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Molecular Pathology and Genetics |
1
Thalassemia Research Center, Institute of Science and Technology for Research and Development, Division of Hematology, Department of Medicine, Mahidol University, Nakornpathom 73170, Thailand.
2
Department of Obstetrics and Gynecology, Faculty of
Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700,
Thailand.
3
Department of Pathology, Faculty of Medicine Ramathibodi
Hospital, Mahidol University, Bangkok 10400, Thailand.
4
Bio-Rad Laboratories, 9810 Nazarette, Belgium.
a Address correspondence to this author at: Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Puttamonthon 4 Rd., Nakornpathom 73170, Thailand. Fax 662-889-2559; e-mail grsfc{at}mahidol.ac.th.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-thalassemia and
ß-thalassemia syndromes in Thailand. The beta-thalassemia short
program is applicable to the diagnosis of -thalassemia and
ß-thalassemia disorders, including Hb H, EA Bart's disease, and EF
Bart's disease, in adults, newborns, and fetuses. The system cannot
quantify accurately certain Hb molecules, such as Hb H and Hb Bart's.
The alpha-thalassemia short program was therefore developed and used to
quantify Hb Bart's to detect -thalassemia genotypes in cord blood.
This automated HPLC system is an alternative approach to the diagnosis
of complicated thalassemia syndromes in Thailand and Southeast Asia. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thalassemia 1/-thalassemia 1),
homozygous ß-thalassemia, and ß-thalassemia/Hb E. Diagnoses of
thalassemia and abnormal Hbs can be performed at three different stages
of development: the prenatal, neonatal, and adult stages. Conventional
methods, including erythrocyte indices and morphology, Hb
electrophoresis, quantitation of Hb A2, Hb E, and Hb F, and
detection of erythrocytes containing Hb H inclusion bodies, provide an
accurate diagnosis (1)(2)(3)(4). Although measurement of Hb
A2 and Hb E by cellulose acetate electrophoresis and
elution (5) and microcolumn chromatographic (6)
techniques are reproducible and accurate, they are labor-intensive and
time-consuming.
HPLC is a sensitive and precise method for detecting thalassemia and
abnormal Hbs (7)(8)(9)(10). It has become the preferred
method for thalassemia screening because of its speed and reliability.
An automatic HPLC system (VARIANT(TM), Bio-Rad) has been
developed primarily for the detection of ß-thalassemia disorders such
as ß-thalassemia carriers, Hb S and Hb C. But information is quite
limited about using such a system to study the complicated
-thalassemia and ß-thalassemia syndromes in Southeast Asia
(11). In this study, we used the automatic HPLC system set
up with the alpha-thalassemia short (ATS) program that we developed and
with the beta-thalassemia short (BTS) program to detect various types
of thalassemias in both prenatal and postnatal specimens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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procedures
Erythrocyte indices and Hb concentrations were determined
using an automatic cell counter, the Sysmex NE 1500 (TOA). Quantitation
of Hb A2, Hb E, Hb A, and Hb F was performed by the
BTS program; quantitation of Hb Bart's was performed by the ATS
program on the Bio-Rad VARIANT, a fully automated HPLC system that uses
double wavelength detection (415 and 690 nm). The ATS program,
developed by the manufacturer, has been designed to separate and
quantitate Hb Bart's from all other Hbs in a 6 min run. Hb Bart's and
total Hbs elute at 1.70 and 3.15 min. This program uses a 3 cm x
0.46 cm cartridge packed with a 5-µm porous silica-based weak cation
exchange material. The analytes are eluted at a flow rate of 2 mL/min,
using a step gradient of two phosphate buffers that differ in pH and
ionic strength. An in-line prefilter is installed between the injector
and the column. Quantitation is based on a specific Hb Bart's
calibrator. Samples of whole blood (5 µl in 2 mL of buffer) are
hemolyzed before injection. The hemolysate (20 µL) is injected onto
the cartridge for analysis. All reagents were provided by the
manufacturer and used according to the manufacturer's instructions.
