Background: We have recently explored the detection of circulatory male fetal cells directly in maternal whole blood samples by fluorescence in-situ hybridization (FISH). In order to improve the efficacy of fetal cell detection, we have now examined whether this could be enhanced by the use of two different Y chromosome-specific FISH probes (α-satellite and classical satellite III regions) in combination with an X chromosome-specific FISH probe. Methods: Nineteen maternal blood samples (median gestational age=28weeks, range=12-37weeks) were examined in a blinded manner. No enrichment procedure was performed. Following hypotonic treatment and Carnoy's fixation, total nucleated cells were examined by two color FISH with a single X and two Y chromosome-specific probes. Nine cases were examined in parallel by conventional XY-FISH. Results: Fetal cell detection was superior when using two Y chromosome-specific probes (specificity=75%; sensitivity=91%) when compared to the conventional XY-FISH approach (specificity=50%; sensitivity=60%). Conclusions: Male fetal cells can be detected in most maternal blood samples examined. Specificity and sensitivity is improved when using a combination of single X and two Y chromosome-specific probes when compared to a conventional XY-FISH protoco

Babochkina, Tatiana

Dinges, Tatyana

Hahn, Sinuhe

Holzgreve, Wolfgang

Mergenthaler, Susanne

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ORIGINAL ARTICLETatiana Babochkina Æ Susanne MergenthalerTatyana Maier Dinges Æ Wolfgang HolzgreveSinuhe HahnDirect detection of fetal cells in maternal blood: a reappraisalusing a combination of two different Y chromosome-specific FISHprobes and a single X chromosome-specific probeReceived: 14 June 2005 / Accepted: 17 June 2005 / Published online: 20 August 2005 Springer-Verlag 2005Abstract Background: We have recently explored thedetection of circulatory male fetal cells directly inmaternal whole blood samples by ﬂuorescence in-situhybridization (FISH). In order to improve the eﬃcacy offetal cell detection, we have now examined whether thiscould be enhanced by the use of two diﬀerent Y chro-mosome-speciﬁc FISH probes (a-satellite and classicalsatellite III regions) in combination with an X chromo-some-speciﬁc FISH probe. Methods: Nineteen maternalblood samples (median gestational age=28 weeks,range=12–37 weeks) were examined in a blinded man-ner. No enrichment procedure was performed. Followinghypotonic treatment and Carnoy’s ﬁxation, total nucle-ated cells were examined by two color FISH with a singleX and two Y chromosome-speciﬁc probes. Nine caseswere examined in parallel by conventional XY-FISH.Results: Fetal cell detection was superior when using twoY chromosome-speciﬁc probes (speciﬁcity=75%; sensi-tivity=91%) when compared to the conventional XY-FISH approach (speciﬁcity=50%; sensitivity=60%).Conclusions: Male fetal cells can be detected in mostmaternal blood samples examined. Speciﬁcity and sen-sitivity is improved when using a combination of single Xand two Y chromosome-speciﬁc probes when comparedto a conventional XY-FISH protocol.Keywords Circulatory fetal cells Æ Maternal blood ÆFISHIntroductionEven though fetal genetic loci such as RhD status or sexcan now be reliably determined by the analysis ofcell-free fetal DNA in maternal plasma, this approach iscurrently not suited for the analysis of complex fetalgenetic traits, such as those involved in the case of fetalaneuploidies [4, 5]. For this reason the enrichment andanalysis of fetal cells from maternal blood samples iscurrently still the most promising strategy for the non-invasive determination of numerical anomalies of fetalchromosomes, such as those involved in Down’ssyndrome [5].Large scale studies, using either MACS (magnetic cellseparation) or FACS (ﬂuorescent activated cell sorting),have however indicated that fetal cells, speciﬁcally fetalerythroblasts, cannot be reliably retrieved and analyzedfrom maternal blood samples [2, 3]. These results haveraised the question of whether the fetal cells, especiallyerythroblasts, were indeed present in each pregnancyexamined [9]. Furthermore, it appears that the fetalerythroblast