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Articles |
-, 
-, and 
-Globin mRNA in Blood Samples and CD71+ Cell Fractions from Fetuses and from Pregnant and Nonpregnant Women, with Special Attention to Identification of Fetal Erythroblasts
1
Departments of Clinical Biochemistry 339, and
2
Gynecology and Obstetrics, Copenhagen University Hospital, H:S Hvidovre Hospital, 30 Kettegaard Allé, DK-2650 Hvidovre, Denmark.
3
The Chromosome Laboratory, Prenatal Research Unit, and
4
Department of Obstetrics and Gynecology, Juliane Marie
Center, H:S Rigshospitalet, University of Copenhagen, 9 Blegdamsvej,
DK-2100 Copenhagen Ø, Denmark.
5
Dako A/S, 42 Produktionsvej, DK-2600 Glostrup, Denmark.
a Author for correspondence. Fax 45-3675-0977; email
thomas.hviid{at}hh.hosp.dk or hviid{at}dadlnet.dk.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-, 
-, and
-globin mRNAs in fetal erythroblasts during gestation and about the
presence and amounts of these mRNAs in pregnant and nonpregnant women
is important from the perspective of using these molecules as a marker
of fetal erythroblasts. A specific marker is necessary for isolation
and identification of fetal nucleated red blood cells from maternal
blood samples for use in antenatal diagnosis of fetal genetic or
chromosomal abnormalities.
Methods: We used a very sensitive reverse transcription-PCR
(RT-PCR) method, coamplification analysis of - and -globin cDNA,
and quantitative analysis of -globin mRNA based on competitive
RT-PCR to investigate these aspects.
Results: All adult whole-blood samples were negative for - and
-globin mRNA. Analyses of CD71+ cell fractions showed
that specimens from 19 of 20 nonpregnant and 10 of 14 pregnant women
(at 9–13 weeks of gestation) were positive for -globin mRNA
(Fisher’s exact test, P = 0.13), and those from 3
of 20 nonpregnant and 5 of 14 pregnant women were positive for
-globin mRNA (Fisher’s exact test, P = 0.23).
No -globin mRNA was detected in CD71+ cell fractions
from 1-mL blood samples from adults. CD71+ cell fractions
from eight fetal blood samples (at 17–20 weeks of gestation) were
positive for all three globin mRNAs. We found no statistically
significant difference between the amounts of -globin mRNA in
pregnant and nonpregnant women.
Conclusions: This study indicates that -globin mRNA might
function as a marker for fetal CD71+ cells early in
pregnancy. Although -globin mRNA can be detected in
CD71+ cell fractions from most adults, these transcripts
also may be of use because of a marked difference between adult and
fetal values.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-, 
-, -, and to some degree -globin have
been detected in adults (1)(2)(3), but the number of studies is
few, and to our knowledge, investigations of the above forms and
comparisons between pregnant and nonpregnant women have not been
performed.
In the embryo, the earliest globin chains are the and chains,
and 22 is the major
hemoglobin until 5–6 weeks of gestation. The
22 form has been
found at 4 weeks but is absent after 13 weeks of gestation. As the
liver replaces the yolk sac as the main site of erythropoiesis,
synthesis of the and chains decreases and that of the and
chains increases. Although hemoglobin F
(22) is present in
very young embryos, it is not the major hemoglobin of fetal life until
10–12 weeks of gestation (4)(5)(6)(7).
The possibility of isolating fetal nucleated red blood cells from maternal blood samples for use in antenatal diagnosis of fetal genetic or chromosomal abnormality has gained much attention in recent years (8)(9). Although several studies have been somewhat successful, further development of this approach of prenatal diagnosis into a broader clinical setting has been hampered by the lack of fetal-specific markers for use in cell isolation and identification procedures. Embryonic and fetal hemoglobin forms are obvious candidates for such markers, and monoclonal antibodies against some of these forms have already been used (10)(11). However, the use of specific globin mRNA molecules as targets for probes in cell identification procedures will require further studies to clarify the possibility of low transcription of these mRNAs in adults, which might be great enough to interfere with probe hybridization detection procedures.
