1 Harris Birthright Research Centre For Fetal Medicine, 2 Department of Haematological Medicine and 3 Department of Molecular Medicine, Kings College London School of Medicine, Denmark Hill, London SE5 9RS, UK
4 To whom correspondence should be addressed.
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Abstract |
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Key words: embryonic (,
) and fetal (
) globins/fetal cells/maternal blood/multifetal pregnancy
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Introduction |
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In multifetal pregnancies, there is an increase in the level of maternal serum alpha-fetoprotein (AFP), which is proportional to the number of fetuses. This may be due to associated increase in feto-maternal haemorrhage (Abbas et al., 1994) or twinning by itself. The aim of this study is to investigate further feto-maternal cell trafficking in multifetal pregnancies.
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Materials and methods |
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Isolation of fetal cells and staining
Maternal venous blood (20 ml) samples were collected into Lithium heparinized vacutainers (Beckton Dickinson, Franklin Lakes, NJ, USA), stored at 4°C, and processed within 24 h of collection. Triple density gradient centrifugation was carried out as previously described and the middle layer containing the erythroblasts was separated and isolated ((Al-Mufti et al., 1999b, 2000a,b, 2001a,b). Cells were incubated with magnetically-labelled CD71 antibody to the transferrin receptor antigen (Miltenyi Biotech, Bergisch Gladbach, Germany) for 15 min at 4°C. Magnetic cell sorting was then performed to enrich these erythroblasts as previously described (Al-Mufti et al., 1999b
, 2000a,b, 2001a,b). In each case, four aliquots were obtained from the positively selected fractions, cells were cytocentrifuged at 14.3 g for 10 min (Shandon, Frankfurt, Germany) and were cytospun onto four slides. The fetal cells were detected and quantitated using two methods; firstly by morphology staining by the KleihauerBetke method (GTI, North Patrick Boulevard, Brookfield, Wisconsin, USA) and counterstaining with methylene blue (Gurr-Giemsa, BDH Merck Ltd, Poole, UK), and secondly by immunocytochemistry using monoclonal fluorescein isothiocyanate (FITC) conjugate fluorescent antibody against zeta (
), epsilon (
) and gamma (
) haemoglobin chains. Cells were fixed and permeabilized, as previously described (Al-Mufti et al., 2000a
,b,c, 2001a,b) using a commercial Fix and Perm reagents (Caltac Burlingame, CA, USA). Slides were then incubated with monoclonal FITC conjugate fluorescent antibody for the
,
and
chain respectively ((Al-Mufti et al., 2000a
,b,c, 2001a,b). After antibody incubation, the slides were washed in phosphate buffered saline solution and mounted with 4,6-diamidino-2-phenylindole (DAPI). The slides were examined under fluorescence microscope (Zeiss Axioskop microscope, Carl Zeiss, Gottingen, Germany), using DAPI/FITC/TRITC (tetramethyl rhodamine isothiocyanite) triple band pass filter set. Image capture and processing was by a Microsoft computerized system (Vysis Inc., Downers Grove, Illinois, USA). Nucleated cells that showed specific staining above the DAPI background stain were counted as positive. At least 100 nucleated cells were counted.
The remaining cells in the positive fractions were centrifuged, treated with KCl and fixed with methanol/glacial acetic acid. Cells were transferred to glass slides and fluorescence in-situ hybridization (FISH) was carried out using a dual chromosome-specific DNA probes kit (Vysis Inc.) to detect chromosomes X and Y as previously described (Al-Mufti et al., 1999b, 2000a,b, 2001a,b). The number of nucleated cells and Y-signal positive cells were calculated. At least 100 nucleated cells were examined on each slide and the percentage of cells with one signal for the Y chromosome probe, and one, two and three signals for the X chromosome probe were calculated. Only intact cells that were not overlapping were chosen for the analysis. The slides were examined under fluorescence microscope (Zeiss Axioskop microscope; Carl Zeiss), using DAPI/FITC/TRITC triple band pass filter set. Image capture and processing was by a Microsoft computerized system (Vysis Inc.). Enrichment of fetal cells and analysis were carried out without knowledge of the clinical details of the patients.
Statistical analysis
For morphology and immunocytochemistry, the number and percentage of fetal cells was calculated out of the total nucleated cells for each of the globin chains and for the KleihauerGiemsa stain. On FISH analysis, the number and percentage of cells positive for Y- and X-signals were calculated. Comparison was made between the proportion of cells positive for each staining method in the multifetal and singleton pregnancies using MannWhitney U-test. Spearman correlation coefficient analysis was used to determine the significance of the association between the different methods of staining.
