FISH Analysis of All Fetal Nucleated Cells in Maternal Whole Blood : Improved Specificity by the Use of Two Y-chromosome Probes
Laboratory for Prenatal Medicine, University Women's Hospital (OL,WH) / Department of Research (SM,TB,VK,WH,SH), Basel, Switzerland
Correspondence to: Dr. Sinuhe Hahn, Laboratory for Prenatal Medicine, University Women's Hospital / Department of Research, Spitalstr. 21, 4031 Basel, Switzerland. E-mail: shahn{at}uhbs.ch
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: noninvasive prenatal diagnosis entire fetal cell population Carnoy FISH
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Not only has the suitability of both enrichment strategies been questioned but also the use of the fetal NRBC as a sole target cell, because this cell appears to be refractory to fluorescence in situ hybridization (FISH) analysis (Babochkina et al. unpublished data). For this reason, the question has been raised whether a different target cell should be chosen or whether the analysis should be performed on all fetal cell types present in the maternal circulation. Evidence that the latter approach may be feasible was described in non-blinded studies for the first time by Hamada et al. (1993) and more recently by Krabchi et al. (2001)
. Hamada et al. (1993)
published parallel investigations by FISH and polymerase chain reaction (PCR) in 50 pregnancies in which both methods used repetitive and single-copy sequences specific for the Y-chromosome. Analyzing
100,000 nucleated cells per maternal blood sample, these investigators reported an overall sensitivity of 82.8% for Y-chromosome-specific FISH as well as increasing fetal cell numbers during pregnancy. At term, they detected up to 1 nucleated fetal cell in 10,000 maternal nucleated cells.
Krabchi et al. (2001) described the quantification of the entire fetal nucleated cell population in maternal blood in a morphology-independent manner using FISH. Their study indicated that 26 fetal nucleated cells were present per ml of maternal blood in 12 healthy pregnancies. Their procedure, which required minimal manipulation of cells, was based on a hypotonic treatment combined with Carnoy's fixation. This treatment removes the cell cytoplasm and causes swelling of the nuclei, thereby rendering all cell types readily amenable to FISH analysis.
To test this procedure in a larger study population and to assess the number of fetal cells per ml of maternal blood, we performed a blinded analysis of 40 samples from normal pregnancies with unknown fetal gender using two different sets of FISH probes.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Equal volumes of the cell suspension representing the entire cell population of 125 µl maternal whole blood were cytocentrifuged (four slides per case). Dual-color FISH using the chromosome enumeration probe cocktail CEPX spectrum green/CEPY [alpha ()-satellite] spectrum orange (Vysis/Abbott Diagnostics; Baar, Switzerland), further referred to as XY-FISH, was performed on two slides per case. Pretreatment of slides encompassed 0.005% pepsin digestion (10 min) and fixation in 1% formaldehyde (10 min). The FISH probe cocktail was diluted 1:100 in cDenHyb-1 (Insitus Biotechnologies, Albuquerque, NM) and 10 µl per slide was applied. Co-denaturation at 72C for 8 min was followed by hybridization at 37C for 6 hr. Posthybridization washes were performed in 50% formamide/1.5 x SSC at 45C (5 min) and in 1.5 x SSC at 45C (2 min). After a final wash with distilled water, slides were counterstained with 0.01% DAPI (Sigma; Fluka Chemie, Buchs, Switzerland)/glycerol.
In addition, we performed dual-color FISH with two different Y-chromosome probes [CEPY -satellite (spectrum orange) and CEPY III-satellite (spectrum aqua) (Vysis/Abbott), further referred to as YY-FISH] on the remaining two slides per case. Per cytospin, 1 µl of the centromeric Y-chromosome probe was applied, together with 1 µl of a 1:10 dilution of the III-satellite probe in chromosome enumeration probe (CEP) hybridization buffer (Vysis/Abbott). One microliter of distilled water and 7 µl CEP hybridization buffer were added, to a final volume of 10 µl. Co-denaturation was performed at 72C for 8 min, and hybridization followed at 37C for 16 hr.
In every hybridization, a male cord blood control slide was run in parallel to monitor the quality of the FISH performance. According to the manufacturer, the efficiency of the Y-chromosome-specific probe in the XY cocktail is 97.5%. In our hands, the efficiency on male cord blood control slides was calculated to be 98.5%, on average. Likewise, the manufacturer-described 99.5% efficiency of both single Y-chromosome probes was obtained in our controls.
Blinded evaluation of the FISH signals was performed by manually screening two cytospins per FISH probe combination using single bandpass filters. In the case of XY-FISH, the identification of any cell with a Y-chromosome -satellite signal prompted us to check for the further presence of an X-chromosome signal. Identification of any cell with a Y-chromosome
-satellite signal in the case of YY-FISH was followed by checking for a second Y-chromosomal III-satellite signal. The latter hybridizations were also re-scanned to detect first the Y-chromosomal III-satellite signal and then re-checked for the presence of the Y-chromosomal
-satellite-signal. A cell was accepted to be of male origin only in cases of two Y-chromosomal signals, one of each color.