Therefore, information about these embryonic and fetal hemoglobin
mRNAs, especially in pregnant and nonpregnant women, is important from
the perspective of using these molecules as markers of fetal
erythroblasts. To investigate whether these embryonic and fetal globin
transcripts are present in nonpregnant or pregnant women, we analyzed
whole-blood samples and CD71+ cell fractions with
a sensitive reverse transcription-PCR (RT-PCR) method and with
quantitative analysis of -globin mRNA based on competitive RT-PCR.
The CD71 receptor for transferrin is highly expressed on fetal
erythroblasts, and monoclonal antibodies against CD71 have been used
for cell sorting of these cells in several studies
(9)(12).
The amounts of the mRNA for a possible specific fetal marker sequence might be important when using probe hybridization technology (e.g., peptide nucleic acid probes) to identify fetal cells. If the number of specific mRNA copies in adult cells is less than a certain threshold limit, their presence may not lead to positive results in a detection assay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 h). The concentration of human choriogonadotropin
measured in the samples from women who claimed never to have been
pregnant was in all cases <5 IU/L and therefore negative.
isolation of cd71 (transferrin receptor)-positive cells and
extraction of poly(a)+ rna from cd71+ cells
CD71+ cells were isolated from 1 mL of whole
blood using Dynabeads M-450 CD71 (Dynal) according to the
manufacturer’s instructions. The CD71+ cells
were then lysed with 500 µL of Lysis/Binding Buffer from the
Dynabeads mRNA DIRECT reagent set. Poly(A)+ RNA
from CD71+ cells was extracted using the
Dynabeads mRNA DIRECT reagent set according to the manufacturer’s
instructions; 50 µL (250 µg) of Oligo(dT)25
Dynabeads was used.
extraction of poly(a)+ rna from whole blood
Whole blood (1 mL) was centrifuged in a benchtop centrifuge at
maximum speed. The plasma phase was discarded, and the pellet was lysed
with 500 µL of Lysis/Binding Buffer. The
poly(A)+ RNA was extracted as described above,
except that 250 µL (1250 µg) of Oligo(dT)25
Dynabeads was used per sample. The concentration of the extracted
poly(A)+ RNA was calculated from absorbance
measurements at 260 and 280 nm.
qualitative rt-pcr
The presence of -, -, or -transcripts in the
poly(A)+ RNA preparations of the whole-blood
samples was analyzed by three individual, specific RT-PCR reactions.
The PCR step was performed in duplicate. Both steps included negative
controls with no template in the reaction mixture. Reverse
transcription was performed as follows (per reaction): 1.0 µL of
Oligo-dT primer (500 ng/µL; Life Technologies), 200 ng of
poly(A)+ RNA, 4.0 µL of 5x First Strand Buffer
(Life Technologies), 2.0 µL of 0.1 M dithiothreitol (Life
Technologies), 2.0 µL of 8.0 mM dNTPs (2.0 mM each dNTP; Amersham
Pharmacia Biotech), 1.0 µL of Superscript II RNase
H- Reverse Transcriptase (200 U/µL; Life
Technologies), and nuclease-free water (Promega) in a total volume of
20 µL. The mixture was incubated at 42 °C for 55 min and then
inactivated at 70 °C for 15 min.
