Is time to use Non-Invasive Prenatal Diagnosis using Maternal Blood for some
Genetic Disorders?
Kalantar SM1., Sitar GM2., Calaberse G3., Baldi M4., Sheikhha MH1., Kalantar S. Milad5
1Research
& Clinical Centre for Infertility, Yazd Medical Sciences University.
2Ppliclinico
. atteo Pavia University-Italy. 3Chieti-Italy. 4Laboratorio agenoma Rome
Italy. 5Khorasgan Azad University-Isfahan-Iran.
Introduction:
Detection of chromosomal aneuploidies using fetal cells isolated from maternal blood is a long-sought goal
of clinical genetics to eliminate Invasive Prenatal Diagnosis (IPND) and pregnancy loss. However the
retrieval of circulating fetal cells has proved difficult1 due to the small number of fetal cells present in the
maternal blood (2-6/ml). We have previously described a procedure to isolate these rare cells from
maternal blood, but any test for an accurate diagnosis using the limited number of fetal cells retrieved
from maternal blood must achieve a high level of sensitivity and specificity before replacing IPND.
Different strategies have been employed for chromosomal analysis of circulating fetal cells. FISH protocols
are quite diverse and small modifications in the protocol, thereby interfering with hybridization specificity
and hybridization sensitivity and affecting the accuracy of any method aimed at chromosome counting with
diagnostic efficiency.
Targeting high sensitivity and specificity in the genetic diagnosis of this isolated cell sample, we have used
the following two approaches: a) FISH-only analysis using two independent probes for the same
chromosome; b) sequential combination of cell immunolabeling and interphase FISH analysis
Materials & Methods:
Samples: We analyzed 14 peripheral blood specimens from women carrying a trisomic fetus (nine cases
with trisomy 21, four cases with trisomy 18, and one case with trisomy 15), as previously diagnosed by
invasive procedures. After receiving written informed consent from pregnant women, and institutional
review board approval, we obtained 25-mL of maternal blood one hour before pregnancy termination
As controls, blood samples from five pregnant women with a normal fetus as determined by amniocentesis,
two non-pregnant women and two men were also investigated.
Fetal cell isolation
Fetal cells, including both nucleated red blood cells (NRBCs) and CD34+ cells, were isolated from
maternal blood according to the procedure previously described in details with some modifications.
Dual-labeling FISH-based detection
Dual-labeled FISH was performed on nuclei obtained from the isolated cell samples. The two probes were
co-denatured with slide cells, followed by overnight hybridization.
FISH procedure on immunostained cells
When FISH was performed on the immunostained cell fraction, slides were postfixed in 2% formaldehyde,
washed and dehydrated in an ethanol series. Fluorescently labeled probes (Cytocell-Celbio) were used to
identify chromosomes 21 and 18 in the nuclei of the cells. The nuclei were located using 4-6-diamidino-2phenylindole (DAPI) counterstain.
General scanning and analysis approach
Slides were observed under a fluorescence microscope either by direct visualization or scanned
by
automated image analyzer using a Duet BioView system equipped with a color CCD camera. At least 899
cells per sample were scored (899-1700 scored cells by direct visualization; 4000 scored cells by automated
scanning) using an appropriate triple pass-band filter (Zeiss, Jena, Germany). Cells showing a twogreen/two-red signal FISH pattern were classified as normal, while cells with a three-green/three-red signal
pattern were classified as trisomic. All other patterns of hybridization were not taken into account, although
recorded for hybridization quality control.
Statistics: Mann-Whitney U test was applied to evaluate the difference in the percentage of trisomic cells
between the two groups (aneuploid pregnancies vs controls).
Results:
With the modified protocol for fetal cell enrichment, 50.000 -100.000 (mean 71.000) cells were recovered
from the maternal blood samples. Co-localization of i-antigen/glycophorin A and i-antigen/CD34 proved
that some of i-antigen positive cells were erythroblasts while others were CD-34 stem cells. When FISH
hybridization was performed on slides immunostained with anti-i directly labeled with FITC, it was
possible to easily observe the probes for chromosome 21, but there was a large heterogeneity in fluorescent
immunostaining which is a source of very subjective signal interpretation (Fig. 1d). Using dual-probe FISH
only, in 13 out of 14 cases, 4-8 trisomic cells /1000 scored cells per slide were found by manual screening
evaluation (1/211 on average, range 0.36-0.76% trisomic cells/sample( In one case, with a +15 fetus, only
2/1700 abnormal cells (0.12%) were observed. Automated FISH analysis was carried out in four of the 14
cases, two with trisomy 21 (pats. no. 9, and 10) and two with trisomy 18 (pats. no.2, and 3), by scoring
4000 cells per sample. Automated FISH scoring was carried out within 120 min on average (range 109-137
min). The images of target nuclei were acquired in multiple focal planes and cells showing three red and
three green spots further analyzed by direct visual observation to be classified as true trisomic cells.
DISCUSSION:
Herein we have compared the combination of FISH with cell staining by a fetal-specific marker, i-antigen,
and FISH analysis of interphase nuclei using two differently-labeled probes specific for different loci of
chromosomes 21, 18, and 15. To date the first approach has made use of either anti- or anti- globinchains-Hb monoclonal antibodies, which recognize only fetal NRBCs, while entirely missing fetal CD34
stem cells. The primitive hemopoietic stem and progenitor cells are largely represented in fetal blood and
circulate early in pregnancy. Which fetal cell type is more highly represented in maternal blood is so far
unknown. In the present study, when FISH was performed on nuclei obtained from cells isolated from
maternal blood , we counted a higher number of trisomic cells than the cumulative number of ε/γ-chain-Hb-
positive erythroblasts we had previously observed6. This finding suggests that the fetal cells detected, based
on the presence of trisomic nuclei in maternal samples, might represent cell types other than, or in addition
to, fetal hemoglobin-expressing erythroblasts. This same observation has been performed by other
investigators who provocatively suggested that "most fetal cells found in maternal blood by FISH methods
may not be NRBCs". In our experience a strict adherence to Yan’s protocol, including a 5 min KCl
hypotonic treatment, was found to be optimal providing intact nuclei and unambiguous FISH signals.
This modified protocol for fetal cell enrichment and the dual-probe FISH approach allow automated FISH
analysis for rare cell detection to be completed within 120 min (360 min for chromosomes 21, 18 and 13
full panel prenatal aneuploidy analysis).
Furthermore, a manual search for fetal cell detection by FISH also becomes feasible. This procedure is easy
and relatively inexpensive, therefore appropriate both for high- and low-income contexts, many samples
per week can be completed and might provide a routine diagnostic test clinically useful when confirmed in
large-scale studies and in other laboratories.