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Technical Briefs |
1 Laboratory for Prenatal Medicine, Department of Obstetrics and Gynecology, University of Basel, Spitalstrasse 21, CH-4031 Basel, Switzerland
aauthor for correspondence: fax 41-61-325-9399, e-mail shahn{at}uhbs.ch
The presence of fetal cells, especially fetal nucleated red blood cells (NRBCs), in the blood of pregnant women is now a widely demonstrated phenomenon and is being considered as the basis for a novel noninvasive means for prenatal diagnosis. However, at present little is known about the fate of fetal cells once they enter the maternal circulation.
Fetal cells could be removed from the maternal circulation by the maternal immune system or by the induction of apoptosis by other means (1)(2)(3). These proposals are supported by reports indicating the presence of terminal dUTP nuclear end labeling (TUNEL)-positive fetal NRBCs in the maternal circulation, a feature that may be attributable to the increased oxygen concentration in the maternal circulation (4), as well as by the description of apoptotic fetal cells or their remnants in the maternal plasma.
Elimination of fetal cells by apoptosis from the maternal periphery may not be as widespread as suggested, or it may not affect all fetal cell types equally. Fetal leukocytes or hemopoietic progenitor cells may persist in the maternal circulation for years to decades after delivery (5)(6), and fetal cells with stem cell-like characteristics may even contribute to the regeneration of maternal tissue (7). Furthermore, microchimerism of the order seen in pregnancy frequently also occurs in solid organ transplant recipients and has, in these instances, been suggested to promote tolerance toward the graft (8). By analogy, it is therefore conceivable that the passage of fetal cells into the maternal periphery may promote tolerance against the semiallogeneic fetus.
A caveat of previous studies on the status of fetal NRBCs in the maternal periphery is that they studied only nuclear events (TUNEL positivity) as indicators of the apoptotic status of trafficking fetal cells. Sekizawa et al. (2) reported that >40% the fetal NRBCs detected in blood samples taken immediately after termination were TUNEL positive. Although this result may serve to indicate that exposure to the maternal environment leads to the induction of apoptosis in fetal NRBCs, it fails to take into account several recent reports suggesting that the terminal differentiation of erythroid cells uses apoptotic mechanisms to facilitate enucleation (9)(10). The pathway(s) used for erythroid differentiation may involve only nucleus-associated apoptotic features but not membrane-associated apoptotic alterations. In this manner, erythroid differentiation and enucleation may involve chromatin condensation, DNA breakage (detected by the TUNEL assay), and degradation of nuclear components, as well as the apoptotic signals triggering these changes, such as caspase activation and mitochondrial potential reduction. In contrast, membrane-associated apoptotic characteristics that facilitate the rapid engulfment of apoptotic cells, such as phosphatidylserine (PS) exposure, are lacking in this maturation process. This appears logical because the erythroblasts need only to get rid of their nuclei and leave their enucleated erythrocytes intact to perform the important task of oxygen transport.
In a previous study we showed that >50% of the NRBCs in the fetal circulation and cord blood (i.e., before exposure of these cells to the maternal circulation) are TUNEL positive (11). We therefore concluded that the detection of TUNEL-positive erythroblasts may reflect a normal physiologic stage of erythroid differentiation associated with chromatin condensation and subsequent enucleation. Therefore, the TUNEL assay may not serve as a marker for the apoptotic death of these cells (11).
Consequently, the purpose of the present study was to examine whether fetal NRBCs that have entered the maternal circulation exhibit only nuclear apoptotic phenotypes (TUNEL positivity) or whether they display other membrane-associated apoptotic traits (PS exposure), which would serve as a signal facilitating rapid cell engulfment. Other assays, such as those for the determination of caspase activity, are currently not feasible because of the extreme scarcity of these fetal cells, as at least several thousand cells are required for these assays (e.g., immunoblotting or enzyme immunoassay for the detection of cleaved caspase substrate). In addition, because caspases are also activated during erythroid maturation and enucleation (9), assays detecting such activity will not serve to distinguish between apoptosis and differentiation.
After receiving informed consent and institutional review board approval, we obtained 20-mL samples of blood from 25 women at weeks 11–38 of gestation. The samples were processed immediately after phlebotomy by density-gradient centrifugation at 680g for 20 min (Ficoll-Paque PLUS; Amersham Pharmacia Biotech AB). Because fetal NRBCs in the maternal blood are very rare (in the range of 1 in 106), they need to be enriched for. Our strategy was to enrich for all PS-exposing cells, using the Annexin V Microbead Kit (Miltenyi Biotec) and then to examine for the presence of NRBCs in the positively enriched fraction. To assess the fraction of NRBCs that were not PS-positive, we further analyzed the PS-negative fraction by CD71 enrichment. All enrichment steps were performed according to the manufacturers’ instructions and as used previously in our laboratory (12). After enrichment, the positively enriched cells were then transferred by cytocentrifugation on Superfrost PLUS microscope slides (Mentzel-Gläser, Germany), and NRBCs were identified microscopically after May–Grünwald–Giemsa staining (12).
The mean number of NRBCs recovered from 20 mL of maternal peripheral blood was 30 (range, 0–213). A total of 649 NRBCs were detected in 23 of 25 samples examined. Of these NRBCs, 626 (>96%) were found in the CD71-positive fraction enriched from the Annexin V-negative fraction. Only 23 NRBCs were identified in the Annexin V-positive fraction.