PCR was performed as follows (per reaction): 5.0 µL of MgCl2 (25 mM; MBI Fermentas), 5.0 µL of 10x PCR buffer (MBI Fermentas), 1.0 µL of Taq polymerase (1 U/µL; MBI Fermentas), 5.0 µL of 8.0 mM dNTPs (2.0 mM each dNTP), 2.5 µL of forward primer (10 µM; TAGC), 2.5 µL of reverse primer (10 µM; TAGC), 1.0 µL of cDNA from the reverse transcription reaction above, and nuclease-free water (Promega) in a volume of 50 µL. Thermocycling conditions were as follows: 94 °C for 3 min; 40 cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min; and 72 °C for 5 min, followed by holding at 4 °C. PCR primers were as follows:
-globin: forward, 5'-TTT TAC TGC TGA GGA GAA GGC TGC C-3';
reverse, 5'-CTT GCC AAA GTG AGT AGC CAG AAT AA-3' -globin: forward, 5'-ACG CCA TGG GTC ATT TCA CAG A-3'; reverse,
5'-GAG CTC AGT GGT ATC TGG AGG A-3' -globin: forward, 5'-CCC TGC CGC CAT GTC TCT GAC CAA G-3';
reverse, 5'-AGG CGG CGT GGG CCT CGG CC-3'
The RT-PCR products were analyzed by electrophoresis on a 2%
agarose gel. Representative RT-PCR products were cloned and sequenced.
Cloning was performed by using the TOPO TA Cloning reagent set with
pCR-2.1 vector (Invitrogen) according to the manufacturer’s
instructions. Sequencing was performed with the Thermo Sequenase
dye-primer 7-deaza cycle seq reagent set (Amersham Pharmacia Biotech)
according to the manufacturer’s instructions. The sequencing primers
were as follows: , 5'-Cy5 ACT CAG CTG GGC AAA GGT GCC-3'; ,
5'-Cy5 ACT CAG CTT AGC AAA GGC GGG-3'; , 5'-Cy5 AGT TGC GCG CGC ACG
GCT CC-3'.
coamplification analysis of - and -globin cDNA
The coamplification analysis was based on the simultaneous
amplification of - and -cDNA (Fig. 1
). The same set of primers could be used for both templates
because of the extensive sequence homology between - and -globin.
One primer was labeled with Cy5, which was incorporated into the PCR
products during amplification. Products of similar length were produced
by this amplification but were cut to different lengths with
sequence-specific restriction enzymes and then analyzed on a denaturing
polyacrylamide gel, where the ratios between the areas under the peaks
corresponding to the restriction products were determined. To verify
the validity of the method and to establish its detection window, we
used different mixtures of and sequences cloned into
pCR2.1-TOPO vectors as templates for coamplification. The ratios in
these mixtures were 1:1–1:100.
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For one set of experiments, K562 cells (from the human chronic
myelogenous leukemia cell line), stimulated with hemin chloride
(Sigma-Aldrich) dissolved in dimethyl sulfoxide (25 µmol/L hemin
chloride was included in the culture medium for 48 h) to induce
the expression of globin mRNAs, was used as starting material.
Poly(A)+ RNA from K562 cells was extracted using
the Dynabeads mRNA DIRECT reagent set according to the manufacturer’s
instructions. cDNA was synthesized as described above. The
coamplification RT-PCR was performed using the reaction mixture
described and thermocycling conditions of 94 °C for 3 min; 30 cycles
of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min; and
72 °C for 5 min, followed by holding at 4 °C. PCR primers for
- and -globin mRNA were as follows: forward, 5'-Cy5 TGG IGC AAG
ITG AAT GTG GGA A-3' (where I = inosine); reverse, 5'-GCT TGA AGT
TCT CAG GAT CCA CA-3'. This RT-PCR created two amplification products
of similar lengths but different sequences. These products were cut to
different lengths by restriction endonucleases specific for each RT-PCR
product. The reaction mixture consisted of 1.6 µL of 10x NE2 buffer
(New England Biolabs), 0.8 µL of nuclease-free water (Promega), 1.6
µL of 10x bovine serum albumin (New England Biolabs), 1.1 µL of
MseI (4 U/µL; New England Biolabs), 0.6 µL of
PvuII (5 U/µL; New England Biolabs), and 10 µL of the
PCR products from the PCR reaction described above. After incubation at
37 °C for 90 min, the restriction fragments were separated on a
denaturing polyacrylamide gel (Amersham Pharmacia Biotech) using the
ALF Express Sequencer (Amersham Pharmacia Biotech) with a run
temperature of 40 °C and a run time of 300 min. The results were
analyzed by Allele Links software (Amersham Pharmacia Biotech).
