1 Harris Birthright Research Centre for Fetal Medicine and 2 Department of Haematological Medicine, King's College Hospital, London, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Doppler ultrasound/fetal cells in maternal blood/intrauterine growth restriction/pre-eclampsia/screening
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pre-eclampsia is thought to be the consequence of abnormal trophoblast invasion of the maternal spiral arteries (Brosens et al., 1972; Sheppard and Bonnar, 1976
), leading to impaired placental perfusion and placental insufficiency. Doppler ultrasound studies have shown that impaired placental perfusion at 2226 weeks gestation is associated with high impedance to flow in the uterine arteries, which is characterized by the presence of an early diastolic notch in the waveform from these vessels. The presence of this abnormal waveform identifies a group of women at high risk of subsequent development of PET and/or IUGR (Steel et al., 1990
; Bower et al., 1993
; Harrington et al., 1996
; Frusca et al., 1997
).
The aim of this study was to determine if the number of fetal cells in maternal blood is increased before the onset of PET and/or IUGR by examining pregnancies with abnormal uterine artery Doppler results before the onset of these complications.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Details of pregnancy outcome were obtained from the patient notes. Pre-eclampsia was diagnosed as maternal blood pressure >140/90 mmHg on more than one occasion with `+' or more protein on reagent strip urinalysis or urine protein collection >300 mg/24 h. The diagnosis of IUGR was made if the birth weight was below the fifth centile of the normal range for gestation (Yudkin et al., 1987).
Isolation and detection of fetal cells
Maternal venous blood (20 ml) was collected into lithium heparinized vacutainers (Becton Dickinson, Franklin Lakes, NJ, USA) after the ultrasound examination, and stored at 4°C. The samples were processed within 24 h of collection. Triple density gradient centrifugation was carried out as described previously and the middle layer containing the erythroblasts was separated and isolated (Ganshirt-Ahlert et al., 1993; Al-Mufti et al., 1999b
). 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 described previously (Ganshirt-Ahlert et al., 1993
; Al-Mufti et al., 1999b
). Aliquots of the positively selected cells were cytocentrifuged at 14.3 g for 10 min (Shandon, Frankfurt, Germany), and the cells were cytospun onto slides. The fetal cells were detected and quantified using two methods: (i) by morphology after staining by the KleihauerBetke method (GTI, Brookfield, WI, USA) and counterstaining with methylene blue (GurrGiemsa; BDH Merck Ltd, Poole, UK); and (ii) by immunocytochemistry using monoclonal fluorescein isothiocyanate (FITC) conjugate fluorescent antibody against the
haemoglobin chain (Caltac, Burlingame, CA, USA). Cells were fixed and permeabilized using commercial `Fix and Perm' reagents (Caltac); slides were incubated with the fluorescent antibody and mounted with 4,6-diamidino-2-phenylindole (DAPI), and then visualized under a fluorescence microscope (Zeiss Axioskop; Carl Zeiss, Gottingen, Germany). Nucleated cells that showed specific staining above the background stain were counted as positive. In each staining method at least 100 nucleated cells were counted on each slide for each case.
The remaining cells in the positive fraction were centrifuged, treated with KCl and fixed with methanol/glacial acetic acid, and frozen at 20°C. Cells were transferred to glass slides and fluorescent in-situ hybridization (FISH) was carried out as described previously (Al-Mufti et al., 1999b, 2000
), using a dual chromosome-specific DNA probe (Vysis Inc., Downers Grove, IL, USA) to screen for X and Y chromosomes, as recommended by the manufacturers. 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 a fluorescence microscope (Zeiss Axioskop), using a DAPI/FITC/TRITC triple band pass filter set. Image capture and processing was by a Microsoft computerized system (Vysis). Enrichment of fetal cells and analysis were carried out without knowledge of the clinical details of the patients.
