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The Fetal Erythroblast Is Not the Optimal Target for Non-invasive Prenatal Diagnosis : Preliminary Results

Steen Kølvraa, Britta Christensen, Lene Lykke-Hansen and John Philip

Prenatal Research Unit, Juliane Marie Center, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark (BC,JP,LL-H), and Institute of Human Genetics, University of Århus, Århus, Denmark (SK)

Correspondence to: Prof. Steen Kølvraa, Inst. of Human Genetics, University of Århus, Bartholin Building, Universitetsparken, 8000 Århus C, Denmark. E-mail: steen{at}humgen.au.dk


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
Fetal cells, present in the blood of pregnant women, are potential targets for non-invasive prenatal diagnosis. The fetal erythroblast has been the favorite target cell type. We investigated four methods of enrichment for fetal erythroblasts, identifying only three fetal erythroblasts in 573 ml of maternal blood. This is much less than the expected two to six fetal cells per ml of maternal blood. Hamada and Krabchi used a cell type-independent marker, i.e., the Y chromosome in maternal blood from male pregnancies after Carnoy fixation, leaving the nuclei for hybridization with X-and Y-chromosome-specific probes. We found with a similar technique 28 fetal cells in 15 ml of maternal blood. The fetal origin of cells was confirmed by hybridizing the nuclei with X- and Y-chromosome-specific probes, using two consecutive hybridizations with the two probes in opposite colors (reverse FISH). Candidate fetal cells were inspected after each hybridization. Only cells that were found to change the color of both probe signals from first to second hybridization were diagnosed as fetal. To reduce the labor-intensive slide screening load, we used semiautomated scanning microscopy to search for candidate cells. We conclude that erythroblasts form only a small fraction of fetal cells present in maternal blood.

(J Histochem Cytochem 53:331336, 2005)

Key Words: fetal cells • maternal blood • semiautomated scanning • erythroblast • reverse-color FISH • Carnoy fixation


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
FOR ALMOST FOUR DECADES it has been known that fetal cells pass from fetus to mother during pregnancy. This has caused researchers to investigate if these cells could be used as a basis for prenatal diagnosis, thereby avoiding the abortion risk of amniocentesis and chorionbiopsy (Tabor et al. 1986Go; Smidt-Jensen et al. 1992Go). Three fetal cell types have been suggested as targets for diagnosis: lymphoblasts, erythroblasts, and trophoblasts. The lymphoblast was rejected as a possible target because this cell type was shown to survive for years in the maternal circulation (Bianchi et al. 1996Go), making it difficult to distinguish between cells from present and earlier pregnancies. For erythroblasts and trophoblasts, a variety of selection methods and supposedly fetal markers have been presented (Jackson 2003Go; Oudejans et al. 2003Go). For erythroblasts, embryonic and fetal hemoglobins have been used and for trophoblasts a number of different placental antibodies have been suggested as markers.

The rarity of fetal cells has made enrichment necessary, and the choice of enrichment method depends on the cell type(s) present. Two groups have focused their investigations on the total number of fetal cells in maternal blood determined by methods that probably do not favor certain nucleated cell types (Hamada et al. 1993Go; Krabchi et al. 2001Go). Both groups used Carnoy fixation on whole blood, thereby obtaining enrichment by lysis of most erythrocytes. Nuclei are usually conserved by this treatment, making subsequent X- and Y-chromosome-specific FISH analysis possible, but the fixative removes the cytoplasm from the nucleated cells, making it impossible later to identify the cell type. Both groups used the X- and Y-chromosome FISH signals as markers for fetal cells and found frequencies in the range of 1–6 fetal cells/ml of maternal blood.

We have investigated a range of enrichment methods aimed at selecting erythroblasts, ending up with the most limited and gentle one (i.e., CD71-positive selection on whole blood) using {zeta}-hemoglobin and X- and Y-chromosomes as markers (Christensen et al. 2003Go,in pressGo). In these series we only found very few fetal cells despite having analyzed more than 560 ml of maternal blood. Parallel to these investigations we have also performed a series using the methods of Krabchi et al. (2001)Go and Hamada et al. (1993)Go aimed at verification of the frequencies of fetal cells in maternal blood found by these authors. Because the method applied by these authors removes only erythrocytes, the slides to be analyzed still contain a large number of nucleated cells. In contrast to Krabchi et al. (2001)Go, we have applied semiautomated scanning for Y-chromosome signals, a method that in our hands is more reliable and less time-consuming than manual scanning. We have in a limited number of samples found a similar number of fetal (X- and Y-chromosome-positive) cells as Hamada et al. (1993)Go and Krabchi et al. (2001)Go. We therefore conclude that erythroblasts can account for only a fraction of the fetal cells identified by those investigators.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
Patients
Forty-three pregnant women volunteered to give a peripheral blood sample. All blood samples were taken before an invasive procedure such as chorion villus sampling (CVS) or amniocentesis, and they were collected in EDTA-containing Vacutainer tubes.