may not be the optimal target cell, and thatit may be necessary to examine for other more suitablefetal cell types [1, 6].In this context a recent study has indicated that fetalcells can be detected by the use of ﬂuorescence in-situhybridization (FISH) in maternal whole blood sampleswithout any form of enrichment [7). These results are ofconsiderable importance as they yield vital informationconcerning the number of circulating fetal cells inmaternal blood. It is also hoped that by the analysis ofthese cells, other more optimal target fetal cells typesmay be identiﬁed.Prompted by these analyses we have recently exam-ined the feasibility of detecting fetal cells directly with-out any form of enrichment in maternal blood samples[8]. This study indicated that fetal cells were indeedpresent in the majority of samples examined, andthat they were present with astonishingly high frequency(12–20 fetal cells/ml maternal blood) [8]. In ourT. Babochkina Æ S. Mergenthaler Æ T. M. DingesW. Holzgreve Æ S. Hahn (&)Laboratory for Prenatal Medicine, Department of Research,University Women’s Hospital/Department of Research,Spitalstr. 21,4031 Basel, SwitzerlandE-mail: shahn@uhbs.chTel.: +41-61-2659224Fax: +41-61-2659399Arch Gynecol Obstet (2005) 273: 166–169DOI 10.1007/s00404-005-0049-3examination, we also observed that the speciﬁcity ofdetecting male fetal cells could be improved by the use oftwo diﬀerent Y chromosome-speciﬁc FISH probes (aand III satellite: speciﬁcity=89.5%) when compared toa conventional XY-FISH protocol (speciﬁcity=68.4%)[8]. As the sensitivity of the latter approach was howeverquite poor (42.9%), we have attempted to further en-hance the eﬃcacy of this approach. For this reason, wehave examined the use of two similar Y chromosome-speciﬁc FISH probes (a and III satellite), but now incombination with a conventional centromeric X chro-mosome-speciﬁc FISH probe (referred to as XYY-FISH). We have also compared this approach to con-ventional XY-FISH.Materials and methodsSample recruitment and processingNineteen healthy pregnant women between the 12th and37th weeks of gestation were recruited for this study,which was approved by the Ethical Review Board of theUniversity of Basel. Written informed consent was ob-tained in all instances. The maternal whole blood sam-ples, anti-coagulated with EDTA, were processed within6 h as described previously [7, 8]. In brief, 500 ll ofEDTA-blood was washed with 8 ml RPMI 1640 med-ium (Invitrogen, Basel, Switzerland), incubated with10 ml prewarmed 0.4% KCl for 20 min at 37C, ﬁxedseveral times with freshly prepared pre-chilled Carnoy‘sﬁxative (methanol:glacial acetic acid, 3:1), washed with1% BSA/PBS and transferred by cytocentrifugationonto glass slides (four slides per case). Prior to FISHanalysis the slide preparations were digested with0.005% pepsin for 10 min at RT and then ﬁxed for10 min in 1% formaldehyde.FISH analysisXYY-FISHAll 19 samples were examined by FISH analysis usingcentromeric and III satellite Y chromosome-speciﬁcprobes (Ya satellite rhodamine and Yqh classical sa-tellite III rhodamine) in combination with a conven-tional centromeric probe for the X chromosome (Xasatellite ﬂuorescein) (Qbiogene, Basel, Switzerland).XY-FISHConventional dual-color FISH for the X and Y chro-mosomes was accomplished using a commercial chro-mosome enumeration cocktail (CEPX spectrum green(a-satellite)/CEPY spectrum orange (a-satellite), (Vysis/ABBOTT Diagnostics, Baar, Switzerland).All FISH analyses were performed according to themanufacturer’s instructions, using FISH probes diluted1:100 in cDenHyb-1 (Insitus Biotechnologies,Alberquerque, NM, USA) [8]. For XYY-FISH, co-denaturation was performed at 80C for 2 min, whilethat for XY-FISH was carried out at 72C for 8 min. Allhybridizations were carried out at 37C for 6 h. Post-hybridization washes were performed in 50% formam-ide/1.5x SSC at 45C (5 min) and in 1.5x SSC at 45C(2 min). After a ﬁnal wash with distilled water, the slideswere counterstained with 0.01% DAPI/Glycerol (Sigma,Fluka Chemie GmbH, Buchs, Switzerland).For each hybridization analysis, male cord bloodcontrol slides were examined in parallel to monitor thequality