1
REVIEW ARTICLE
-------------------------------------------------------------------------------------------------
Use of fetal nucleated red blood cells in maternal
circulation, for noninvasive prenatal diagnosis of
chromosomal and single gene disorders.
Dr Maitreyee
Faculty member in Embryology and Medical Genetics
University of Seychelles
American Institute of Medicine
Seychelles
------------------------------------------------------------------------------------------------KEYWORDS:
Nucleated red blood cells (NRBCs)
Non-invasive prenatal diagnosis (NI-PND)
single gene disorders
cytogenetic disorders
fetal
maternal circulation
trophoblasts
leukocytes
enrichment
------------------------------------------------------------------------------------------------Currently
available
prenatal
spontaneous
first-trimester
diagnostic
procedures
for
abortions, and in about 2% of
detection of fetal aneuploidy and
pregnancies in women older
single
gene
disorders
are
than 35 years who undergo
‘invasive’.
These
procedures
prenatal diagnosis. (1)
In a
include chorionic villus sampling
population study of more than 1
(10-12 weeks of gestation),
million live births, the incidence
amniocentesis (15-20 weeks of
of serious single-gene disorder
gestation) or umbilical blood
was estimated to be 0.36%. (2)
sampling (after 18 weeks of
gestation).
The
scientists
have
been
constantly in search of a better
Along with the need for skill and
method for prenatal diagnosis. A
expertise, they also have low but
reliable noninvasive method will
well-documented risk to the
be the right answer for it, as it
mother and the fetus. They are
will minimize the risk, and may
generally not offered to all
be - will be available at a lesser
pregnant women. These are
cost; and then a larger number
indicated only in high risk
of pregnant women would be
pregnancies - e.g. advanced
benefited by it.
maternal age (> 35 years),
abnormal
maternal
serum
One of the methods that has
screening results, ultrasound
been under research for more
detection of fetal anomalies or
than 3 decades, is to enrich the
likelihood
of
single
gene
fetal cells in the maternal blood.
disorder.
Historically, the presence of fetal
cells in the maternal circulation
Cytogenetic
disorders
are
during pregnancy has been
present in nearly 1% of live
observed since the end of the 19th
births, in almost 50% of all
century.
Schmorl in 1893
2
showed through the autopsy
findings, the presence of trapped
fetal cells in the lungs of
pregnant women. Similarly, the
presence of fetal cells was
demonstrated by Douglas in
1959. (3)
These were the
trophoblasts
traversing
the
placental
barrier
into
the
maternal circulation.
By now, multiple investigators
have confirmed the existence of
fetal cells in maternal blood.
Several types of fetal cells
including
trophoblasts,
lymphocytes, granulocytes, stem
cells, and nucleated erythrocytes
can be found in maternal blood
at as early as 8 weeks gestation,
but in extremely low abundance.
(4)
Three different fetal cells have
been the focus of research:
trophoblasts, fetal leukocytes,
and fetal erythrocytes (fetal
NRBCs). But the researches on
these cell types have been
hampered in several ways.
A. Trophoblasts: These were the
first fetal cells found to traverse
the
placental
barrier
into
maternal circulation. But there
are certain drawbacks associated
with these cells. They do not
appear in maternal blood in all
pregnancies. Because of their
multinucleated nature, they are
rapidly
cleared
from
the
maternal circulation. They are
heterogeneous
because
of
multinucleated status as well as
due to placental mosaicism. (5, 6)
There is difficulty in enrichment
for these cells because of lack of
specific antibodies.
B. Leukocytes: They were first
demonstrated to appear in the
maternal blood in 1969 by
Walknoska. (7)
This was done
by showing that a Y chromosome
could be detected in some of the
lymphocytes
isolated
from
pregnant women bearing male
fetuses.
They are the first cells to be
successfully
enriched
from
maternal blood. This approach
used differences in human
leukocyte
antigen
(HLA)
expression, which required HLA
type of father, thereby raising
the
issue
of
paternity.