All women were pregnant with a male fetus. The gender of the fetus was determined either by ultrasound scanning performed before the blood sample was drawn or by interphase X- and Y-chromosome-specific FISH on a piece of villus material obtained by CVS performed after the blood was drawn. Informed consent was given by all participants, and the project was approved by the local Danish science ethics comittee.

Enrichment and Analysis of Blood Samples
Samples 1–18
A detailed description of the analysis of samples 1–18 (Table 1) is given in Christensen et al. (2003)Go. Briefly, samples 1–12 were enriched for nucleated cells by bulk separation (BS) and sample 13–18 were enriched for nucleated cells by density gradient centrifugation (GC). Both enrichment methods remove a major part of the erythrocytes and enrich for specific subfractions of nucleated blood cells according to buoyant density. Slides from these 18 samples were all stained with a monoclonal antibody against the {varepsilon}-globin chains in embryonic hemoglobin. {varepsilon}-Positive erythroblasts were identified by semiautomated scanning.The male fetal origin of {varepsilon}-positive cells was confirmed by dual-color FISH using X- and Y-chromosome-specific probes (Vysis; Downers Grove, IL).


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Table 1

Number of fetal cells identified in peripheral blood from pregnant women carrying a male fetus

 
Samples 19–43
A detailed description of the analysis of samples 19–43 (Table 1) is given in Christensen et al. (in press)Go. Briefly, samples 19–38 were enriched by density gradient centrifugation followed by CD45 depletion and CD71 positive selection on CD45-negative cells using MACS (double MACS). Samples 39–43 were enriched exclusively by CD71 positive selection on whole blood using Immunicon technology (Immunicon; Huntingdon Valley, PA). Both procedures are designed to cause enrichment of erythroblasts. Slides from these 25 samples were analyzed manually for the presence of male fetal cells by two rounds of dual-color X- and Y-chromosome-specific FISH. After the first X- and Y-chromosome-specific FISH, cells with one signal specific for the label carried by the Y-chromosome-specific probe and one signal specific for the label carried by the X-chromosome-specific probe were identified by manual screening. These cells are classified as candidate fetal cells. To discriminate between false-positive and true-positive Y-chromosome-specific probe signals, candidate fetal cells were re-hybridized with the same probes in reverse colors, and candidate fetal cells were relocated and analyzed with respect to the presence and localization of reverse-color X- and Y-chromosome-specific probe signals.

In addition to the reverse-color X- and Y-chromosome-specific FISH analysis, slides from samples 39–43 were analyzed by staining with a monoclonal antibody against {zeta}-globin chains in embryonic hemoglobin. {zeta}-Positive erythroblasts were identified by automated scanning .

Samples 44–48
Samples 44–48 (Table 1) were enriched by hypotonic treatment and Carnoy fixation and analyzed by reverse-color FISH as described by Krabchi et al. (2001)Go with the following modifications. After the hypotonic treatment with 5 ml of 75 mM KCl at 37C for 5 min, 1 ml of Carnoy fixative (3 volumes ethanol:1 volume acetic acid) was added to the hypotonic solution before the cells were centrifuged and fixed twice in Carnoy. This treatment lyses erythrocytes and results in a preparation of nuclei derived from all nucleated cells in the blood. The spreading of 15 µl or 2 x 15 µl of the suspension of these nuclei was performed at room temperature, and before the first XY FISH procedure the slides were aged by incubation in 2 x SSC at 37C for 1 hr and dehydrated in ethanol. The reverse-color FISH procedure and analysis were the same as described above and in Christensen et al. (in press)Go, except that candidate male fetal cells were identified by automated scanning. In addition, one third of the slides (corresponding to 1 ml of whole blood) were also screened manually.

Analysis of Slides
Fetal cells from the various enrichment experiments were (apart from samples 19–38, which were only scanned manually) identified mainly (see above) by microscope-based automated scanning. The fetal cell-specific stains used were either cytoplasmic immunostaining using antibodies specific for embryonic hemoglobins or Y-chromosome-specific FISH staining.