In general, all identified spots were counterchecked for nonspecificity in all other filters at a magnification of x1000. FISH results predicting fetal gender were compared with birth outcome only after all slides had been evaluated. For seven cases, the birth outcome was not available due to delivery outside of our clinic. Therefore, real-time PCR for glyceraldehyde 3-phosphate dehydrogenase (forward primer 5'CCCCACACACATGCACTTACC3', reverse primer 5'CCTAGTCCCAGGCCTTTGATT3', probe 5'AAAGAGCTAGGAAGGACAGGCAACTTGGC3') and SRY (forward primer 5'TCCTCAAAAGAAACCGTGCAT3', reverse primer 5'AGATTAATGGTTGCTAAGGACTGGAT3', probe 5'CACCAGCAGTAACTCCCCACAACCTCTTT3') on maternal plasma was performed as described by Zhong et al. (2001).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results for the XY-FISH in Table 1 show a high percentage of false-positives in the case of female fetuses (31.6%) together with a low sensitivity (52.4%) in the pregnancies with male fetuses.
|
|
In regard to the quality of FISH signals, we obtained easily identifiable spots for all fluorochromes on male cord blood control slides and for the maternal X-chromosomes in the patients' samples. In contrast, the signals in the fetal cells were much smaller and significantly less intense, which renders both identification and counterchecking more difficult (Figures 1 and 2).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Applying the described cell treatment protocol of hypotonic treatment and Carnoy's fixation after MACS enrichment (CD13, CD5, CD 71+), Hromadnikova et al. (2002) reported complications in evaluation. These were caused by lack of cells or damage to the isolated cells in seven of eight analyzed cases. Because we used the same experimental set-up for the preparation of the FISH slides as Krabchi et al. (2001)
, we suggest that the lower values we obtained for specificity and sensitivity (89.5% and 42.9%, respectively) might be due to procedure-inherent factors influencing the FISH efficiency (Hromadnikova et al. 2002
). However, the lower sensitivity and specificity levels in our experimental conditions mirror much better the situation of a real noninvasive diagnostic setting, because all samples were evaluated in a blinded manner, which is in contrast to the studies performed by Hamada et al. (1993)
and Krabchi et al. (2001)
. In our more realistic scenario, a significant increase in specificity has been achieved by substituting for the X-chromosome probe a second Y-chromosome probe from a different region of the Y-chromosome. Notwithstanding the slight reduction in sensitivity, this probe combination renders the assay for detection of male fetal cells much more reliable. This positive effect is also shown by the low correlation of pregnancy outcome to the predicted outcome of cases with cells showing one Y-chromosome signal of either color only (Table 2). Owing to the incorporation of the second Y-chromosome probe, 78.9% of the female pregnancies are correctly identified; on the basis of only one Y-chromosome probe of either color, these would have been wrongly counted as male pregnancies.
Because this study comprised not only erythroblasts but all kinds of fetal cells, it is possible that fetal cells persisting from previous pregnancies could contribute to some of the false-positive cases. Contamination of the samples with Y-chromosome material during handling of the probes can be excluded because only female examiners processed the samples.
In an interstudy comparison regarding fetal cell numbers, the average concentration of 2 fetal cells/1 ml maternal blood found by Krabchi et al. (2001) matches approximately the concentration of 1.2 fetal cell equivalents per 1 ml of maternal blood found by Bianchi et al. (1997)
using PCR without prior enrichment. However, our results exceed these average cell numbers to a rather large extent. The reasons for these differences might be the much broader range of gestational age represented in our patients than in the study populations of Krabchi et al. (2001)
and Bianchi et al. (1997)
, as well as the differences in our treatment protocol compared with the procedure described by Bianchi et al. (1997)
.
In our study, no male fetal cells could be detected in 9/21 (42.8%) pregnancies with a male fetus. Therefore the amount of 500 µl of maternal whole blood does not appear to be sufficient to guarantee the presence of a small but reliable number of fetal cells of any morphology for use in noninvasive prenatal diagnosis.
Because the nuclei of fetal NRBCs are tightly condensed, the observed differences in the quality of the FISH signals between control cells and maternal cells versus fetal cells were not completely unexpected. However, because in the present cell preparation, fetal cells of any origin and morphology are present, we also expected to find some XY- and YY-chromosome signals of normal size and fluorescence intensity. The lack of these cells suggests that all fetal cells that migrate into the maternal circulation undergo physiological changes that leave the cells in a status not amenable to reliable FISH analysis.
![]() |
Footnotes |
---|
Received for publication May 18, 2004; accepted August 26, 2004
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bianchi DW, Romero R (2003) Biological implications of bi-directional fetomaternal cell traffic: a summary of a National Institute of Child Health and Human Development-sponsored conference. J Matern Fetal Neonatal Med 14:123129[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. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn 22:609615[CrossRef][Medline]
Bianchi DW, Williams JM, Sullivan LM, Hasnon FW, Klinger KW, Shuber AP (1997) PCR quantification of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 61:822829[Medline]
Hahn S, Holzgreve W (2002) Prenatal diagnosis using fetal cells and cell-free fetal DNA in maternal blood: what is currently feasible? Clin Obstet Gynecol 45:649656[CrossRef][Medline]
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 91:427432[Medline]
Hamada H, Arnami T, Sohda S, Hamaguchi H, Kubo T (1995) Mid-trimester fetal sex determination from maternal peripheral blood by fluorescence in situ hybridization without enrichment of fetal cells. Prenat Diagn 15:7881[Medline]
Hromadnikova I, Karamanov S, Houbova B, Hridelova D, Jofer J, Mrstinova M (2002) Non-invasive fetal sex determination on fetal erythroblasts from the maternal circulation using fluorescence in situ hybridisation. Fetal Diagn Ther 17:193199[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 60:145150[CrossRef][Medline]
Zhong XY, Laivuori H, Livingston JC, Ylikorkala O, Sibai BM, Holzgreve W, Hahn S (2001) Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am J Obstet Gynecol 184:414419[CrossRef][Medline]