Furthermore, these results were
not reproduced with the same
degree of success. (8)
Not
only
that
but
two
observations caused obstacles in
further progress while using
these cells:
1. The
persistently
high
number of female-bearing
pregnancies
with
Y
chromosome positive cells,
(9, 10, 11, 12) and –
2. The possible longevity of
fetal leukocytes in the
maternal circulation. (13)
Women
who
had
previously borne a son,
had fetal leukocytes in the
peripheral blood for a
period of up to 1 year and
in some instances 5 years
postpartum.(14) In 1996,
Bianchi et al showed
persistence
of
male
progenitor cells for 27
years
postpartum.
(15)
Because of this, there
always
remains
a
possibility of getting fetal
cells
from
previous
pregnancy, and increase in
the chances of wrong
diagnosis.
3
In spite of the above obstacles,
the
research
regarding
leukocytes definitely suggested
that fetal cells did migrate into
the peripheral blood of pregnant
women, from which they could
be enriched for subsequent
diagnostic procedures.
C. Fetal NRBCs: They have been
always considered as promising
cells for this technique.
A small number of NRBCs occur
in the peripheral venous blood of
women
during
normal
pregnancy, but they are less
common in the blood of healthy
nulligravid women. (16, 17, 18) It
was therefore believed that these
cells were of fetal origin and
hence could be a promising
source for non-invasive prenatal
diagnosis, especially since the
short lifespan of erythrocytes
eliminates the possibility of such
cells persisting from previous
pregnancies. (19)
In
1957,
Kleihauer
et
al
demonstrated the presence of
NRBCs in pregnant women.(20)
Clayton et al in 1964 confirmed
this finding by showing increase
in these cells in the maternal
blood when there is rhesus
incompatibility,
as
well
as
following
terminations
or
amniocentesis. (21) But in these
studies,
there
was
no
independent confirmation that
the NRBCs detected were indeed
of fetal origin.
One of the methods to give a
proof of fetal origin of these cells
is to show the presence of Y
chromatin, which limits it to the
pregnancies with male fetuses.
Bianchi et al (1990) were the
first to enrich for nucleated
erythrocytes containing fetal
DNA (on the basis of CD71
expression, a molecule highly
expressed on erythrocytes). (22)
Price et al (1991), Bianchi et al
(1992), and Ganshirt-Ahlert et al
(1993) then successfully showed
the correct determination of
fetal aneuploidy by this method.
(23, 24, 25)
Fetal erythrocytes are among the
first hemopoietic cells formed
during fetal development and
they are detectable early during
pregnancy.
These cells have an advantage
over leukocytes that these cells
are short-lived cells, of limited
replicating capacity, thereby
preventing the chance of getting
fetal
cells
from
previous
pregnancies.
But use of these cells also was
found to be associated with
certain drawbacks:
Anna Slunga-Tallberg et al
(1995) showed that pregnancy
induces NRBCs of maternal
origin to appear in the maternal
venous blood. A rapid increase
in erythropoiesis during normal
pregnancy raised the number of
red cells and reticulocytes in the
maternal venous blood, and this
may be associated with a
concomitant increase in the
number of NRBCs in the
peripheral blood. They found
that the NRBCs in the maternal
circulation were predominantly
maternal in origin. (26)
This finding that majority of
these
cells,
even
after
enrichment for fetal cells, are of
maternal origin is demonstrated
4
by other workers also. (27, 28, 29,
30)
Most of the times it is difficult to
confirm that the NRBCs detected
are indeed of fetal origin, and
not of maternal origin.
In the pregnancies with male
fetuses; however, the probable
fetal nucleated erythrocytes will
have Y chromosome.
In the
same way, detection of fetal
NRBCs is also possible if mother
is Rhesus negative and fetus is
Rhesus positive.
Some studies proposed that the
presence of fetal cells in
maternal blood is rare in normal
pregnancy. They are 1 in 105 to
107 or even less, early in
gestation i.e. less than 10 weeks
(31, 32, 33, 34, 35), when the
prenatal diagnosis is most
desirable.