Statistical analysis
Comparison between groups was carried out using MannWhitney U-Wilcoxon rank sum tests. Spearman correlation coefficient was carried out to examine the association between results obtained by the different methods of fetal cell detection.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Possible causes for the increase in fetal erythroblasts in maternal blood in pregnancies with abnormal uterine artery Doppler waveform that subsequently developed PET and/or IUGR include increased fetal production of erythroblasts or increased transfer across the placenta. Certainly, in severely growth-restricted hypoxaemic fetuses the fetal erythroblast count is increased (Nicolaides et al., 1987; Soothill et al., 1987
; Snijders et al., 1993
). However, in our pregnancies at the time of maternal blood sampling, fetal growth and fetal arterial Doppler results were normal and the mothers were normotensive. Increased transfer across the placenta may be due to placental damage secondary to impaired perfusion and placental oxygenation. However, the concentration of maternal serum AFP in the pregnancies that subsequently developed PET and/or IUGR was not significantly different from normal, and there was no significant association between the number of fetal erythroblasts and the concentrations of AFP. Since the half-life of AFP is only 57 days (Crandall, 1981
; Caballero et al., 1997
), whereas the life-span of erythroblasts is about 6070 days (Hann et al., 1991
), it is possible that the placental damage unmasked by the abnormal Doppler results at 2226 weeks may have occurred several weeks previously. Indeed, there is evidence that elevated concentrations of AFP at around 1520 weeks are associated with the subsequent development of pregnancy complications (Crandall, 1981
; Walters et al., 1993; Morssink et al., 1995
).
Previous reports have demonstrated that PET is associated with an increase in trophoblasts, free fetal DNA and fetal erythroblasts in the maternal circulation (Chua et al., 1991; Holzgreve et al., 1998
; Lo et al., 1999
). In the previous study examining fetal erythroblasts in maternal blood in pregnancies with PET (Holzgreve et al., 1998
), it was suggested that the increased influx into the maternal circulation of either allogeneic immune effector cells or those that can develop into them could contribute to disease progression or at least exacerbate the condition. Although our findings that the increase in fetal erythroblasts in maternal blood precedes the onset of PET is compatible with the above hypothesis, our patients had Doppler evidence of impaired placental perfusion. To demonstrate a causative association between increased feto-maternal cell trafficking and PET, it would be necessary to show that the increase in fetal erythroblasts precedes the onset of abnormal Doppler results.
In a previous study (Holzgreve et al., 1998), despite there being a good correlation between the number of erythroblasts which stained positive with Giemsa and the number of cells positive for Y chromosome on FISH, the number of Y chromosome-positive cells was much lower than those cells stained by Giemsa. This difference was attributed to the presence of enriched maternal erythroblasts. Our data showed a similar pattern but not such a marked difference between the number and percentage of erythroblasts positive for KleihauerGiemsa staining and cells positive for Y chromosome on FISH.
Early diastolic notches in the Doppler waveform of the uterine arteries at about 24 weeks gestation identifies a group of pregnancies at high risk of subsequent development of PET and/or IUGR (Steel et al., 1990; Bower et al., 1993
; Harrington et al., 1996
; Frusca et al., 1997
). The potential clinical application of our finding is in reducing the false-positive rate in patients with abnormal Doppler waveforms. However, the complexity and expense of investigating the presence of fetal erythroblasts in maternal blood at present are such to make it unlikely that this technique would have a useful role in screening for PET and/or IUGR. Nevertheless, the finding of increased feto-maternal cell traffic preceding the onset of the clinical manifestations of the disease provides further insight into the pathophysiology of impaired placentation.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Al-Mufti, R., Hambley, H., Farzaneh, F. and Nicolaides, K.H. (1999b) Investigation of maternal blood enriched for fetal cells: role in screening and diagnosis of fetal trisomies. Am. J. Med. Genet., 85, 6675.[ISI][Medline]
Al-Mufti, R., Lees, C., Albaiges, G. et al. (2000) Fetal cells in maternal blood of pregnancies with severe fetal growth restriction. Hum. Reprod., 15, 218221.
Bower, S., Bewley, S. and Campbell, S. (1993) Improved prediction of pre-eclampsia by two-stage screening of uterine arteries using the early diastolic notch and colour Doppler imaging. Obstet. Gynecol., 82, 7883.[Abstract]
Brosens, I.A., Robertson, W.B. and Dixon, H.G. (1972) The role of the spiral arteries in the pathogenesis of pre-eclampsia. Obstet. Gynaecol., 1, 177191.