The VARIANT system was compared with an electrophoresis/elution method (5) for Hb A2 (E) measurement and against the alkali denaturation method of Betke et al. (12) for Hb F measurement in a total of 245 healthy, ß-thalassemia trait, Hb E trait, or ß-thalassemia/Hb E disease samples.
In the cord-blood study Hb types, including Hb Bart's and Hb Constant
Spring (Hb CS), were also confirmed by isoelectric focusing (Isolab).
The quantitation of Hb Bart's by HPLC was also compared with
microcolumn chromatography by analyzing 50 cord-blood samples with a
commercial kit, Quik-Sep (Isolab). The presence of ß-thalassemia and
Hb E in cord-blood samples was confirmed by dot blot hybridization
between the amplified DNA and the allele-specific oligonucleotide
probes (13)(14); in fetal blood samples,
ß-thalassemia and Hb E were confirmed by reverse dot blot
hybridization (15). Detection of -thalassemia 1,
-thalassemia 2 (both the rightward, -3.7, and the
leftward, -4.2, deletion types), and Hb CS was by PCR,
as described previously (16)(17)(18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. Hb F was eluted at 1.38 min, Hb A at 2.64 min, and Hb
A2 at 3.84 min; the total analysis time was 6.5 min. Fig. 1B
illustrates the ability of BTS program to distinguish various Hbs in
the Lyphochek Hemoglobin A2/F control (Bio-Rad Laboratories). In
samples with the ß-thalassemia trait, the chromatogram is similar to
that of the nondiseased sample, but the concentration of Hb
A2 was increased. Mutations causing ß-thalassemia produce
a deficit of ß-globin production that ranges from minimal
(ß-thalassemia) to a complete absence of ß-globin
(ß-thalassemia). In homozygous ß-thalassemia, the
chromatogram shows the prominence of Hb F; the absence of or minor
amounts of Hb A, depending on the genotype of ß-thalassemia
(ß-thalassemia or ß-thalassemia,
respectively); and an unaffected or increased Hb A2
concentration (Fig. 1C
). Hb E was eluted at the same retention time as
the Hb A2 found in healthy samples or samples with the
ß-thalassemia trait. Usually 25–30% was detected in Hb E
heterozygotes, and almost 100% was detected in Hb E homozygotes (Fig. 1
, D and E). The chromatogram also shows the presence of a Hb A peak in
ß-thalassemia/Hb E disease, whereas there was no Hb A in
ß-thalassemia/Hb E disease (Fig. 1F
).
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In Hb H disease, there were one or two abnormal peaks present at the
retention time of 1 min, before the Hb F peak. The two peaks represent
Hb Bart's and Hb H, respectively (Fig. 1G
). For EA Bart's disease
(-thalassemia 1/-thalassemia 2-EA), the chromatogram showed
increased concentrations of Hb E (~15%) and Hb F (~2–3%; Fig. 1H
). For EF Bart's disease (-thalassemia 1/-thalassemia 2-EE or
-thalassemia 1/-thalassemia 2-EF), the chromatogram illustrates
the predominance of Hb F and Hb E, with minor amounts of Hb Bart's
(Fig. 1I
)
In healthy newborns, the major Hb was Hb F (>80%), and Hb A was found
in small amounts (Fig. 2
A). The Hb E peak was also detected in newborns with Hb E
syndromes (Fig. 2B
). In newborns with -thalassemia diseases, the Hb
Bart's peak was also detected (Fig. 2
, C and E). The pattern of the
chromatogram of Hb Bart's hydrops fetalis was very specific, and the
major Hb eluted at almost 0 retention time (Fig. 2C
). Because the BTS
program cannot quantitate Hb Bart's, the ATS program was developed to
identify and quantitate Hb Bart's in -thalassemia (Fig. 2
, D and
F). The presence of Hb Bart's (first peak) was confirmed by an
isoelectric focusing method. The second peak in the chromatogram
represents other Hb derivatives.