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Results |
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In the multifetal pregnancies, positive erythroblasts were detected in all cases using the KleihauerGiemsa, and
-chains stains and in 16 of the 31 (52%) cases with the
-chain stain, while in the singleton pregnancies positive erythroblasts were identified in 49 of the 50 (98%) cases with the KleihauerGiemsa,
and
-chains and in 10 (20%) cases with the
-chain. The proportion of fetal erythroblasts was significantly higher in the multifetal than in the singleton pregnancies, being three times higher in the twin than in singleton pregnancies (KleihauerGiemsa,
and
-chains, P < 0.0001;
-chain, P < 0.004; Figure 1), twice higher in the triplet than in twin pregnancies (KleihauerGiemsa, P < 0.01;
-chain, P < 0.02;
-chain, P = 0.45;
-chain, P = 0.48; Figure 1), and five times higher in the triplet than in singleton pregnancies (KleihauerGiemsa and
-chain, P < 0.0001;
-chain, P < 0.001;
-chain, P < 0.02; Figure 1). The median percentages of positive-erythroblasts for KleihauerGiemsa,
,
and
-chains for the singleton pregnancies were 2 (range 15), 2 (range 0.54), 1 (range 0.34) and 0.5 (range 0.31) respectively, for twin pregnancies were 6 (range 39), 4 (range 28), 3 (range 17) and 1 (range 0.42) respectively and for triplet pregnancies were 10 (range 511), 7 (range 49), 5 (range 29) and 2 (range 0.23) respectively. There was no significant difference in the percentage of positive erythroblasts between the monochorionic and dichorionic twin pregnancies (KleihauerGiemsa, monochorionic = 4.7, dichorionic = 5.9;
-chain, monochorionic = 4, dichorionic = 4.5;
-chain, monochorionic = 2.5, dichorionic = 3.4;
-chain, monochorionic = 0.7, dichorionic = 1.1%).
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Using FISH for Y chromosome, the proportion of fetal erythroblasts positive for Y-signals was significantly higher in the multifetal than in the singleton pregnancies. Of the 50 singleton pregnancies, 28 were carrying male fetuses. In these 28 males, 11 cases had positive signals for Y chromosome with a median percentage of 3 (range 14). In the 31 multifetal pregnancies there were 28 pregnancies with twins and triplets. Of these 28 pregnancies 14 were carrying male fetuses. In the six monochorionic twins, two were with male fetuses. In the dichorionic twins, seven were with male fetuses, of which four cases had both fetuses being male and three had one male and one female fetus. In the six twin pregnancies with both fetuses being male (two of monochorionic and four of dichorionic), the median percentage of cells positive for Y-signals was 8 (range 39). This was significantly higher than the singleton pregnancies (P < 0.001). In the three cases of dichorionic twins with one male and one female fetus, the median percentage of positive cells for Y-signals was 4 (range 14), which is similar to that in singletons. In triplet pregnancies, five of the seven cases studied were carrying male fetuses. Of these five male pregnancies, two cases had all three fetuses being male and three cases had one female and two male fetuses. In the two cases with all three fetuses being male, the percentages of positive cells for Y-signals were 12 and 11 respectively. This was significantly higher than singleton (P < 0.02) and twin pregnancies (P < 0.05). In the three triplet pregnancies with one female and two male fetuses, the median percentage of positive cells for Y-signals was 8 (range 310), which is similar to the values in twin pregnancies with both fetuses of male gender.
There was a significant correlation between Y-signal FISH and the KleihauerGiemsa (r = 0.85, P < 0.001), -chain (r = 0.75, P < 0.001),
-chain (r = 0.7, P < 0.001) and
-chain (r = 0.5, P < 0.003).
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Discussion |
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We previously reported the use of embryonic and fetal haemoglobins as fetal cell markers to detect the presence of fetal erythroblasts enriched from maternal blood in the first trimester of pregnancy and confirmed these findings by the presence of Y-signals on FISH in the male cases (Al-Mufti et al., 2001b). We demonstrated the variation in the normal frequency of the embryonic and fetal haemoglobin chains in these normal singleton pregnancies (Al-Mufti et al., 2001b
). We have also shown that the embryonic haemoglobin chains were absent in the non-pregnant females and male adults, providing strong evidence of the specificity of the staining and utility of this technique in identifying fetal cells by immunocytochemistry techniques (Al-Mufti et al., 2000c
, 2001b). In this study we demonstrated the distribution of embryonic and fetal haemoglobins in multifetal pregnancies and found an increase in cells positive for the embryonic and fetal haemoglobin chains and an increase in the percentage of cells positive for Y-signals from pregnancies with male fetuses. The data of the current study and previous study therefore exclude the possibility of the enriched erythroblasts that positively stained for embryonic haemoglobins are maternal in origin. In addition, all the pregnancies included in this study were normal and therefore the increase in fetal cell number cannot be attributed to an abnormal pathology such as chromosomal defects (Bianchi et al., 1997
; (Al-Mufti et al., 1999b
) or utero-placental insufficiency (Holzgreve et al., 1998
; (Al-Mufti et al., 2000a
;b) that result in an increase in the proportion of fetal cells in maternal blood.
In the twin pregnancies, the type of placental chorionicity might hypothetically have an effect on transfer of fetal cells across the placenta into the maternal circulation. However, our data showed that chorionicity did not appear to influence the number of fetal cells enriched from maternal blood, with the proportion of enriched erythroblasts being similar in monochorionic and dichorionic twins.
The increase in fetal cells enriched from maternal blood in multifetal pregnancies is most likely to be simply related to the number of fetuses, as well as the increased placental mass, resulting in a larger feto-maternal cell trafficking.
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Acknowledgement |
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References |
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Submitted on March 31, 2003; accepted on May 20, 2003.