Caballero, C., Vekemans, M., Lopez-del-Campo, J.G. and Robyn, C. (1997) Serum alpha fetoprotein in adults, in women during pregnancy, in children at birth, and during the first week of life: a sex difference. Am. J. Obstet. Gynecol., 127, 384389.
Chua, S., Wilkins, T., Sargent, I. and Redman, C. (1991) Trophoblast deportation in pre-eclamptic pregnancy. Br. J. Obstet. Gynaecol., 98, 973979.[ISI][Medline]
Crandall, B.F. (1981) Alpha-fetoprotein: a review. Crit. Rev. Clin. Lab. Sci., 15, 127185.[Medline]
Frusca, T., Soregaroli, M., Valcamonico, A. et al. (1997) Doppler velocimetry of the uterine arteries in nulliparous women. Early Hum. Dev., 48, 177185.[ISI][Medline]
Ganshirt-Ahlert, D., Borjesson-Stoll, R., Burschyk, M. et al. (1993) Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting. Am. J. Reprod. Immunol., 30, 194201.[ISI][Medline]
Ganshirt-Ahlert, D., Garritsen, H.S.P. and Holzgreve, W. (1995) Fetal cells in maternal blood. Curr. Opin. Obstet. Gynaecol., 7, 103108.[ISI][Medline]
Hann, I.M., Gibson, B.E.S. and Letsky, E.A. (1991) (eds) The normal blood picture in neonates. In Fetal and Neonatal Haematology. Ballière-Tindall, p. 40.
Harrington, K., Cooper, D., Lees, C. et al. (1996) Doppler ultrasound of the uterine arteries: the importance of bilateral notching in the prediction of pre-eclampsia, placental abruption or delivery of a small-for-gestational-age baby. Ultrasound Obstet. Gynecol., 7, 182188.[ISI][Medline]
Holzgreve, W., Ghezzi, F., Di Naro, E. et al. (1998) Disturbed feto-maternal cell traffic in pre-eclampsia. Obstet. Gynaecol., 91, 669672.
Lo, Y.M.D., Leung, T.N., Tein, M.S.C. et al. (1999) Quantitative abnormalities of fetal DNA in maternal serum in pre-eclampsia. Clin. Chem., 45, 184188.
Morssink, L.P., Kornman, H., Beekhuis, J.R. et al. (1995) Abnormal levels of maternal serum human chorionic gonadotropin and alpha-fetoprotein in the second trimester: relation to fetal weight and preterm delivery. Prenat. Diagn., 15, 10411046.[ISI][Medline]
Nicolaides, K.H., Clewell, W.H. and Rodeck, C.H. (1987) Measurement of human fetoplacental blood volume in erythroblastosis fetalis. Am. J. Obstet. Gynecol., 157, 5053.[ISI][Medline]
Sheppard, B.L. and Bonnar, J. (1976) The ultrastructure of the arterial supply of the human placenta in pregnancy complicated by fetal growth retardation. Br. J. Obstet. Gynaecol., 83, 948959.[ISI][Medline]
Snijders, R.J.M., Abbas, D.M., Melby, O. et al. (1993) Fetal plasma erythropoietin concentration in severe growth retardation. Am. J. Obstet. Gynecol., 168, 615619.[ISI][Medline]
Soothill, P.W., Nicolaides, K.H. and Campbell, S. (1987) Prenatal asphyxia, hyperlacticaemia, hypoglycaemia, and erythroblastosis in growth retarded fetuses. Br. Med. J., 294, 10511053.[ISI][Medline]
Steel, S.A., Pearce, J.M., Mcparland, P. and Chamberlain, G.V. (1990) Early Doppler ultrasound screening in prediction of hypertensive disorders of pregnancy. Lancet, 335, 15481551.[ISI][Medline]
Walters, C. (1993) Poor pregnancy outcome associated with elevated maternal serum alpha-fetoprotein in combination with increased risk for Down syndrome. Prenat. Diagn., 13, 221222.[ISI][Medline]
Yudkin, P.L., Aboualfa, M., Eyre, J.A. and Redman, C.W.G. (1987) New birth weight and head circumference centile for gestational ages 24 to 42 weeks. Early Hum. Dev., 15, 4552.[ISI][Medline]
Submitted on November 10, 1999; accepted on April 13, 2000.