The RCDetect scanner (Metasystems, Altlussheim, Germany; see Christensen et al. 2003Go) was used in scanning for cytoplasmic stains. The system was also used for manual analysis, allowing detailed inspection of whole slides by special software so that no cells on the slide were missed. The Y-chromosome FISH signals were searched for with the MDS scanner (Applied Imaging, Newcastle, UK; see Christensen et al. 2003Go).


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
After protocol development and modifications the various identification markers gave intense, easily readable signals. The automated scanning results also seem reliable on the basis of many slides that were inspected both manually and scanned automatically. Therefore, in case of antibody-stained slides, manual and semiautomated scanning yielded identical results. For XY hybridized slides, however, manual and automated scanning did not always give identical results. On rare occasions both methods might result in a few fetal cells being missed (see below). Furthermore, the scanner picked up many false-positive candidate cells. After the first FISH treatment, some cells were found by the scanner (as well as by manual microscopy) to have both a red and a green signal. Typically, the number of candidate fetal XY cells per slide was less than 10 cells. These apparently fetal cells were relocated after renewed FISH using the X- and Y-chromosome-specific probes with opposite colors (reverse-color FISH). In false-positive cells the green X-signal became red, but the red Y remained red, while in true-positive cells both signals changed color. All candidate fetal cells were therefore always inspected in the microscope after both the first and the second FISH and rejected or classified as true fetal cells according to the results of reverse FISH or antibody staining combined with XY FISH. The nature of the false positive red signals is not known. They might originate from unspecific binding of labeled probe or might be small particles present in the wash solutions.

The results for all patients are shown in Table 1 and are summarized in Tables 2A and 2B. The number of fetal cells found after Carnoy fixation in each of the five samples analyzed by this method ranged from three to 12 (Table 1; samples 44–48, total 28). A typical example of how such cells appear after the first and second FISH is shown in Figure 1. Note that true-positive fetal cells vary in morphology. The appearance of a false- positive cell before and after the reverse FISH procedure is also illustrated.


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Table 2

Number and frequency of fetal cells identified in peripheral blood from pregnant women

 


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Figure 1

Digitized images of true-positive and false-positive male fetal cells identified by reverse-color FISH using sex chromosome-specific probes. (A,D) A male fetal nucleated red blood cell identified by anti-{zeta} hemoglobin staining, visualized in green. (A) FISH signals after the first hybridization with the X-chromosome-specific probe in aqua and the Y-chromosome-specific probe in red. (D) Same cell after rehybridization with the same probes in reverse colors. (B and E, C and F, G and K, H and L) Four different true-positive male fetal cells. (B,C,G,H) FISH signals after the first hybridization with the X-chromosome-specific probe in green and the Y-chromosome-specific probe in red. (E,F,K,L) FISH signals in the same four cells after rehybridization with the same probes in reverse colors. The X-chromosome-specific signal has changed color from green to red and the Y-chromosome-specific signal has changed from red to green. (J,M) A false-positive male fetal cell and a maternal cell. (J) FISH signals after the first hybridization with the X-chromosome-specific probe in green and the Y-chromosome specific probe in red. (M) FISH signals after rehybridization with the same probes in reverse colors. The X-chromosome-specific signals have changed color from green to red. The supposed red Y-chromosome-specific signal remains red. Images A and D were captured with the imaging system ISIS (Metasystems) using a x40 objective. All other images were captured using the multifilter acquisition setup in the MDS software (Applied Imaging) using a x40 objective.

 
Eleven of the 28 fetal male cells identified in the five samples analyzed after Carnoy fixation were found on slides that were scanned both manually and automatically. Three of these cells were identified by manual scanning but not by automated scanning, and nine cells were identified by automated scanning but not by manual scanning. Of the three fetal cells that were identified exclusively by manual scanning, one cell was located outside the automated scan area and the remaining two cells were rejected by the scanner because of the elongated shape of their nuclei. These two cells were identified in the very first patient analyzed, and they led to an expansion of the scanning parameters.