of the FISH performance, yielding average eﬃ-ciencies of greater than 98% for each FISH protocol.All samples were analyzed in a blinded manner usinga triple pass ﬁlter for the ﬁrst scan and the subsequentconﬁrmation of individual X and Y chromosome-spe-ciﬁc FISH signals using the appropriate single band passﬁlters at 630· magniﬁcation (Axiovision 3.1, Carl Zeiss,Zu¨rich, Switzerland).In order for a cell to be classiﬁed as fetal, the clearpresence of speciﬁc signals for both X and Y FISHchromosomes had to be present. Results from the FISHanalysis were only compared to the fetal gender asdetermined from birth outcome once the evaluation wascomplete.ResultsOur analysis of the 19 maternal blood samples by thecombination of two Y chromosome-speciﬁc FISHprobes in combination with a FISH probe for the Xchromosome (XYY-FISH) indicated that male fetalcells could be detected with 75% speciﬁcity and 91%sensitivity (Table 1), indicating that male fetal cellscould be detected in 10 out of the 11 pregnancies withmale fetuses. Our parallel analysis of nine of thesesamples by conventional XY-FISH indicated that malefetal cells could be detected with 50% speciﬁcity and60% sensitivity (Table 1), which is in good agreementwith our previous results [8]. On the other hand, whenthe same samples were analyzed by our new XYY-FISH approach, male fetal cells could be detected with100% speciﬁcity and 100% sensitivity. Therefore, theuse of two diﬀerent Y chromosome-speciﬁc FISHprobes in combination with an X chromosome-speciﬁcFISH does appear to yield a better result than thatobtained by conventional XY-FISH. Furthermore, itappeared that more male fetal cells were detected bythe XYY-FISH approach, where 1 to 4 fetal cells wererecorded per slide, (yielding an average concentrationof between 8 to 32 fetal cells per ml of maternalblood), compared to the 1 to 2 fetal cells that wererecorded per slide using conventional XY-FISH. Wealso observed that the signals for the Y chromosomewere not as bright and readily identiﬁable using theconventional XY-FISH approach (Fig. 1a) whencompared to those detected by the novel XYY-FISHprocedure (Fig. 1b).167DiscussionPrevious studies have indicated that male fetal cells canbe detected directly in maternal whole blood samples byconventional XY-FISH or PRINS (primed in-situlabelling) [7]. Although these studies yielded importantinformation concerning the number of circulating fetalcells in maternal blood, the results are possibly skewedby the fact that they were not conducted in a blindedmanner.In a recent study, we have used a similar approach tothat described by Krabchi and colleagues, in that we alsoused conventional XY-FISH to detect the presence ofsuch circulatory male fetal cells, but performed ouranalysis in a blinded manner concerning the sex of thefetus [8]. This analysis indicated that male fetal cellscould be detected with a speciﬁcity of 68.4% and sen-sitivity of 52.4%. By switching to a protocol in whichmale fetal cells were detected solely on the basis of twoindependent Y chromosome-speciﬁc FISH signals (a-and III-satellite), we achieved a speciﬁcity of 89.5% [8].However, the sensitivity was slightly decreased to 42.9%[8]. Although this study did indicate that male fetal cellswere present in a large proportion of pregnancies withmale fetuses, we were anxious to improve upon theaccuracy of our method. For this reason we have againused two similar Y chromosome-speciﬁc FISH probes(a- and III-satellite), but now in combination with an Xchromosome-speciﬁc FISH probe.Our current investigation indicates that circulatorymale fetal cells can now be detected with 75% speciﬁcityTable 1 Detection of male fetalcells in maternal blood by twodiﬀerent FISH strategiesCase Number ofmale fetal cellsdetected byXYY-FISH/mlmaternal bloodNumber of malefetal cells detectedby XY-FISH/mlmaternal bloodGender offetus1 24 12 Male2 16 12 Male3 0 8 Female4 0 0 Female5 0 16 Female6 0 0 Female7 32 0 Male8 32 0 Male9 32 20 Male10 8 n.t. Female11 0 n.t. Male12 0 n.t. Female13 8 n.t. Male14 16 n.t. Male15 32 n.t. Male16 8 n.t. Male17 0 n.t. Female18 24 n.t. Male19 8 n.t. FemaleSensitivity 91% 60%Speciﬁcity 75% 50%Fig. 1 a