competitive (quantitative) rt-pcr analysis of -globin
transcripts
The amount of a specific mRNA transcript in a sample can be
determined by the use of competitive RT-PCR analysis. We used a
modification of the method of Diviacco et al. (13) to design
a -globin DNA internal standard for the analysis. In addition to
inserting an artificial linker sequence in the internal standard as
devised by Diviacco et al., we incorporated 50 bp of intron 1 in the
genomic sequence of -globin to increase the difference in length
between the natural transcript and the internal standard. The insertion
of an intron sequence into the DNA sequence of the internal standard
was achieved by a combination of two separate PCR reactions, A and B. A
third PCR joined the products of reactions A and B and generated the
internal standard. The following reaction conditions were similar for
reactions A and B: 5.0 µL of MgCl2 (25 mM; MBI
Fermentas), 5.0 µL of 10x PCR buffer (MBI Fermentas), 1.0 µL of
Taq polymerase (1 U/µL; MBI Fermentas), 5.0 µL of 8.0 mM dNTPs (2.0
mM each dNTP), 7.5 µL of forward primer (10 µM; TAGC), and 2.5 µL
of reverse primer (10 µM; TAGC). The template for reaction A was 2.0
µL of human genomic DNA (100 ng/µL), whereas that for reaction B
was 1.0 µL of K562 cDNA. For both reactions nuclease-free water
(Promega) was added to give a volume of 50 µL. Thermocycling
conditions were as follows: 94 °C for 3 min; 2 cycles of 94 °C
for 1 min, 50 °C for 1 min, and 72 °C for 1 min; 33 cycles of
94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min; and
72 °C for 5 min, followed by holding at 4 °C. The primers were as
follows:
The PCR products were analyzed on a 1% agarose gel. Separate
pipette tips were dipped into the band for each product, A and B. The
tips were incubated in 50 µL of sterile water for 2 min at room
temperature. Five microliters of this mixture was used as template with
the following PCR reaction mixture: 10.0 µL of
MgCl2 (25 mM; MBI Fermentas), 10.0 µL of 10x
PCR buffer (MBI Fermentas), 2.0 µL of Taq polymerase (1 U/µL; MBI
Fermentas), 10.0 µL of 8.0 mM dNTPs (2.0 mM each dNTP), 5.0 µL of
forward primer (10 µM; TAGC), 5.0 µL of reverse primer (10 µM;
TAGC), and nuclease-free water (Promega) to give a volume of 100 µL.
Thermocycling conditions were as follows: 94 °C for 1 min, 90 °C
for 1 min, 85 °C for 1 min, 80 °C for 1 min, 75 °C for 1 min,
65 °C for 1 min, 60 °C for 1 min, 55 °C for 1 min, 50 °C
for 2 min, and 72 °C for 1 min, followed by 30 cycles of 94 °C
for 1 min, 60 °C for 1 min, and 72 °C for 1 min, and finally
72 °C for 5 min. The PCR product was analyzed by gel electrophoresis
on a 1% agarose gel. The PCR product was then cloned using the TOPO TA
Cloning reagent set with pCR-2.1 vector (Invitrogen) according to the
manufacturer’s instructions. The plasmid carrying the internal
standard sequence was used as a template (diluted 1:5000) in a PCR
reaction with primers for the -globin sequence, as described
previously. Before quantification by absorbance measurements, the PCR
product was cleaned up using the spin column technique with Sephacryl
S-400 HR (Amersham Pharmacia Biotech). The PCR product was henceforth
used as a DNA internal standard.