As shown in Table 2, only three fetal cells were found after an enrichment procedure optimized for NRBCs in 573 ml of maternal blood. In contrast, 28 fetal cells of unidentified type were found in 15 ml of blood after hypotonic treatment and Carnoy fixation.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
The purpose of our studies during the past few years has been identification and isolation of fetal cells from maternal blood. The first approach was the use of fetus-specific hemoglobins as markers for fetal erythroblasts. We developed a PNA probe for {gamma}-hemoglobin mRNA (Larsen et al. 2003Go). However, this probe could not discriminate between fetal and maternal {gamma}-hemoglobin-positive NRBCs (Christensen et al. unpublished data). We also tried to develop PNA probes specific for {varepsilon}-hemoglobin, but with no success. Consequently, we used commercially available antibodies against {varepsilon}- and {zeta}-hemoglobin. Because of the rarity of fetal cells in maternal blood, we (Christensen et al. 2003Go, in pressGo), like others (cf. Jackson 2003Go), enriched the blood samples before analysis. We tried four different methods, which yielded different degrees of enrichment and presumably placed different degrees of stress on the fetal cells, which probably are more fragile than the maternal cells. Of the four methods we have tried, we believe that the CD71 enrichment in whole blood is the least damaging procedure. In accordance with this, we have previously presented evidence that this procedure gives by far the highest recovery of fetal cells from maternal samples taken after CVS (Christensen et al. in pressGo). In agreement with this, two of the three fetal cells found after enrichment procedures in pre-CVS samples were found after CD71 enrichment in whole blood. Others have found larger numbers of erythroblasts (cf. Jackson 2003Go). Estimates vary from 1 fetal cell/104–109 maternal nucleated cells. We believe that some of these presumed fetal erythroblasts could be either false-positive XY cells in studies where fetal cells are identified by only one FISH procedure (Bianchi et al. 2002Go; Hromadnikova et al. 2002Go) or {gamma}-positive fetal NRBCs where fetal cells are identified by, e.g., Kleihauer staining combined with FISH (Rodriguez de Alba et al. 2001Go). We have found that it is necessary to use two independent markers for fetal origin, either in the form of two FISH procedures (X- and Y-chromosome-specific probes with reverse colors in male pregnancies) or specific fetal markers such as embryological hemoglobin antibodies combined with X- and Y-chromosome-specific FISH.

The number of erythroblasts found in our investigation is orders of magnitude lower than the number of fetal cells found by Hamada et al. (1993)Go and Krabchi et al. (2001)Go. These authors did not use enrichment procedures based on density gradient centrifugations and antibodies against cell surface epitopes. They performed hypotonic treatment and Carnoy fixation, which primarily destroy the erythrocytes. Using the Y chromosome as a fetal marker in male pregnancies, these investigators found 2–6 fetal cells/ml maternal blood. Using almost the same methodology, we have found fetal cells in comparable numbers. These numbers are also comparable to the number of genomic equivalents (1–4/ml of blood) obtained by quantitative PCR with Y-chromosome- specific primers (Bianchi et al. 1997Go; Ariga et al. 2001Go). It therefore appears possible that only a small fraction of the fetal cells present in maternal blood are of erythroblastic origin. Another possible cell type is the trophoblast (cf. Oudejans et al. 2003Go). Specific placental trophoblast antibodies have been scarce. We have analyzed a limited amount (9 ml) of blood from three pregnant women using either an HLA-G antibody (kindly supplied by Susan Fischer; University of California, San Francisco) or a mixture of antibodies against various types of cytokeratin. Both types of antibodies were first tested on CVS washings and were found to stain trophoblast cells of fetal origin. However, when they used on maternal blood samples no fetal cells were found. This suggests that trophoblasts may constitute only a very small fraction of the fetal cells in maternal blood or that circulating trophoblasts must be stained by a different protocol from the one used for CVS washings.

Using the Y-chromosome as a fetal specific marker has enabled us to conclude that the erythroblast can contribute only a small fraction of the fetal cells in maternal blood. The Y-signal is not useful if the method is to obtain clinical importance because it does not identify female fetal cells. Therefore, other markers must be identified. However, Carnoy fixation cannot be a useful fixation procedure with epitope-based markers because it removes the cytoplasm from the nucleated cells. The next step in our project is to develop an alternative fixation method that will allow X- and Y-chromosome-specific FISH to be performed and, at the same time, will preserve the cytoplasmic morphology and cellular epitopes.

Fetal cells in maternal blood will continue to be rare regardless of the marker used. We therefore believe that the automated scanning system which, in our hands, is less time-consuming and more reliable than manual analysis will be an integrated part of the identification process, both in research or at some later stage in the development of a prenatal diagnostic method based on fetal cells isolated from maternal blood.