Detection of a malefetal cell in maternal blood byconventional XY-FISH. bDetection of a male fetal cell inmaternal blood by the use oftwo Y chromosome-speciﬁc (a-and III-satellite) FISH probesin combination with a FISHprobe speciﬁc for the Xchromosome (XYY-FISH)168and 91% sensitivity, which is a considerable improve-ment over previous results. The eﬃcacy of this approachalso becomes readily apparent in our parallel assessmentof the same samples by conventional XY-FISH, wheremale fetal cells could only be detected with 50% speci-ﬁcity and 60% sensitivity.Our data are also quite encouraging with regard tothe number of male fetal cells detected per ml ofmaternal blood, in that these may also be slightly higherthan our previous estimates (8–32 vs. 12–20). Akin toour previous study, male fetal cells could be detected inthe majority of maternal blood samples examined. Al-though encouraging, it is clear that these results willhave to be conﬁrmed in a larger study. It will be par-ticularly important to determine at what stage of preg-nancy circulatory cells can be detected reliably, since inour current study we examined a broad spectrum ofgestational ages. It will also be important to determinetheir frequency early in pregnancy at those stages wherethey may be useful as the basis for a non-invasive pre-natal diagnosis, particularly in the ﬁrst trimester (5).Furthermore, the fetal source (placental or hemopoietic)of these cells is also of considerable interest, as this couldlead to the development of new tools permitting theireﬃcacious isolation from maternal blood samples.Acknowledgements We thank Dr. Dorothy Huang for her carefulappraisal of this manuscript and critical comments.References1. Babochkina T, Mergenthaler S, Lapaire O, Kiefer V, Yura H,Koike K, Holzgreve W, Hahn S (2005) Evaluation of a soybeanlectin-based method for the enrichment of erythroblasts. J His-tochem Cytochem 53:329–3302. Bianchi DW, Simpson JL, Jackson LG, Elias S, Holzgreve W,Evans MI, Dukes KA, Sullivan LM, Klinger KW, Bischoﬀ FZ,Hahn S, Johnson KL, Lewis D, Wapner RJ, de la CF (2002)Fetal gender and aneuploidy detection using fetal cells inmaternal blood: analysis of NIFTY I data. National Institute ofChild Health and Development Fetal Cell Isolation Study.Prenat Diagn 22:609–6153. Bianchi DW, Simpson JL, Jackson LG, Evans MI, Elias S,Holzgreve W, Sullivan LM, de la CF (1999) Fetal cells inmaternal blood: NIFTY clinical trial interim analysis. DM-STAT. NICHD fetal cell study (NIFTY) group. Prenat Diagn19:994–9954. Chiu RW, Lo YM (2004) The biology and diagnostic applica-tions of fetal DNA and RNA in maternal plasma. Curr Top DevBiol 61:81–1115. Hahn S, Holzgreve W (2002) Prenatal diagnosis using fetal cellsand cell-free fetal DNA in maternal blood: what is currentlyfeasible? Clin Obstet Gynecol 45:649–6566. Kolvraa S, Christensen B, Lykke-Hansen L, Philip J (2005) Thefetal erythroblast is not the optimal target for non-invasiveprenatal diagnosis: preliminary results. J Histochem Cytochem53:331–3367. Krabchi K, Gros-Louis F, Yan J, Bronsard M, Masse J,Forest JC, Drouin R (2001) Quantiﬁcation of all fetal nucle-ated cells in maternal blood between the 18th and 22nd weeksof pregnancy using molecular cytogenetic techniques. ClinGenet 60:145–1508. Mergenthaler S, Babochkina T, Kiefer V, Lapaire O, HolzgreveW, Hahn S (2005) FISH analysis of all fetal nucleated cells inmaternal whole blood: improved speciﬁcity by the use of two Y-chromosome probes. J Histochem Cytochem 53:319–3229. Slunga-Tallberg A, el Rifai W, Keinanen M, Ylinen K, Kurki T,Klinger K, Ylikorkala O, Knuutila S (1995) Maternal origin ofnucleated erythrocytes in peripheral venous blood of pregnantwomen. Hum Genet 96:53–57169

Direct detection of fetal cells in maternal blood: a reappraisal using a combination of two different Y chromosome-specific FISH probes and a single X chromosome-specific probe

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Direct detection of fetal cells in maternal blood: a reappraisal using a combination of two different Y chromosome-specific FISH probes and a single X chromosome-specific probe

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