Before subjecting the samples to competitive RT-PCR analysis, we had to establish an interval in which the amount of amplification product from the sample transcript roughly equaled the amount of amplification product of the added internal standard. This was accomplished by amplifying serial dilutions of the internal standard in a fixed amount of sample cDNA. The actual competitive RT-PCR analysis was subsequently carried out at the concentration at which the amounts of the internal standard and the sample products were equal and at the concentrations immediately bracketing this value. The reaction mixture for the competitive RT-PCR was similar to that described for qualitative RT-PCR except for addition of the standard template. The cycling conditions were also similar, except that the amplification steps were repeated for 35 cycles instead of 40. It was also necessary to verify that the amplification kinetics for the two templates were the same. To do so, we set up a 60-µL PCR reaction using nearly equal amounts of sample transcript and internal standard as templates. Aliquots (10 µL) were removed from the reaction mixture after 20, 25, 30, 35, and 40 cycles and analyzed to determine whether the original ratio between the two templates was conserved through the course of the reaction, as would be expected.
statistical analyses
The number of samples positive for a specific globin mRNA in the
groups of pregnant and nonpregnant women was compared by Fisher’s
exact test. Differences in the amount of -globin mRNA in samples
from pregnant and nonpregnant women were compared by the Mann–Whitney
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-globin
rt-pcr analysis-globin
DNA internal standard (453 bp) for use in competitive RT-PCR vs the
sample -globin RT-PCR product (523 bp), obtained using the same set
of primers and having considerable sequence similarity, which is a
requirement for use in competitive RT-PCR. The sequence of the DNA
internal standard fragment was verified by DNA sequencing and included
both the intron sequence and the linker sequence. The sequence of the
sample -globin mRNA RT-PCR product was also verified by sequencing.
kinetics of amplification of -globin internal standard and
sample cDNA
cDNA from the mRNA was coamplified with a nearly equal amount of
the corresponding internal standard over a range of PCR amplification
cycles (20–40). The concentration of (RT)-PCR products was determined
by densitometry. In Fig. 2
the amount of (RT)-PCR product is plotted as a function of the
cycle number, and the ratio (internal standard:sample) from the
densitometry results is plotted against the cycle number. As shown, the
internal standard and the sample sequence were amplified with the same
efficiency for all numbers of amplification cycles. The CV of the
ratios of the areas of two (RT)-PCR product bands in an agarose gel as
measured by densitometry was 9%. The CV of the whole procedure from
isolation of CD71+ cells to quantification of
-globin mRNA by the competitive RT-PCR, determined with six
replicates, was 17%. We consider this an acceptable CV, given that the
procedure is divided into several parts, each containing many steps.
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amounts of - and -globin mRNA transcripts in
induced k562 cells
To verify the validity of the method and to establish its
detection window, we performed coamplification analysis on different
ratios of the artificially mixed templates of cloned - and
-globin sequences, and the electropherograms correctly reflected the
ratios of the mixture down to a ratio of 1:25 (: or :);
below this only the dominant template was detected. Use of RT-PCR
coamplification analysis showed that the relative proportion of -
and -globin mRNA was 3:1 in the induced K562 cells. An example of an
electropherogram of the PCR fragments is shown in Fig. 3
. The quantitative -globin mRNA RT-PCR procedure indicated
that the amount of -globin mRNA in induced K562 cells was 
1200
copies/cell (or 920 pg/µg of total mRNA). An example of -globin
mRNA internal standard titrated in a fixed amount of cDNA derived from
K562 cells followed by PCR amplification is shown in Fig. 4
, together with a plot of the data. Given that the proportion of
- to -transcripts is 1:3, the amount of -globin mRNA in the
induced K562 cells must be 400 copies per cell.
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-, -, and -mRNA in fetal blood samples and
fetal cd71+ cell fractions
The qualitative RT-PCR results are listed in Table 1
. In all fetal samples (17–20 weeks of gestation), -, -, and
-mRNA could be detected in the CD71+ cell
fraction; in some whole-blood samples, however, - and -mRNA could
not be detected. The quantitative -globin RT-PCR analysis showed
that the median amount of -globin mRNA in fetal
CD71+ cells (at 17–20 weeks of gestation) was
151 ng/µg of total mRNA (range, 87–810 ng/µg of total mRNA; n
= 5). This corresponded to 1.6 x 1011
copies/µg of total mRNA. Positive results for RT-PCR analyses or the
quantities of -globin mRNA showed no correlation with the number of
weeks of gestation in the narrow gestational interval investigated.
