CORD BLOOD ANALYSIS FOR RAPID PRENATAL CONFIRMATION OF Hb BART’S DISEASE USING THE SEBIA CAPILLARY ELECTROPHORESIS SYSTEM Liao2011 phân tích máu cuống rốn để khẳng định chẩn đoán nhanh trước sinh bệnh Hb Bart's bằng cách sử dụng hệ thống điện di mao quản bs võ tá sơn bsvotason bác sĩ võ tá sơn

1. Hemoglobin, 36(2):186–191, (2012)
Copyright © Informa Healthcare USA, Inc.
ISSN: 0363-0269 print/1532-432X online
DOI: 10.3109/03630269.2011.644409
SHORT COMMUNICATION
CORD BLOOD ANALYSIS FOR RAPID PRENATAL CONFIRMATION
OF Hb BART’S DISEASE USING THE SEBIA CAPILLARY
ELECTROPHORESIS SYSTEM
Can Liao, Jian-Ying Zhou, Xing-Mei Xie, Ru Li, and Dong-Zhi Li
Prenatal Diagnostic Center, Guangzhou Maternal & Neonatal Hospital, Guangzhou Women & Chil-
dren Medical Center, Guangzhou Medical College, Guangzhou, Guangdong, People’s Republic of
China
Prenatal diagnosis of severe α-thalassemia (α-thal) diseases is usually performed by DNA anal-
ysis. To establish a simple and rapid method, we evaluated the reliability of cord blood hemoglobin
(Hb) analysis using an automated capillary electrophoresis (CE) system. Our results demonstrated
that analysis of fetal Hb using the Sebia CapillaryS 2 is an effective, accurate and simple alternative
for prenatal diagnosis of Hb Bart’s (γ4) disease.
Keywords α-Thalassemia (α-thal), Hb Bart’s disease, Fetal blood, Prenatal diagnosis,
Capillary electrophoresis (CE)
Hb Bart’s (γ4) disease is the result of the complete absence of functional
α-globin genes because of the homozygosity for α0-thalassemia (α0-thal).
Individuals with this disease usually die in utero at late gestation or soon
after birth. Most of the hemoglobins (Hbs) present in the affected fetuses is
Hb Bart’s, which is unable to deliver oxygen to tissues, is relatively unstable,
and can precipitate and cause shortened red cell survival and possibly inef-
fective erythropoiesis. These fetuses are usually severely anemic and have
massive total body edema due to high output heart failure, pallor, massive
hepatomegaly secondary to extramedullary hematopoiesis and edematous
Received 30 May 2011; Accepted 3 August 2011.
Address correspondence to Dong-Zhi Li, M.D., Prenatal Diagnostic Center, Guangzhou Women
& Children Medical Center, Jinsui Road 9, Zhujiang New Town, Guangzhou, Guangdong 510623,
the People’s Republic of China; Tel./Fax: +86-20-8185-4671; E-mail: dongzhi3@yahoo.com.cn;
lidongzhi2011@hotmail.com
186
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2. Rapid Diagnosis of Hb Bart’s Disease by a CE System 187
friable placenta. Pregnancy involving Hb Bart’s disease is associated with an
increased risk of maternal complications such as hydromnios, preeclampsia,
antepartum or postpartum hemorrhage and difficult vaginal delivery (1,2).
With carrier detection, timely genetic counseling, and the availability
of prenatal diagnosis during early pregnancy, many at-risk couples will be
spared serious medical and psychological consequences. To accomplish this,
these genetic disorders should be recognized in early or before pregnancy,
so that the appropriate maternal health care and preventive measures can
be provided to the affected couples. The earlier method for the prenatal
diagnosis involves fetal tissue samples for a DNA-based test obtained by
chorionic villus sample (CVS) biopsy at 10 to 11 weeks of gestation. How-
ever, these procedures need previous studies of at-risk couples, and also
the molecular analysis results are available only after 5-7 days. This rela-
tively long wait for the test results might be unacceptable to couples when
their fetuses are disclosed by ultrasound to present hydropic changes. In this
study, we reported on the feasibility of prenatal confirmation of Hb Bart’s
disease by fetal blood electrophoresis analysis in mid- or late-pregnancy.
