumes
of a normal cord blood (Hb A2 undetectable; Hb F, 84.8%)
and of a blood from a healthy adult (Hb A2, 2.9%;
Hb F, 1.0%) to obtain various Hb A2 and Hb F
concentrations between these limits. Seven mixtures were prepared in
duplicate and analyzed. Because accurate expected concentrations of Hb
A2 and Hb F were unknown (they are related to hematocrit
and the mean corpuscular Hb concentration of each sample), the
results obtained for Hb F were plotted against those obtained for Hb
A2 in each mixture. Linear regression was applied, and
results were graphically interpreted.
Limit of quantification for Hb F.
Packed red cells from a
blood displaying high Hb F concentrations (7.4% with CZE) were
serially diluted with packed red cells from a blood having undetectable
Hb F concentrations. Six mixtures displaying theoretical Hb F
concentrations ranging from 0.2% to 7.4% were analyzed in triplicate
by CZE. The limit of quantification was defined as the least amount of
Hb F that could be quantified reproducibly (CV <10%).
Interference.
The influence of Hb S on Hb A2 and
Hb F quantification was tested by analyzing samples containing Hb S,
using all three methods.
comparison of methods
Fifty-seven samples containing no abnormal Hbs were selected and
analyzed by all three methods.
reference values
The reference range for Hb A2 was determined
by calculating the 2.5–97.5 percentile interval obtained in the 107
blood donors. The 97.5 percentile for Hb F in the same population was
considered as the high reference value.
statistical analysis
Comparisons of methods were made with the Passing and Bablok
method using the Analyse-It for Microsoft Excel (Analyse-It Software
Ltd.). Linear regression was used in the linearity studies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The separation of Hb fractions was achieved within 6 min. The
hemolyzer marker, Hb A2, Hb S, Hb F, and Hb A
displayed migration times of 4.59, 4.92, 4.99, 5.12, and 5.27 min,
respectively. These correspond to relative migration times (calculated
as the Hb fraction migration time divided by the hemolyzer marker
migration time) of 1.00, 1.07, 1.09, 1.12, and 1.15, respectively.
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imprecision
The total CVs for the migration times are summarized in Table 1
, and the total CVs for quantification of the Hb fractions are
summarized in Table 2
. The imprecision was always <2% for migration times and <1%
for relative migration times. The CV for Hb F quantification was 5%
for concentrations ranging from 1.8% to 18.9%; the CV for Hb
A2 was 3–6% for concentrations ranging from
2.0% to 5.6%.
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linearity
The measured concentrations of Hb A2 and Hb
F obtained in mixtures prepared as explained in Materials and
Methods are shown in Fig. 2
. The relationship between Hb A2 and Hb F
seemed linear (r2 = 0.9934; Fig. 2A
).
The same procedure applied to HPLC gave also good results
(r2 = 0.9839; Fig. 2B
).
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limit of quantification for Hb F
The Hb F results in serial dilutions (given as the mean
concentration and the CV) were as follows: mean, 7.4%, CV, 2%; mean,
3.4%, CV, 4%; mean, 1.7%, CV, 6%; mean, 0.9%, CV, 3%;
undetectable; and undetectable. The value of 0.9% was thus
considered as the quantification limit for Hb F.
comparison of methods
The comparisons for Hb A2 quantification are
shown in Fig. 3
. All three methods gave similar results but significant
proportional biases were observed between CZE and other methods, and a
significant constant bias was observed between CZE and MAEC as well as
between HPLC and MAEC (Table 3
). The comparison of Hb F concentrations obtained with HPLC and
CZE is shown in Fig. 4
. Only samples with Hb F concentrations above the CZE
quantification limit were included (n = 33). Results of both
methods were comparable despite constant and proportional biases (Table 3
).
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reference values
The distributions of Hb F and Hb A2 are
shown in Fig. 5
. The calculated reference values were 2.7–3.8% for Hb
A2 and <1.2% for Hb F.
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interference
We tested the effect of Hb S on Hb
A2 and Hb F quantification, according to the
method of Suh et al. (10), by assaying samples that
contained [Hb AS (n = 8) and Hb SS (n = 7)] or did not
contain Hb S [Hb AA (n = 57)] by all three methods. The biases
in Hb A2 values obtained between HPLC or CZE and
MAEC were compared in samples with or without Hb S (Table 4
). Using the Student t-test, we determined that the
mean bias in Hb A2 values between MAEC and CZE
was not significantly higher in Hb S than in Hb AA samples. On the
other hand, the bias between MAEC and HPLC was significantly higher
(P <0.000001) in Hb S than in Hb AA samples. The bias in Hb
F values between CZE and HPLC was not statistically different in
samples with Hb S and samples without Hb S. Nevertheless, in S
heterozygotes, measurement of low Hb F concentrations (
3%) was not
possible, because of an incomplete return to the baseline of the
electropherogram between Hb A and Hb S (Fig. 1C
).