-Globin mRNA should correspond to 15% of the total mRNA pool in the
fetal CD71+ cells. Furthermore, the
coamplification RT-PCR assay was not useful for coamplifing - and
-globin mRNA transcripts in the CD71+ cells
from the fetal blood samples because expression of -globin mRNA has
ceased or decreased to a very low amount by 17 weeks of gestation.
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-, -, and -globin mRNA in blood samples and in
cd71+ cell fractions from pregnant and nonpregnant women
All adult whole-blood samples were negative for - and
-globin mRNA (Table 1
). Analyses of CD71+ cell
fractions showed that 19 of 20 nonpregnant and 10 of 14 pregnant women
were positive for -globin mRNA (Fisher’s exact test,
P = 0.13), and 3 of 20 nonpregnant and 5 of 14 pregnant
women were positive for -globin mRNA (Fisher’s exact test,
P = 0.23); any difference between the pregnant and the
nonpregnant women was not significant. The amount of -globin mRNA in
CD71+ cells from pregnant and nonpregnant women
was in the same range: median, 85 pg/µg of total mRNA (range,
26–1185 pg/µg of total mRNA; n = 7) for pregnant women vs 161
pg/µg of total mRNA (42–790 pg/µg of total mRNA; n = 6;
P = 0.37, Mann–Whitney test) for nonpregnant women.
This corresponds to a median of 9.0 x 107
copies/µg of total mRNA for nonpregnant women and 1.7 x
108 copies/µg of total mRNA for pregnant women.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and -globin mRNA in the reticulocytes
of healthy adults and patients with sickle cell disease, chronic
myelogenous leukemia, and polycythemia vera (1). One approach to identifying fetal erythroblasts in the maternal blood for use in prenatal diagnosis could be based on a crude preselection of, e.g., CD71+ cells in a maternal blood sample, followed by identification of the fetal cells with a hybridization probe directed against the mRNA of an embryonic or fetal globin sequence. A highly specific probe based on recognition of a nucleic acid sequence could be more specifically compared with antibodies directed against protein epitopes.
In this study, we investigated the presence of - and -globin
mRNAs and the amounts of -globin mRNA in whole-blood samples and
CD71+ cell fractions from nonpregnant women,
pregnant women (at 9–13 weeks of gestation), and fetal blood (at
17–20 weeks of gestation). We had no access to fetal samples from the
first trimester of pregnancy.
We undertook the examination of a possible background transcription of
fetal hemoglobin mRNAs in adult blood cells. The transferrin receptor
CD71 was chosen as a marker for the preselection because it is highly
expressed on nucleated red cells in early fetal blood (12).
Having examined whole-blood samples and the CD71+
fractions of these samples with RT-PCR, we found that a preselection
step is necessary to increase the prevalence of erythroblasts in a
sample to permit detection of the embryonic and fetal mRNAs. All adult
whole-blood samples were negative for - and -globin mRNA. Perhaps
isolating the mRNA from 10–20 mL of whole blood and using more RT-PCR
amplifications for each sample might increase the possibility of
obtaining positive results. One adult (nonpregnant) whole-blood sample
was positive for -globin mRNA. All adult samples were negative for
-globin mRNA, whereas the fetal samples were positive. Albitar et
al. (1) previously identified low amounts of - and
-globin mRNA in the reticulocytes of patients with sickle cell
disease, chronic myelogenous leukemia, and polycythemia vera. They also
detected - and -globin mRNA by radioactively labeling RT-PCR
products from reticulocyte RNA from four apparently healthy adults.
Only trace amounts of -globin mRNA were detected in these samples
after several days of exposure. This is consistent with our own
findings, although we did not detect - or -globin mRNA in all
blood samples. Perhaps the content of these hemoglobin mRNAs in the
heterogeneous cell fractions falls below the detection limit of the
RT-PCR assay, or perhaps our method of detection (ethidium
bromide-stained RT-PCR products on an agarose gel) is less sensitive
than with RT-PCR radioactive labeling. Or perhaps no - or -globin
mRNA was present in the particular samples we examined.