This is a retrospective descriptive study designed to include women
undergoing cordocentesis for prenatal diagnosis of Hb Bart’s disease at our
Prenatal Diagnostic Center at the Guangzhou Maternal & Neonatal Hos-
pital, Guangzhou, Guangdong, the People’s Republic of China. Between
January 2008 and December 2010, 127 pregnant women were referred to
our center for evaluation because the fetuses were suspected to carry Hb
Bart’s disease by ultrasound examination. None of the couples had ever had
a thalassemia screen test. After a full blood count and Hb electrophoresis
suggested an α-thal trait in both partners, cordocentesis under ultrasound
guidance was performed. The cord blood samples were sent for Hb anal-
ysis using the CapillaryS 2 system (Sebia, Lisses, France) according to the
manufacturer’s instructions.
The mean age of the 127 women was 25.3 years (range 20-41 years).
The mean gestational age was 24.5 weeks (range 18-34 weeks). Of the 127
fetuses studied, 123 were diagnosed as Hb Bart’s disease, three carried Hb
H hydrops fetalis, and one α-thal trait. Diagnosis of the Hb Bart’s disease is
easy due to the specific electropherogram by the CapillaryS 2 (Sebia) system
with a major peak of Hb Bart’s and a few minor peaks of embryonic Hbs
(Figure 1A). The mean levels of Hb Bart’s were 79.7% (74.5–90.4%). Hb
F and Hb A were not detected. Small amounts of Hb H (β4) were also
observed in these affected fetuses. Fetuses of Hb H hydrops fetalis demon-
strated a major peak of Hb F, a minor peak of Hb A and decreased amounts
of Hb Bart’s (Figure 1B) compared to those of Hb Bart’s disease. The
α-thal trait fetus showed a small amount of Hb Bart’s in addition to Hb F
and Hb A (Figure 1C). The levels of Hb Bart’s in cord blood could be used
for differential diagnoses of these α-thal disorders.
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3. 188 C. Liao et al.
(A)
Hb Bart’s
Hb H Epsilon 4
Gower 1
Portland
(B)
Hb Bart’s
Hb H
Epsilon 4
Hb A
Hb F
(C)
Hb F
Hb A
Hb Bart’s
FIGURE 1 Representative capillary electropherograms of fetal blood samples: (A) Hb Bart’s hydrops
fetalis, (B) Hb H hydrops fetalis, (C) heterozygous α0-thal.
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4. Rapid Diagnosis of Hb Bart’s Disease by a CE System 189
All of the women carrying fetuses with Hb Bart’s disease were immedi-
ately admitted and underwent termination of their pregnancies. Molecular
diagnosis on cord blood disclosed that all of the fetuses without the pres-
ence of Hb F had a genotype of – –SEA/– –SEA. The three fetuses with Hb
H hydrops fetalis had a genotype of Hb H-Quong Sze [α128(H8)Leu→Pro,
CTG>CCG (α2)] disease (– –SEA/αTα). Their families opted to terminate
the pregnancy after being counseled about the prognosis of these fetuses.
The fetus with α-thal trait (– –SEA/αα) was referred for further evaluation of
the cause of the hydropic changes.
Carriers of α-thal were screened on the basis of a low MCV and a nor-
mal or reduced Hb A2 level, and were confirmed to have α0-thal (– –SEA)
by polymerase chain reaction (PCR). For an at-risk pregnancy, ultrasound
examination can readily detect the many hydropic changes found in fetuses
with Hb Bart’s disease. Cardiomegaly and placentomegaly are the two main
characteristics. By the 12th week, these measurements could identify most of
the affected pregnancies (3,4). By the 18th week, they could identify almost
all affected pregnancies with a high specificity (5). In our local practice, for
an at-risk pregnancy with gestational age beyond 14 weeks, invasive testing
is seldom indicated directly, instead a sonographic examination is offered
to detect signs of fetal anemia. A normal scan suggests that the fetus is
not affected with homozygous α-thal, and invasive testing can be avoided.