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Comparison of concentrations of Hb A
2, Hb E,
HB F, and Hb Bart's measured by HPLC and conventional methods.
Precision studies were performed using blood samples with different
concentrations of Hb A2 and Hb F. Each sample was analyzed
in 20 replicates for each HPLC assay for the within-run precision study
and analyzed once a day for 10 consecutive days for the between-days
precision study. The results showed low within-run variability in the
measurement of Hb A2 concentrations within the reference
range or of increased concentrations of Hb A2 in samples
with ß-thalassemia and with the Hb E trait (CV = 0.46–0.84%);
the results of the measurement of Hb F concentrations within the
reference range or of the raised Hb F concentrations in the two
ß-thalassemia/Hb E samples also showed little within-run variability
(CV = 0.31–4.70%). An increase in imprecision (CV =
2.32–10.66%) was observed in the between-days precision study,
especially in the measurement of Hb F concentrations within the
reference range.
Quantitative values for Hb A2, Hb E, Hb F, and Hb
Bart's by HPLC were in agreement with values obtained using the
conventional methods (Table 1
). The linear regression equation of Hb A2 and Hb E
measured by HPLC (VARIANT BTS program) vs those measured by
electrophoresis/elution was y = 0.274
1.019x, with a correlation coefficient of 0.99. Hb F
measured by HPLC (BTS program) was also compared with the method of
Betke et al. (12); the linear regression equation was
y = 0.218 1.380x, with a correlation
coefficient of 0.99. Although linear regression analysis of Hb F
measured by HPLC showed excellent correlation with values obtained by
the alkali denaturation method, the mean values of Hb F detected by
HPLC were substantially higher than those determined by the alkali
denaturation method (Table 1
). The amounts of Hb Bart's measured by
the ATS program was also comparable with those obtained by microcolumn
technique; the linear regression line was y = 0.416
0.915x, with a correlation coefficient of 0.99.
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thalassemia screening by hplc
Postnatal screening with adult blood.
A total of 4147 adult
blood specimens were screened for thalassemias and hemoglobinopathies.
Subjects were from the routine hematology clinic. Apparently healthy
individuals with Hb concentrations <120 g/L were excluded to avoid the
effect of iron deficiency anemia. Table 2
shows that the concentration of Hb A2
increased with the ß-thalassemia trait, and the Hb F concentration
increased (
1%) in 40% of those cases. Hb concentrations >10% at
the position of Hb A2 were assumed to be Hb E. The
concentration of Hb E was 27.8 ± 7.5% in the heterozygous state
and 90.2 ± 4.9% in the homozygous state. In EA Bart's disease,
the Hb E concentration was 14.9 ± 1.6%, with the appearance of
an abnormal peak at the Bart's position. A presumptive diagnosis was
made for the -thalassemia 1 trait when patients had a low mean
corpuscular volume (MCV, 70 ± 6.9 x 10-15 L)
and concentrations of Hb A2 within the reference range
without anemia. Although the VARIANT BTS program cannot measure the
concentration of Hb Bart's and Hb H in Hb H disease, the abnormal
peaks representing these Hbs were shown (Fig. 1G
). Diagnosis of Hb H
disease was also confirmed by the finding of erythrocytes containing
inclusion bodies.
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Neonatal screening with cord blood.
A total of 521 cord-blood
samples were screened with the two VARIANT programs. Of these, 195
cases (37.4%) were found to have thalassemias and hemoglobinopathies.