    Conclusion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
The number of fetal cells isolated from maternal blood after hypotonic treatment and Carnoy fixation has been shown to be several orders higher than the number of fetal erytroblasts found after enrichment.


    Acknowledgments
 
Supported by the Alfred Benzon Foundation, the Brødrene Hartmann Foundation, the Chromosome Research Foundation, the Steenbeck Foundation, the Danish Medical Research Council, the Ivan Nielsen Foundation, the Novo Nordisk Foundation, the Lundbeck Foundation, the Carl and Ellen Hertz Foundation, the Danish Agency for Trade and Industry, the Copenhagen Hospital Corporation, and the Frode Nygaard Foundation.

Parts of the results described here have been published in Fetal Diagnosis and Therapy. We acknowledge Karger Publications for the permission to use these results, and our co-authors of the two articles published in Fetal Diagnosis and Therapy. We thank MetaSystems for placing the automated scanning instrument at our disposal.


    Footnotes
 
Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich Schiller University, Jena, Germany, April 17–18, 2004.

Received for publication May 17, 2004; accepted August 26, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 

Ariga H, Ohto H, Busch MP, Imamura S, Watson R, Reed W, Lee TH (2001) Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis. Transfusion 41:1524–1530[CrossRef][Medline]

Bianchi DW, Simpson JL, Jackson LG, Elias S, Holzgreve W, Evans MI, Dukes KA, et al. (2002) Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. Prenat Diagn 22:609–615[CrossRef][Medline]

Bianchi DW, Williams JM, Sullivan LM, Hanson FW, Klinger KW, Shuber AP (1997) PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 61:822–829[Medline]

Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA (1996) Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 93:705–708[Abstract/Free Full Text]

Christensen B, Kølvraa S, Lykke-Hansen L, Lörch T, Gohel D, Smidt-Jensen S, Bang J, et al. (2003) Studies on the isolation and identification of fetal nucleated red blood cells in the circulation of pregnant women before and after chorion villus sampling. Fetal Diagn Ther 18:376–384[CrossRef][Medline]

Christensen B, Philip J, Kølvraa S, Lykke-Hansen L, Hromadnikova I, Gohel D, Lörch T, et al. (in press) Fetal cells in maternal blood: a comparison of methods for cell isolation and identification. Fetal Diagn Ther

Hamada H, Arinami T, Kubo T, Hamaguchi H, Iwasaki H (1993) Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age. Hum Genet 91:427–432[Medline]

Hromadnikova I, Karamanov S, Houbova B, Hridelova D, Kofer J, Mrstinova M (2002) Non-invasive fetal sex determination on fetal erythroblasts from maternal circulation using fluorescence in situ hybridisation. Fetal Diagn Ther 17:193–199[CrossRef][Medline]

Jackson L (2003) Fetal cells and DNA in maternal blood. Prenat Diagn 23:837–846[CrossRef][Medline]

Krabchi K, Gros-Louis F, Yan J, Bronsard M, Massé J, Forest J-C, Drouin R (2001) Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques. Clin Genet 60:145–150[CrossRef][Medline]

Larsen RD, Schønau A, Thisted M, Petersen KH, Lohse J, Christensen B, Philip J, et al. (2003) Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization. Prenat Diagn 23:52–59[CrossRef][Medline]

Oudejans CBM, Tjoa ML, Westerman BA, Mulders MAM, Wijk IJV, Vugt JMGV (2003) Circulating trophoblast in maternal blood. Prenat Diagn 23:111–116[CrossRef][Medline]

Rodriguez de Alba M, Palomino P, Gonzalez-Gonzalez C, Lorda-Sanchez I, Ibanez MA, Sanz R, Fernandez-Moya JM, et al. (2001) Prenatal diagnosis on fetal cells from maternal blood: practical comparative evaluation of the first and second trimesters. Prenat Diagn 21:165–170[CrossRef][Medline]

Smidt-Jensen S, Permin M, Philip J, Lundsteen C, Zachary JM, Fowler SE, Gruning K (1992) Randomised comparison of amniocentesis and transabdominal and transcervical chorionic villus sampling. Lancet 340:1237–1244[CrossRef][Medline]

Tabor A, Philip J, Madsen M, Bang J, Obel EB, Nørgaard-Pedersen B (1986) Randomized controlled trial of genetic amniocentesis in 4606 low-risk women. Lancet 1:1287–1293[CrossRef][Medline]





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