The Fetal Erythroblast Is Not the Optimal Target for Non-invasive Prenatal Diagnosis : Preliminary Results
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
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Summary |
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(J Histochem Cytochem 53:331336, 2005)
Key Words: fetal cells maternal blood semiautomated scanning erythroblast reverse-color FISH Carnoy fixation
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Introduction |
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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. 1993; Krabchi et al. 2001
). 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 16 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 -hemoglobin and X- and Y-chromosomes as markers (Christensen et al. 2003
,in press
). 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)
and Hamada et al. (1993)
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)
, 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)
and Krabchi et al. (2001)
. We therefore conclude that erythroblasts can account for only a fraction of the fetal cells identified by those investigators.
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Materials and Methods |
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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 118
A detailed description of the analysis of samples 118 (Table 1) is given in Christensen et al. (2003). Briefly, samples 112 were enriched for nucleated cells by bulk separation (BS) and sample 1318 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
-globin chains in embryonic hemoglobin.
-Positive erythroblasts were identified by semiautomated scanning.The male fetal origin of
-positive cells was confirmed by dual-color FISH using X- and Y-chromosome-specific probes (Vysis; Downers Grove, IL).
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In addition to the reverse-color X- and Y-chromosome-specific FISH analysis, slides from samples 3943 were analyzed by staining with a monoclonal antibody against -globin chains in embryonic hemoglobin.
-Positive erythroblasts were identified by automated scanning .
Samples 4448
Samples 4448 (Table 1) were enriched by hypotonic treatment and Carnoy fixation and analyzed by reverse-color FISH as described by Krabchi et al. (2001) 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)
, 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 1938, 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. 2003) 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. 2003
).
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Results |
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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 4448, 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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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.
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Discussion |
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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) and Krabchi et al. (2001)
. 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 26 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 (14/ml of blood) obtained by quantitative PCR with Y-chromosome- specific primers (Bianchi et al. 1997
; Ariga et al. 2001
). 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. 2003
). 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.
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Conclusion |
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Acknowledgments |
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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.
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Footnotes |
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Received for publication May 17, 2004; accepted August 26, 2004
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Literature Cited |
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