The presence of Hb E, Hb Bart's, Hb H, and an abnormal Hb was
confirmed by isoelectric focusing. Dot blot hybridization with the
allele-specific oligonucleotide probes detected the
ßE-globin gene in 141 cord blood samples in
which the Hb concentration at the Hb A2 position was >2%
(Table 3
). Of these, 12 cases were found to be homozygous for Hb E; one
case was later found to be EF Bart's disease (-thalassemia
1/-thalassemia 2-EE), with a Hb E concentration of 30.1% and a Hb
Bart's concentration of 17.55%. The Hb E concentration in the
remaining cases of homozygous Hb E ranged between 3.9% and 14.9% with
a mean ± SD = 8.0 ± 3.55%. Two newborns carrying one
allele of ßE were also found to have EA Bart's disease
(-thalassemia 1/-thalassemia 2-EA) and homozygous Hbcs
(CS/CS-EA), respectively. A newborn with Hb A2 (E) (2% of
total Hb) was also found to have ß-thalassemia/Hb E
disease by dot blot hybridization, which demonstrated the presence of a
mutation in the IVS II nucleotide 654, a mutation of C
T in one
allele and of the ßE-globin gene in the other.
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PCR detected -thalassemia determinants, including -thalassemia 1,
-thalassemia 2, and Hb CS in 70 cord-blood specimens, of which 15
cases also had Hb E. Of these, two were found to have Hb H disease, one
had EA Bart's disease, one had EF Bart's disease, and two were
homozygous for Hb CS (Hb CS/Hb CS). The concentrations of Hb Bart's
determined by the ATS program of VARIANT were all >15% except for a
case of homozygous Hb CS coinherited with Hb E, in which the Hb Bart's
was 8.3% (Table 3
). Because the Hb Bart's concentrations were
similar, DNA analysis was necessary to distinguish -thalassemia
syndromes among the -thalassemia 1 heterozygotes, the
-thalassemia 2 homozygotes, and compound heterozygotes for
-thalassemia 2/Hb CS and among Hb CS heterozygotes, -thalassemia
2 heterozygotes, and healthy individuals. Although Hb Bart's in
-thalassemia 2 heterozygotes ranged between 1.2% and 2.6%, 40
cases (7.6%) of newborns with the nondiseased -globin genotype also
had Hb Bart's (1.2–2.6%).
Prenatal diagnosis with fetal blood.
Cordocentesis was
performed in the pregnancies at risk of having thalassemic fetuses at
the gestational ages of 16–24 weeks. Chromatograms of thalassemia
syndromes prenatally diagnosed by the VARIANT BTS program were similar
to those of the cord blood specimens. The fetuses with ß-thalassemia
traits had similar concentrations of Hb F and Hb A to the healthy fetus
(Table 4
). In the Hb E heterozygote, 0.8–1.4% of the Hb E was Hb E at
the Hb A2 position, which was detected in addition to Hb A,
whereas there was no Hb A in fetuses with homozygous Hb E and
ß-thalassemia/HbE disease. The latter two conditions
were distinguished by DNA analysis. Hb Bart's hydrops fetalis also
produced no Hb A, and only Hb Bart's was demonstrated by HPLC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thalassemia diseases,
including Hb H and EA Bart's or EF Bart's diseases (Fig. 1
). The low variability for the measurement of Hb A2 and Hb E was comparable with the other methods for Hb A2 quantitation; the CV of Hb A2 by microcolumn chromatography was 2.5%, and that by elution from cellulose acetate electrophoresis was >3.6% (5). The other automated HPLC, the DIAMAT Analyzer System, gave a CV ranging from 1.8% to 4.1% (20). The within-run precision for Hb F in this study was also appreciably better than that of other means. CVs for Hb F by radial immunodiffusion and alkali denaturation range from 3.95% to 5.74% and from 1.4% to 9.1%, respectively, whereas the CV of the HPLC method was 4.5% (21).