According to the reports by
Ganshirt-Alhert (1994b) (46) and
Bianchi et al (1997) (34), fetal
cells appear to be more frequent
in aneuploid pregnancies.
In the 19th century, Schmorl
(1893) (3) and Clayton et al
(1964) (21) showed altered fetalmaternal trafficking in preeclampsia, but there was no
direct proof that the cells
observed were indeed of fetal
origin.
In 1998, Holzgreve et al by the
exclusive
use
of
male
pregnancies, determined that
significantly more fetal cells
traversed the placental barrier in
pre-eclamptic patients (9/1000)
than in the control group
(2/1000) and also there was
large increase in the number of
NRBCs (38 versus 7) in these
patients, most of which were of
maternal origin. (30)
Along with the difficulties in
enrichment,
the
issues
pertaining to the fetal cell
recognition, cultivation, and
diagnostic efficiency still remain
to be explored to their fullest
extent.
The inability to expand fetal
erythroid
progenitors
successfully from the maternal
circulation in vitro might be
explained by the fact that the
occurrence of expandable fetal
hematopoietic progenitor cells in
the maternal blood are rare to
very rare events (36, 37, 38) or
that they require some as-yet
unidentified culture conditions.
To enhance the enrichment of
fetal NRBCs, several workers
have sought for more specific
antibodies
or
biochemical
markers.
Since most of such studies in
past have focused on the
identification of male fetal cells
(because of presence of Y
chromatin), almost half of the
patient population was not
analyzed. Osamu Samura et al
developed
a
method
to
determine that female gamma
positive NRBCs are fetal (not
maternal) in origin using DNA
polymorphism. They reported
that
this
technique
allows
genetic studies of all fetal
NRBCs, including female fetal
cells. (39)
Polymerase chain reaction (PCR)
was tried to support existence of
fetal cells in maternal blood i.e.
where the gene of interest is
present in the fetal genome but
absent in the maternal one e.g.
in
paternally
inherited
5
hemoglobinopathy (40) and to
determine the fetal RhD status.
(41)
Use
of
zeta
globin
immunophenotyping
for
the
determination
of
beta
thalassemia(42),
fetal
NRBC
identification by epsilon-globin
chain immunophenotyping(43),
use of combined gamma and
epsilon globin antibody (44) are
also some of the steps towards
the goal.
In 2003, Cha Donghyun et al
defined certain parameters to
distinguish fetal NRBCs from
maternal NRBCs. (45) They
defined
four
parameters:
nuclear
roundness,
nuclear
morphology, gamma Hb staining
intensity,
and
peripheral
brightness
of
the
stained
cytoplasm. They suggested that
these defined parameters might
serve
as
computational
classifiers for the automated
detection of fetal NRBCs from
maternal NRBCs.
Jackson Laird in 2003 in a
review article concludes that
research attempts to use fetal
cells from maternal blood for
prenatal diagnosis have not had
any consistent success and that
much work remains to be done
to develop practical technology
for consistent recovery and assay
of the fetal cells or fetal DNA.
(47)
Shinya Masaru et al used
galactose-specific
lectin
to
concentrate fetal nucleated RBCs
from maternal blood (2004). (49)
The same method was used by
Sekizawa Akihiko et al (2007).
(50) This has been thought to be
one of the techniques with high
sensitivity, for the separation of
fetal cells.(55)
Till recently, the workers have
been trying both old and
advanced techniques to isolate
fetal NRBCs from maternal
NRBCs.
In 2007, a new method, a
micromachined device (Biochip)
was used to separate fetal cells
based on differences in size and
deformation characteristics. (51)
The search for the fetal NRBCs in
maternal circulation has been in
progress in many of the ways
described above; however, there
is still a lot that needs to be done
to achieve one of the most
satisfactory means to detect and
enrich the fetal NRBCs in
maternal circulation.