However, although sonographic markers are very sensitive in predicting Hb
Bart’s disease, it does not mean that a fetus with those findings would suf-
fer from Hb Bart’s disease. Indeed, non immune hydrops fetalis is not an
uncommon feature with routine sonograms in clinical practice (6). It can
result from a large number of causes, including chromosomal abnormal-
ities, infections, metabolic diseases, cardiac failure, tumors and idiopathy.
Thus, the sonographic diagnosis should always be confirmed with invasive
and definitive methods.
In our center, as for other centers, prenatal diagnosis of α-thal is
performed using DNA analysis of CVS or amniocytes using PCR-related
techniques to identify the corresponding mutations. This is usually done
during the mother’s first trimester or early second trimester of gestation. If
an abnormal ultrasound scan is found at 18 weeks or beyond and indicates
an hydropic-like fetus, the pregnant women would be too anxious to wait
for the PCR-based DNA diagnosis. In this situation, fetal blood samples can
be obtained by cordocentesis, and Hb electrophoresis can be performed on
these blood samples. The major Hb found in a normal fetus during the sec-
ond trimester of gestation is Hb F (α2γ2), with 10% or less of Hb A (α2β2).
In a fetus with homozygous α-thal, the major Hb is Hb Bart’s, with 10 to 20%
embryonic Hbs, and possibly some Hb H. Hb F or Hb A are not present
in these fetuses. These results can be obtained rapidly by the CapillaryS 2
(Sebia) system.
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5. 190 C. Liao et al.
Several authors have described this approach for prenatal diagnosis of
Hb Bart’s disease using automated high performance liquid chromatogra-
phy (HPLC) to determine the level of Hb Bart’s and the absence of Hb F in
fetal blood (7,8). The results also corresponded with those obtained by DNA
analysis in their studies. Indeed, the CapillaryS 2 (Sebia) system is suitable
for the identification and quantification of both common and unusual Hb
variants, producing results comparable to existing HPLC methods (9,10).
Maternal contamination is a serious issue in the application of fetal Hb
analysis in prenatal diagnosis. For a fetal blood specimen, this could be easily
detected by many simple methods (11,12). In our hands, with an experi-
ence of more than 3,000 cordocentesis procedures, maternal contamination
occurred in less than 1% of cases. Indeed, the fetal Hb electrophoresis itself
could act as a test to detect maternal blood contamination, as the Hb com-
ponents are different between adults and mid-pregnancy fetuses. In real
practice, it is conceivable that even with a small amount of maternal blood,
the diagnosis of Hb Bart’s disease (with as much as 80% Hb Bart’s and no
Hb F) would not be affected. In contrast, this is particularly of concern with
sensitive PCR-based molecular assays in which a maternal contamination-
free sample is an absolute requirement.
Cordocentesis-related fetal loss is the greatest concern for this method.
In our experience, the risk of an unintended abortion after mid-trimester
cordocentesis is about 1%. Despite its advantages and high acceptance, fetal
blood sampling is relatively invasive and can be performed only by a skilled
operator. Currently, in prenatal diagnosis of α-thal at our center, fetal blood
sampling is only reserved for pregnancies with hydrops fetalis having already
been identified by ultrasound in an at-risk fetus after 18 weeks of gestation.
In summary, our results demonstrated that fetal Hb analysis using the
CapillaryS 2 (Sebia) system could be an effective alternative to a DNA
assay for prenatal confirmation of Hb Bart’s disease. This fetal blood anal-
ysis is simple, rapid, fully automated and much less technical effort is
needed.
ACKNOWLEDGMENTS
This study was supported by Guangzhou Technology Bureau Scientic and
Technological Support Project (2009Z1-E131).
Declaration of Interest: The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of this article.
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