In our search for a globin mRNA combination specific for fetal
erythroblasts, we designed a coamplification RT-PCR assay for - and
-globin mRNA to determine whether the ratio of - to -globin
mRNA could be used as a positive identification of a fetal cell. The
assay was initially tested on a model system of stimulated K562 cells
or artificial DNA samples with different known ratios of - to
-globin and - to -globin sequences. Analyses of the artificial
DNA samples showed that the assay detected ratios between 1:1 and 1:25.
Analysis of the stimulated K562 cells showed that in these cells the
ratio of - to -globin mRNA is 3:1. Subsequent analysis of both
fetal whole blood and CD71+ cell fractions did
not detect any -globin mRNA. This indicates that the production of
hemoglobin at gestation week 17 has either ceased or decreased to a
point where the ratio of - to -globin mRNA exceeds 25:1. This is
consistent with an immunocytochemical study of the expression of
and hemoglobin in erythroblasts from fetal blood at 10 weeks of
gestation (11). That study showed that 1–5% of the
erythroblasts express only hemoglobin at that stage, whereas 1–3%
express both and hemoglobin. Furthermore, -positive blasts
were seen in fetal liver tissue at 14 weeks of gestation at a frequency
of 2–3%, and blasts positive for both phenotypes were seen in 1%.
This expression of -globin will have decreased even further by
gestation week 17 and makes our results, which indicated a ratio of
- to -globin mRNA >25:1, very probable. The results from the
immunocytochemical studies, together with our own results, also help
explain why no -globin mRNA was detected in the samples from
pregnant women at 9–13 weeks of gestation. Studies indicate that only
1–10 fetal cells can be detected in 1 mL of maternal blood
(14)(15). If some of these fetal cells contain
only a little (low percentage) -mRNA/hemoglobin, then the
probability of detecting -mRNA in such a sample would be very low.
Although we are aware of the possible limitations of the present study
(RT-PCR amplification based on 1 mL of blood), the lack of detection of
-globin mRNA in pregnant (9–13 weeks of gestation) and nonpregnant
women leads us to speculate that -globin mRNA could be a candidate
marker for fetal erythroblasts in a background of maternal blood cells
at 6–8 weeks of gestation. Questions remain regarding whether fetal
erythroblasts typically are present in maternal blood, and in what
numbers they occur at this early stage of pregnancy.
The coamplification assay described here can be used in the analysis of
- and -globin mRNA ratios in very early fetal blood (weeks 6–8)
with special attention to identifying fetal cells based on globin mRNA
ratios or monitoring the switch from embryonic to fetal globin mRNAs.
Furthermore, the assay might be used to identify fetal erythroblast
colonies in in vitro cell cultures of cell populations isolated from
maternal blood samples. These in vitro cell culture systems are a
possible way to increase the number of fetal cells from maternal blood,
as has been described by some investigators
(16)(17).
To investigate the possible differences between the amounts of
-globin mRNA in the blood cells of pregnant women and those of
nonpregnant women, we designed a quantitative RT-PCR assay with a DNA
internal standard. Essentially, we used the method described by
Diviacco et al. (13) and evaluated the assay as described by
Hayward et al. (18). We initially tested the assay with a
definite number of stimulated K562 cells and found that the amount of
-globin mRNA in these was 1200 copies/cell. Accordingly, the
number of -globin mRNA copies must be 400. In subsequent tests of
the CD71+ cell fractions of whole blood from
pregnant and nonpregnant women, we found that the -globin mRNA was
present in CD71+ cells from nonpregnant women at
a median of 85 pg/µg of total mRNA, whereas the median in pregnant
women was 161 pg/µg of total mRNA. However, the median value in
pregnant women, although higher, was not significantly different from
that of nonpregnant women (P = 0.37). This result seems
reasonable considering that the samples were from women only in the
first trimester of pregnancy. We think that it is possible and rather
likely that some of the -globin mRNA found in the blood samples from
the pregnant women originates from fetal cells. From the results in
this study, however, whether any of the -globin mRNA in the samples
from the pregnant women at 9–13 weeks of gestation originates from
fetal cells cannot be determined. Moreover, the -globin mRNA in
samples from pregnant and nonpregnant women might not be directly
comparable because they represent the amount of -globin mRNA in a
CD71+ cell fraction, not that in whole blood. The
CD71+ cell isolation procedure was used as a
means of enriching for cells expressing the globin mRNAs of interest
and an attempt to obtain a pure cell fraction. Pregnant women have a
greater total white cell count (which in an activated state may express
CD71) in the peripheral blood than do nonpregnant women [see, e.g.,
Ref. (19)]; therefore, the number of
CD71+ erythroblasts isolated from pregnant women
might be relatively fewer than the number isolated from nonpregnant
women because the erythroblasts compete with the activated lymphocytes
for the antibodies to CD71. Thus, the possibility exists that the
peripheral blood cells of pregnant women may contain embryonic and
fetal globin mRNAs in greater numbers than stated above.