Whereas this is the current
status of research in reference to
the fetal NRBCs, just the
presence of NRBCs in maternal
circulation - (irrespective of
whether they are fetal or
maternal in origin) – also has
been shown to be important.
Maternal NRBCs
In 2004, (48) antibody against
gamma chain of HbF was used to
detect fetal erythroblasts in
maternal circulation and they
were used for sex determination
and
genetic
diagnosis
of
Duchenne muscular dystrophy.
Anna Slunga-Tallberg et al found
that there is an increase in the
number of circulating NRBCs in
association with pregnancy, and
moreover
these
are
predominantly
of
maternal
origin. (26)
6
Many researchers have shown
that although the small increase
in the number of NRBCs in
normal pregnancy is definite, the
rise in the number of NRBCs is
especially
remarkable
in
association with certain specific
conditions. (3, 21, 30)
The observations in 2006, by
Mavrou et al were also found to
be on the similar lines. The
number
of
NRBCs
was
determined from 20 ml of
peripheral blood in 351 women
in the second trimester of
pregnancy, after isolation of
magnetic cell sorting (MACS)
with anti-CD71 antibody. They
found following numbers of
NRBCs in the maternal blood, in
association with certain specific
conditions.
No
1
2
3
4
5
Condition
Number of
NRBCs in
maternal
circulation
Women
with 1-12
chromosomally (average
normal fetuses 8)
aneuploid
7-113
fetuses
(average
35)
Trisomy 21
average 71
Beta22-158
thalassemia
(average
42)
pregnancies
2-75
with abnormal (average
Doppler
15)
findings in both
uterine arteries
They concluded, this could be an
additional ‘screening step’ for
fetal aneuplodies, as long as the
anemic status of the mother is
taken into consideration. (52)
In 2005, Ikeya Miki et al (2005)
used
lectin
method
for
separation and recovery of
NRBCs (53) in maternal blood.
They showed that there was
increase in maternal NRBCS in
pregnancies with trisomy 18,
trisomy 21, placenta previa, preeclampsia, and intrauterine fetal
death. They concluded that
NRBC exam may play an
assisting role not only in fetal
diagnosis
but
also
in
fetomaternal diagnosis.
In 2007, Aali Bibi Shahnaz et al
demonstrated the same finding
regarding pre-eclampsia. (54)
Though these studies suggest
that maternal NRBCs could be
useful
for
screening
for
conditions like fetal aneuploidies
and fetomaternal disturbances,
for definitive diagnosis we shall
be still in need of a more reliable
technique.
Promises
and
Unanswered
questions:
Use of the fetal NRBCs would
remain one of the more reliable
non-invasive
techniques
for
prenatal
diagnosis.
This
obviously has a promise of lot of
advantages over routine invasive
procedures. However, there are
many unanswered questions.
The obvious problem is of the
limited number of fetal cells
found to be circulating within
maternal blood. Due to this, the
procedures to enrich the cells
and enable single cell analysis
with
high
sensitivity
are
required. Some of the recent
methods of separation included
a lectin-based method and
autoimage analyzing.
These
methods have improved the
7
sensitivity of genetic analysis.
(55)
The ongoing progress in this
field
has
supported
the
possibility
of
non-invasive
prenatal diagnosis, although a
single,
definitive,
accurate
practicable method is yet to be
established. There is still a wide
scope for many more areas in
research to be explored in this
direction.
--------------------------------------------References:
1. Thompson and Thompson Genetics in
Medicine – 6th edition - Chapter 9
2. Thompson and Thompson Genetics in
Medicine – 6th edition - chapter 5
3. Gussin Helene A Elicha, Elias Sherman –
Culture of fetal cells from maternal blood for
prenatal diagnosis – Human Reproduction
Update – Vol 8 No 6 pp 523-527, 2002
4. Principles of Medical Genetics (Williams and
Wilkins) – 2nd edition - chapter 12
5. Henderson, K.G., Shaw, T.E., Barret, I.J. et
al, (1996) Distribution of mosaicism in human
plancentae. Human Genetics, 97, 650-654)
6. Goldberg, J.D. and Wohlferd, M.M. (1997)
Incidence and outcome of chromosomal
mosaicism found at the time of chorionic villus
sampling. American Journal of Obstetrics and
Gynecology., 176, 1349-1353
7. Walknoska, J., Conte, F.A., and Grumbach,
M.M.