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Hb variants
Analysis of samples containing common Hb variants gave the
following results: Hb S was separated from Hb A and Hb A2,
but not from Hb D-Punjab and Hb G-Philadelphia; Hb C and Hb O-Arab were
separated from Hb A, but not completely from Hb A2; and Hb
E coeluted with Hb A2 (not shown, see
Discussion).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thalassemia trait and in iron
deficiency. Increases in Hb F can be observed in conditions such as
hereditary persistence of fetal Hb, ß-thalassemia intermedia,
ß-thalassemia major, and some drug treatments. Hb F inhibits
polymerization of Hb S and thus prevents painful crises in sickle cell
disease. Hydroxyurea is used to increase Hb F concentrations in sickle
cell patients. The monitoring of Hb F concentrations during the follow
up of these patients is thus mandatory. Ion-exchange chromatography is considered as a reference method for Hb A2 and Hb F measurements (3)(4). The latter is also measured by alkaline denaturation and immunological methods (3). We compared a CZE method developed for Hb A2 and Hb F quantification to commonly used ion-exchange chromatographic methods. This new method compares favorably with the traditional methods. Nevertheless, higher values are obtained, which are also reflected by the higher reference values provided by the manufacturer. The reference values that we determined for Hb A2 are somewhat higher than those provided with the kit. This can be explained by the fact that the manufacturer used blood from patients, potentially including patients with iron deficiency.
The CZE method displays excellent CVs for both migration times and quantification, similar to those published for chromatography (3)(4)(6)(7)(9).
The presence of Hb S was shown to influence Hb A2 quantification with the HPLC method used in this study (10). Spuriously increased concentrations are obtained, probably because of Hb S adducts coeluting with Hb A2 in this system. Hb A2 quantification is not influenced by Hb S in the CZE method, which provides accurate Hb A2 values. This allows the detection of the ß-thalassemia trait and the differentiation of S-ß°-thalassemia patients from S homozygotes, for example. To the same extent, Hb F quantification is accurate in Hb SS patients, allowing the induction of Hb F by hydroxyurea in sickle cell patients to be followed.
The results obtained with samples containing Hb variants showed that this kit might be suitable only for a limited screening. For further characterization of the variant, this method should be combined with another (11). Moreover, in the presence of some of these variants (e.g., Hb C, Hb E, and Hb O-Arab), quantification of Hb A2 becomes impossible because of incomplete resolution. This drawback is seen in almost all methods (3)(7)(9) but seems clinically insignificant.
The last 5 years have seen the appearance of an increasing number of capillary electrophoresis applications in clinical chemistry. In the field of Hb study, several methods have been developed, mainly based on capillary isoelectric focusing (5)(6)(7). Although highly effective, this technique is less suitable for routine use than older chromatographic methods because of unacceptable imprecision at low concentrations of minor fractions, time consumption, and higher costs (7). A recent study described a homemade CZE method for Hb A2 quantification (6). Unfortunately, the CVs are quite high, and the method does not allow Hb F separation and measurement. At the same time, a highly effective commercial CZE method for Hb A1c determination has been released (5). It uses the same original procedure of dynamic coating, which maintains constant electroosmotic flow from run to run and from capillary to capillary and decreases protein adhesion to the capillary wall. Hence, variations in electroosmotic flow are the main source of imprecision encountered in CZE.
This new capillary electrophoresis method is precise, quick, and comparable to well-accepted chromatographic methods for Hb A2 measurement without interference from Hb S; thus, it is suitable for routine use. Moreover, it allows precise estimation of Hb F. This method could be a worthwhile tool in the clinical laboratory studying Hb diseases.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  D. D. Mais, R. D. Gulbranson, and D. F. Keren The Range of Hemoglobin A2 in Hemoglobin E Heterozygotes as Determined by Capillary Electrophoresis Am J Clin Pathol, July 1, 2009; 132(1): 34 - 38. [Abstract] [Full Text] [PDF]
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T. N. Higgins, A. Khajuria, and M. Mack Quantification of HbA2 in Patients With and Without {beta}-Thalassemia and in the Presence of HbS, HbC, HbE, and HbD Punjab Hemoglobin Variants: Comparison of Two Systems Am J Clin Pathol, March 1, 2009; 131(3): 357 - 362. [Abstract] [Full Text] [PDF]
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 A Mosca, R Paleari, G Ivaldi, R Galanello, and P C Giordano The role of haemoglobin A2 testing in the diagnosis of thalassaemias and related haemoglobinopathies J. Clin. Pathol., January 1, 2009; 62(1): 13 - 17. [Abstract] [Full Text] [PDF]
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 K. Zurbriggen, M. Schmugge, M. Schmid, S. Durka, P. Kleinert, T. Kuster, C. W. Heizmann, and H. Troxler Analysis of Minor Hemoglobins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Clin. Chem., June 1, 2005; 51(6): 989 - 996. [Abstract] [Full Text] [PDF]
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A. Joutovsky, J. Hadzi-Nesic, and M. A. Nardi HPLC Retention Time as a Diagnostic Tool for Hemoglobin Variants and Hemoglobinopathies: A Study of 60000 Samples in a Clinical Diagnostic Laboratory Clin. Chem., October 1, 2004; 50(10): 1736 - 1747. [Abstract] [Full Text] [PDF]
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G. M. Clarke and T. N. Higgins Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update Clin. Chem., August 1, 2000; 46(8): 1284 - 1290. [Abstract] [Full Text] [PDF]
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F. Cotton, B. Gulbis, V. Hansen, F. Vertongen, and S. Dash Interference of Hemoglobin D in Hemoglobin A2 Measurement by Cation-Exchange HPLC • Dr. Dash responds: Clin. Chem., August 1, 1999; 45(8): 1317 - 1318. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and Hb A2 (
)
values (%) in 107 healthy blood donors.