To ensure the reliability of the Annexin V enrichment method, freshly isolated mononuclear cells were cultured in presence of 2 µmol/L staurosporine for 4 h at 37 °C in 7.5% CO2, a condition known to induce rapid apoptosis. The PS-exposing cells were then enriched for with use of the Annexin V Microbead Kit. Fluorescence-activated cell-sorting analysis of the Annexin V-positive and Annexin V-negative fractions (Annexin V-Fluos; Roche Molecular Biochemicals) showed that >94% of the cells in the Annexin V-positive and <4% of the cells in the Annexin V-negative fraction were identified as Annexin V-positive (Table 1
). We therefore considered that the Annexin V Microbead Kit is reliable and can be used for enrichment of PS-exposing cells.
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Because most NRBCs were found in Annexin V-negative/CD71-positive fraction, only the cells from this fraction were used to perform the following TUNEL and fluorescent in situ hybridization (FISH) analyses. No statistically reliable analysis could be performed on the few NRBCs obtained in the Annexin V-positive fraction.
The cells in nine cases were examined by the TUNEL assay as described previously (11). Of the total of 425 NRBCs analyzed, 211 (49.6%) were scored as TUNEL positive. This is in good agreement with our previous results for cord- and fetal-blood NRBCs (11).
To determine whether the identified NBRCs were of fetal or maternal origin, we next examined them by FISH for the X and Y chromosomes (CEP X SG/CEP Y (
) SO; Vysis). All hybridization steps were performed according to the manufacturer’s instructions. Of the 14 samples analyzed, 8 were from women carrying male fetuses and 4 were from women carrying female fetuses. In the samples with female fetuses, no Y-positive cells were found. In the samples with male fetuses, we analyzed a total of 103 NRBCs. Of these, 49 NRBCs (48%) were identified as fetal in origin by detection of a XY karyotype. This result is in good agreement with a previous report indicating that approximately one-half of the NRBCs in maternal blood are of fetal origin (12). Because our analysis of all of the NRBCs in the above samples had indicated that 49.6% scored TUNEL positive, this result implies that an equal proportion of the fetal NRBCs will also be TUNEL positive, i.e., almost 50%. In this instance, this result is in good agreement with our previous studies on fetal/cord blood (11) and those made by Sekizawa et al.(2) on post-termination samples, where similar proportions of TUNEL-positive fetal NRBCs were observed.
Our results therefore indicate that the majority of NRBCs (96%) in the maternal circulation cannot be enriched for with Annexin V microbeads. This implies that these NRBCs are not in the process of apoptosis because they do not exhibit the typical membrane-associated apoptotic phenotype of PS exposure, a feature that facilitates the rapid engulfment of apoptotic cells, thereby preventing inflammation.
On the other hand, the analyzed NRBCs do display nuclear TUNEL positivity, an apoptotic feature associated with DNA cleavage. The discrepancy between the two apoptotic features suggests that the detection of TUNEL-positive/PS-negative NRBCs is probably not associated with apoptosis but rather is associated with erythroid terminal differentiation and enucleation as the NRBCs mature into erythrocytes. This is in good agreement with previous reports concerning erythroid differentiation (9)(10).
Furthermore, by FISH we were able to show that almost one-half of the NRBCs were fetal. Because the fetal NRBCs were similarly obtained from the Annexin V-negative fraction, this result implies that these cells are also not undergoing apoptosis, but rather, like the maternal NRBCs, they carry on with terminal differentiation leading to enucleation. Thus, although our results are in good agreement with recent reports indicating the presence of TUNEL-positive fetal cells in the maternal circulation, we do not support the notion that these cells undergo apoptosis after entry into the maternal circulation, as has been suggested previously (1)(2)(3)(4). It is worth noting, however, that the TUNEL assay was the only apoptotic marker used in the earlier studies and that in most of them the fetal cell type was not determined. Our studies differ from these in that we have focused exclusively on fetal NRBCs and have examined their apoptotic status using both nuclear (detected by the TUNEL assay) and cell membrane (PS exposure detected by Annexin V binding) events. This is the first time that fetal NRBCs in the maternal circulation have been examined for PS exposure.
Our analysis also differs from a recent report made by Kolialexi et al. (13), who reported a high percentage of Annexin V-positive fetal cells in the maternal circulation, which were proposed to be of an apoptotic nature. A major technical difference between this study and most other studies examining PS exposure is that these authors used an anti-Annexin V antibody for their analysis and not Ca2+-dependent Annexin V binding to exposed PS, as is the commonly used and well-validated approach. In this regard, it is likely that the use of this antibody can lead to the detection of endogenous Annexin V expression. This is important when considering trophoblast cells, where it is well established that Annexin V is found both in the cell cytoplasm (in undifferentiated cells) and on the cell surface (in differentiated cells) (14)(15). Consequently, it is possible that the use of this anti-Annexin V antibody may have detected nonapoptotic trophoblast cells. This issue may need to be resolved by determining the cellular origin of the fetal cells examined and whether similar results can be obtained by use of Annexin V-/PS- binding-based enrichment systems of the sort we used in this study.
Although our study suggests that trafficking NRBCs do not undergo apoptosis on entry into the maternal circulation, the fate of other trafficking fetal cells, such as lymphocytes, or various progenitor cells is unclear. These studies will, however, be technically extremely challenging because of the scarcity of these fetal cell subpopulations.
References
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