(1969)
Practical
and
theoretical
implication of fetal/maternal lymphocyte
transfer. Lancet, i, 1119-1122
8. Tharapel, A.T., Jaswaney, V.L., Dockter, M.E.
et al. (1993) Inability to detect fetal metaphases
in flow-sorted lymphocyte cultures based on
maternal-fetal HLA differences. Fetal diagnosis
and therapy., 8, 95-101
9. Schindler, A.M., Graf, E., and Martin-du-Pan,
R.
(1972)
Prenatal
diagnosis
of
fetal
lymphocytes in the maternal blood. Obstetrics
and Gynecology., 40, 340-346
10. Schroder, J. and de la Chapelle, A. (1972)
Fetal lymphocytes in maternal blood. Blood, 39,
153-161
11. Grosset, L., Barelet, V., and Ordatchenko, N.
(1974) Antenatal fetal sex determination from
maternal blood during pregnancy. American
Journal of Obstetrics and Gynecology., 120, 6063
12. Sargent, I.L., Choo, Y.S, and Redman,
C.W.G.(1994) Isolating and analyzing fetal
leukocytes in maternal blood. Ann. NY Acad.
Sci., 731, 154-161
13. Schroder, J., Tilikainen, A., and de la
Chapelle, A. (1974) Fetal leukocytes in maternal
circulation after delivery. Transplantation, 17,
346-360)
14. Ciaranfi, A, Curchod, A., and Odartchenko,
N. (1977) Post-partum survival of fetal
lymphocytes in the maternal blood. Schweiz.
Med. Wochensch., 107, 134-138
15. Bianchi Diana W., Zickwolf, Gretchen K.,
Weil, Gary J., et al. (1996) Male fetal progenitor
cells persist in maternal blood for as long as 27
years postpartum. Proc. Natl. Acad. Sci. USA.
93, 705-708
16. Bianchi, D. W., Stewart, J.E., Garber, M.F. et
al. (1991) Possible effect of gestational age on
the detection of fetal nucleated erythrocytes in
maternal blood. Prenatal Diagnosis. 11, 523528.
17.
Ganshirt-Ahlert,
D.,
Burschyk,
M.,
Garritsen, H.S.P., et al. 1992 Magnetic cell
sorting and the transferrin receptor as
potential means of prenatal diagnosis from
maternal blood. American Journal of Obstetrics
and Gynecology. 166, 1350-1355.
18. Slunga-Tallberg, A., Wessman, M., Ylinen,
K. et al (1994) Nucleated erythrocytes in
enriched and unenriched peripheral venous
blood samples from pregnant and nonpregnant
women. Fetal Diagnosis and Therapy. 9, 291295
19. Wessman, M., Ylinen, K., Knuutila, S. (1992)
Fetal granulocytes in maternal venous blood
detected by in situ hybridization. Prenatal
Diagnosis. 12, 993-1000
20. Kleihauer, E., Braun, H., and Betke, K. 1957
Klin. Wochenschr., 35, 637-638
21. Clayton, E.M., Feldhaus, W.D., and
Whiteacre, F.E. 1964 Fetal erythrocytes in the
maternal circulation of pregnant women.
Obstetrics and Gynecology, 23, 915-919
22. Bianchi, D.W., Flint, A.F., Pizzimenti, M. et
al. 1990. Isolation of fetal DNA from nucleated
erythrocytes in maternal blood. Proc. Natl.