Because the CD71+ cell fraction is a
heterogeneous cell population, we chose to quantify the -mRNA globin
per total mRNA rather than per cell. Some cells might contain rather
high amounts of -mRNA and some very little. Accordingly, for
determining the specific amount of -mRNA in single
CD71+ cells or in cells positive for
-hemoglobin protein from nonpregnant and pregnant women, laser
microdissection methods must be used. The methods described here could
form the basis for such a study, which could be important in designing
a detection assay for fetal erythroblasts based on -globin mRNA
contents in single cells as described below. Furthermore, RT-PCR-based
quantification of -globin mRNA in single fetal erythroblasts at 6–8
weeks of gestation would be relevant for use of this marker with in
situ hybridization techniques. Our study indicates that -globin mRNA
is not present (or is present only in a very low amount) in maternal
erythroblasts. Therefore, as stated previously, -globin mRNA might
be a useful marker of fetal erythroblasts in early pregnancy.
Quantification of -globin mRNA in pregnant and nonpregnant women may
seem imminently possible, but it was not performed in this study.
Because -mRNA can be detected in both nonpregnant and pregnant
women, and probably not in high amounts in fetal erythroblasts after 10
weeks of gestation, -mRNA would never be a completely specific fetal
marker; indeed, it is probably present in substantially greater amounts
in fetal erythroblasts than in maternal erythroblasts only during a few
weeks in very early pregnancy.
Examination of the -globin mRNA content of the
CD71+ fraction of fetal blood by the quantitative
RT-PCR method showed a median of 151 ng/µg of total mRNA. Again, this
number may not be directly comparable to the numbers for the women
because of a possible difference in the populations of isolated
CD71+ cells; nonetheless, the -globin mRNA
obviously is expressed to a considerably greater extent in the fetal
cell fraction than in the adult fractions. Therefore, as the results in
this work indicate, further studies on a homogeneous population of
fetal or adult erythroblasts that produce -globin mRNA may reveal
whether the difference in the amount of -globin mRNA produced by
fetal and adult cells can be used as a marker for identifying or
isolating a fetal cell. The amount of -globin mRNA in adult cells
might very well be below the threshold value for a positive signal in,
e.g., an in situ probe hybridization detection system, such that only
fetal nucleated red blood cells would show a positive signal.
In conclusion, using RT-PCR methods and 1-mL blood samples, we
found that - and -globin, but not -globin mRNA, can be
detected in the CD71+ fractions of peripheral whole blood
from pregnant (end of first trimester) or nonpregnant women.
CD71+ fractions of fetal blood (at gestation weeks 17–20)
express all three mRNA forms. On the basis of our results, we speculate
that -globin mRNA might be useful as a positive identification
marker of fetal nucleated red blood cells in a background of maternal
blood cells in early pregnancy.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-globin cluster: fetal/adult pattern of -globin gene expression. Blood 1992;80:1586-1591. and chains. Nature 1975;256:511-513.[Medline]
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) and epsilon () type haemoglobin in fetal red cells. Prenat Diagn 1998;18:1131-1137.[Web of Science][Medline]
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