In adults, an increased Hb A2 in the range of 4–7%
is specific for the ß-thalassemia trait in almost all cases
(22)(23). Our study confirmed the identification
of the ß-thalassemia trait and the Hb E trait, using the Hb
A2 (E) concentration determined by HPLC (Table 2
). However,
iron deficiency anemia remains a problem in differential diagnosis of
-thalassemias, ß-thalassemias, and Hb E carriers. Iron deficiency
anemia has been shown to decrease Hb A2 and E
concentrations and must be ruled out before a diagnosis is made
(24). In addition, coinheritance of the -thalassemia gene
in ß-thalassemia or Hb E also affects their phenotypic expression.
The concentration of Hb E decreased proportionally with the number of
-globin gene deletions, from 25–30% in the heterozygote
with a nondiseased -globin genotype to 19–21% in Hb E with
-thalassemia 1 or to 11–15% in EA Bart's disease
(25)(26)(27)(28). Coinheritance of -thalassemia 1 also affects
MCV in the ß-thalassemia trait. The MCV was found to be increased up
to 80 x 10-15 L, resulting in a misdiagnosis of the
ß-thalassemia trait in some cases. Transfusions also increase the
risks of false-positive and false-negative results, especially in
individuals who have received blood transfusions from Hb E donors or in
Hb E individuals who have received transfusions from healthy donors.
HPLC is also useful for large-scale screening of thalassemias and
hemoglobinopathies in the neonatal period. Although newborns with
ß-thalassemia cannot be distinguished from healthy newborns because
ß-globin gene expression is not fully functional at this
stage of development, the ability of the VARIANT BTS program to detect
all cases of Hb E syndromes and demonstrate the absence of Hb A is a
great advantage for the diagnosis of ß-thalassemia
diseases (Table 3
). However, both homozygous Hb E (asymptomatic) and
ß-thalassemia/Hb E diseases had similar chromatograms
composed of Hb E and Hb F. Although newborns with homozygous Hb E have
a tendency to have higher concentrations of Hb E (3.9–15%) than those
with ß-thalassemia/Hb E disease (2%), DNA analysis is
necessary to differentiate the two syndromes .
Our study also showed that the qualitative demonstration of Hb Bart's
and Hb H using the VARIANT BTS program aids in the diagnosis of
-thalassemia syndromes during the neonatal period. All cases of Hb
Bart's hydrops fetalis are detected, and all cases of Hb H disease and
most cases of -thalassemia trait were also detected. The ATS program
helps to quantitate the amount of Hb Bart's and provides a diagnosis
for newborns with Hb H disease whose Hb Bart's was increased to
20–25% (Table 3
). However, as mentioned in previous studies,
diagnosis of -thalassemia cannot rely solely on the amount of Hb
Bart's because the concentration of Hb Bart's in each phenotype may
be similar (29)(30). It should also be noted
that in the neonatal period, newborns with -thalassemia syndromes,
including Hb H, EA Bart's disease, EF Bart's diseases, the
-thalassemia 1 trait, and homozygous -thalassemia 2, have low MCV
values, whereas those with ß-thalassemia syndromes still have MCV
values within the reference range. This is because full synthesis of
the -globin chain can be attained in the fetal stage, and the
abnormalities would interfere with Hb molecules produced thereafter,
resulting in abnormal erythrocyte production.
In the fetal stage, the patterns of chromatograms with various genotypes of thalassemia were similar to those obtained from cord blood. Although the concentration of Hb A and Hb E were lower than those measured during the neonatal period, the disappearance of Hb A and the specific feature of chromatograms can help in prenatal diagnosis of severe thalassemia diseases.
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Acknowledgments |
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Footnotes |
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-thalassemia short; BTS, ß-thalassemia short; and MCV, mean corpuscular volume. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thalassemia-2 determinants by PCR. Am J Hematol 1994;46:208-213.
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Lys] in a Thai male. Hemoglobin 1993;17:415-425.
-thalassemia. Br Med J 1967;4:29-32.
-thalassemia in Thailand. Hemoglobin 1988;12:581-592.
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-thalassemia. Br J Haematol 1982;151:177-178.
-globin genes from cord blood in Thailand. Birth Defects 1988;23(5A):15-21.
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