Acad. Sci. USA, 87, 3279-3283
23. Price, J.O., Elias, S., Wachtel, S.S. et al. 1991
Prenatal diagnosis with fetal cells isolated from
maternal blood by multiparameter flow
cytometry. American Journal of Obstetrics and
Gynecology. 165, 1731-1737
24. Bianchi, D.W., Mahr, A., Zickwolf, G.K. et
al. 1992 Detection of fetal cells with 47, XY, +21
karyotype in maternal peripheral blood.
Human Genetics, 90, 368-370
25. Ganshirt-Ahlert, D., Borejesson-Stoll, R.,
Burschyk, M., et al. 1993 Detection of fetal
trisomies 21 and 18 from maternal blood using
triple gradient and magnetic cell sorting.
American Journal of Reprod. Immun. 30, 194201
26. Slunga-Tallberg, A., El-Rifai, W., Keinanen,
M., et al. 1995 Maternal origin of nucleated
erythrocytes in peripheral venous blood of
pregnant women. Human Genetics, 96, 53-57
27. Ganshirt, D., Garritsen, H.S.P., Miny, P. et
al. 1994a. Fetal cells in maternal circulation
throughout gestation. Lancet. 343, 1038-1039.
28. Hamada, H, Arinami, T., Sohda, S, et al.
1995. Midtrimester fetal sex determination
from
maternal
peripheral
blood
by
fluorescence in situ hybridization without
enrichment of fetal cells. Prenatal Diagnosis.
15, 78-81.
29. Slunga-Tallberg, A., El-Rifai, W., Keinanen,
M., et al. 1996 Maternal origin of transferring
receptor positive cells in venous blood of
pregnant women. Clinical Genetics, 49, 196-199
30. Holzgreve, W., Ghezzi, F., Di Naro, E., et al.
1998. Feto-maternal cell traffic is disturbed in
8
pre-eclampsia. Obstetrics and Gynecology., in
press.
31. Ganshirt-Ahlert, D., Pohlschmidt, M., Gal,
A., et al. 1990. Ratio of fetal to maternal DNA is
less than 1 in 5,000 in different gestational ages
in maternal blood. Clinical Genetics. 38, 38-43.
32. Hamada, H., Arinami, T., Kubo, T., et al.
1993.
Fetal nucleated cells in maternal
peripheral blood: Frequency and relationship
to gestational age. Human Genetics, 91, 427-432
33. Hamada, H., Arinami, T., Sohda, S, et al,
1995. Mid-trimester fetal sex determination
from maternal peripheral blood by fluroscence
in situ hybridization without enrichment of
fetal cells
34. Bianchi, D. W., Williams, J.M., Sullivan,
L.M., et al, 1997. PCR quantitation of fetal cells
in maternal blood in normal and aneuploid
pregnancies. American Journal of Human
Genetics. 61, 822-829.
35. Sohda, S., Arinami, T., Hamada, H. et al,
1997, The proportion of fetal nucleated red cells
in maternal blood: Estimation by FACS
analysis. Prenatal Diagnosis, 17, 743-752
36. Little M T, Langlois S, Wilson R D et al,
(1997) Frequency of fetal cells in sorted
subpopulations of nucleated erythroid and
CD34+ hematopoietic progenitor cells from
maternal peripheral blood, Blood 89, 23472358
37. Jansen M W J C, Korcer-Hakkennes K, van
Leenen et al, (2000), How useful is the in vitro
expansion of fetal CD34+ progenitor cells from
maternal blood samples for diagnostic
purposes? Prenatal Diagnosis, 20, 725-731
38. Campagnoli C, Roberts I A G, Kumar S, et al,
(2002),
Expandability
of
hematopoietic
progenitors in first trimester fetal and maternal
blood: implications for non-invasive prenatal
diagnosis, Prenatal Diagnosis, 22, 463-469
39. Samura Osamu, Perl Barbara, Sohda
Santoshi et al (2000), Female fetal cells in
maternal blood: use of DNA polymorphisms to
prove origin, Human Genetics, 107, 28-32
40. Camaschella , C., Multhaupt
H A,
Ludomirski A et al (1990) Prenatal diagnosis of
fetal hemoglobin Lepore-Boston disease on
maternal peripheral blood., Blood, 75, 21022106.
41. Lo Y M D, Bowell P J, Selinger M et al (1993)
Prenatal determination of fetal RhD status by
analysis of peripheral blood of rhesus negative
mothers. Lancet, 341, 1147-1148
42. Naro Edoardo Di, Ghezzi Fabio, Vitucci
Angelantonio et al (2000), Prenatal diagnosis of
beta thalasemia using feta; erythroblasts
enriched from maternal blood by a novel
gradient, Molecular Human Reproduction, 6,
571-574
43. Cholani Mahesh, O’Donoghue Keelin,
Talbert David, et al, (2001) Characterization of
first trimester fetal erythroblasts for noninbvasive prenatal diagnosis, Molecular Human
Reproduction, 9, 227-235
44. Christensen Britta, Philip John, LykkeHansen Lene et al (2003), Sensitivity and
specificity of the identification of fetal cells in
maternal blood by combined staining with
antibodies against beta, gamma and epsilon
blobin chains, Fetal Diagnosis and Therapy, 18,
479-484
45. Cha Donghyun, Hogan Brad, Bohmer R M,
Bianchi D M et al, (2003) A simple and sensitive
erythroblast scoring system to identify fetal
cells in maternal blood, Prenatal Diagnosis, 23,
68-73
46.
Ganshirt,
D.,
Borejesson-Stoll,
R.,
Burschyk, M. eta al. (1994b), Successful
prenatal diagnosis from maternal blod bwith
magnetic-activated cell sorting., Ann. NY Acad.
Sci. 731, 103-114
47. Jackson Laird, (2003), Review, Fetal cells
and DNA in maternal blood, Prenatal
Diagnosis, 23, 837-846
48. Liu L, Jin C, Wang Y et al, (2004), Use of
specific antibody to detect fetal erythroblasts in
maternal circulation, Chinese Journal of
Medical
Genetics,
21
(5),
494-497.
http://www.ncbi.nlm.nih.gov/sites/pubmed/15
476180
49. Shinya Masaru, Okamoto Aikou, Sago
Haruhiko et al (2004), Analysis of fetal DNA
from maternal peripheral blood by lectinpolymerase chanin reaction – single strand
conformation
polymorphism,
Congenital
Anomalies, 44, 142-146
50. Sekizawa Akihiko, Purwosunu Yuditiya,
Matsuoka Ryu et al, (2007), Recent advances in
non-invasive
prenatal
diagnosis
through
analysis of maternal blood, The journal of
obstetrics and Gynecology Research, 33, 747764
51. Mohamed Hisham, Turner JN, Caggana
Michele (2007), Biochip for separating fetal
cells from maternal circulation, Journal of
Chromatography A, 1162, 187-192.
52. Mavrou A., Kouvidi E., Antsaklis A. et al,
(2006), Identification of nucleated red blood
cells in maternal circulation: A second step in
screening for fetal aneuploides and pregnancy
complications, Prenatal Diagnosis, 27, 150-153
53. Ikeya Miki, Shinya Masaru, Kitagawa
Michihiro (2005), Basic investigation of the
lectin method for separation and recovery of
nucleated red blood cells in maternal blood,
and a study into the frequency of nucleated red
blood
cells
in
fetomaternal
disorers.,
Congenital Anomalies, 45, 26-31
54. Aali Bibi Shahnaz, Malepour Reza, Sedig
Fatemeh et al, (2007), Comparison of maternal
and cord blood nucleated red blood cell count
between pre-eclamptic and healthy women,
Journal of Obstetrics an Gynecology Research
33, 274-278
55. Sekizawa Akihiko, Purwosunu Yuditiya,
Matsuoka Ryu et al 2007) Recent advances in
non-invasive prenatal DNA diagnosis through
analysis of maternal blood. The Journal of
Obstetrics and Gynecology Research, 33, 747764
--------------------------------------------------------------------------------------------------------------------------------------------