Evaluating the culture of fetal erythroblasts from maternal blood for non-invasive prenatal diagnosis

EVALUATING THE CULTURE OF FETALERYTHROBLASTS FROM MATERNAL BLOODFOR NON-INVASIVE PRENATAL DIAGNOSIS

1,2, . 1, 3, 4, 4 . -1*

1Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, U.K.2Department of Biochemitry and Immunology, Capital Institute of Paediatrics, Beijing, 100020, P.R. China

3Department of Haematology, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ, U.K.4Rosie Maternity Hospital, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ, U.K.

Received 10 October 1997Accepted 14 December 1997

SUMMARY

Fetal erythroblasts circulating in maternal blood are important candidate cells for non-invasive prenatal diagnosis.We have cultured erythroblasts from 16 maternal blood samples, both with and without prior enrichment bymagnetic activated cell sorting (MACS), in a semi-solid medium containing growth factors. Individual colonies wereexamined by PCR with sex chromosome-specific primers and microsatellite marker primers. No conclusiveY-chromosome specific amplification could be demonstrated in any of the 16 cases, even when the mother wasconfirmed to be carrying a male fetus. All colonies tested by microsatellite marker PCR were of maternal origin. Ourresults suggest that the probability of obtaining fetal colonies from fetal erythroblasts circulating in maternal bloodis very low and that approaches for culturing fetal erythroblasts in vitro cannot yet be used reliably for prenataldiagnosis using current methods for fetal cell enrichment. ? 1998 John Wiley & Sons, Ltd.

: fetal erythroblast; in vitro culture; erythropoietin; sex genes; microsatellite PCR

*Correspondence to: M. A. Ferguson-Smith, Department ofPathology, University of Cambridge, Tennis Court Road,Cambridge, CB2 1QP, U.K. E-mail: [email protected]

INTRODUCTION

Non-invasive prenatal diagnosis has receivedmuch attention in recent years as it will eliminatethe small but significant risk to the fetus associatedwith more traditional prenatal samplingapproaches. Methodologies exploiting the pres-ence of fetal cells in the maternal circulation havetargeted three fetal cell types, namely, tropho-blasts, lymphocytes and erythroblasts (Adinolfi,1992). Erythroblasts (nucleated red bloodcells—NRBCs) have received the most attention asthey comprise some 10 per cent of all red blood

Contract grant sponsor: MRC.

CCC 0197–3851/98/090883–10 $17.50? 1998 John Wiley & Sons, Ltd.

Prenat. Diagn. 18: 883–892 (1998)

cells in the 11-week-old fetus and 0·5 per cent of allred blood cells in the 19-week-old fetus (Simpsonand Elias, 1994). Moreover, they have a short lifespan, (approximately 90 days) and thus do notpersist from previous pregnancies.

Four approaches have been used for recoveringfetal erythroblasts from maternal blood: (1) tripledensity gradient Ficoll separation (Bhat et al.,1983); (2) fluorescence activated cell sorting(FACS) (Bianchi et al., 1990; Zheng et al., 1995);(3) magnetic activated cell sorting (MACS) withpositive and negative selection (Zheng et al., 1993;Cheung et al., 1996); and (4) culture of fetalerythroblasts (Lo et al., 1994; Valerio et al., 1996).Although the first three strategies attempt toenrich for erythroid cells, only a small number offetal nucleated red cells are actually isolated.Hence, prenatal diagnosis for single gene disorders
and chromosome abnormalities remains difficult.

884 . .

The ability to culture such fetal cells however,could exponentially increase the number of cellsavailable for diagnosis. Weinberg et al. (1992)compared erythropoiesis from fetuses, newborninfants and adults in semi-solid cultures witherythropoietin (Ep) and indicated that most fetalcultures reached maximal growth at Ep<2 U/ml.Conversely most adult cultures required Ep>2 U/ml. These results suggested that fetal erythroidprogenitors are more sensitive to Ep concen-trations in vitro. Lo et al. (1994) obtained maternalblood from five women (at 11–20 weeks’ gestation)each carrying a male fetus; non-sorted mononu-clear cells were cultured in erythropoietin-enrichedliquid medium and after seven days, Y-positivecells were detected by FISH (one in 4000 to 9500female cells) in all five cases. In Valerio et al.(1996), fetal erythroid progenitors were firstenriched by MACS using biotin-labelled humanerythropoietin ligands and then cultured in a semi-solid medium containing several growth factors. Ineight euploid pregnancies, mixtures of colonieswere obtained comprising 18 per cent fetal cells.This promising observation, if confirmed, suggeststhat it may be possible to obtain sufficient uncon-taminated fetal erythroblasts from single coloniesin culture for the analysis of single gene disordersand chromosome abnormalities.

To evaluate the feasibility of culturing fetalerythroblasts from maternal blood for prenataldiagnosis, we have used two different in vitroculturing methods. One method was designed onthe basis of suggestions made by Weinberg et al.(1992). The second method exactly followed theprocedures of Valerio et al. (1996). Our studydemonstrates some of the problems involvedin using current fetal erythroblast culturingtechniques for non-invasive prenatal diagnosis.

MATERIALS AND METHODS

We obtained, following informed consent,maternal blood samples from 27 pregnancies, at9–17 weeks’ gestation. Five of these pregnancieswere at high risk for Down syndrome andwere followed up with cytogenetic analysis ofamniotic fluids. Blood cultures were also obtainedfrom five non-pregnant women to serve asnormal controls. In all cases, peripheral blood(8–20 ml) was collected in heparin tubes anddiluted 1:2 with phosphate buffered saline(PBS). Mononuclear cells were then isolated

? 1998 John Wiley & Sons, Ltd.

by Ficoll-Paque (Pharmacia Biotech) densitycentrifugation according to the manufacturer’srecommendations.

Cell culture

Two different culture conditions were used inthis study.

(1) Culture medium containing a low concen-tration of erythropoietin: isolated mononu-clear cells were plated in duplicate in 35 mmPetri dishes with 3 ml of semi-solid medium(Stemcell Technologies Inc, Canada) contain-ing 1 U/ml human recombinant erythropoi-etin (Boehringer Mannheim, Germany). Cellswere grown at a final concentration of3#105/ml, at 37)C, 100 per cent humidityand 5 per cent CO2 atmosphere. Colonieswere counted on days 10 and 14.

(2) MACS-complete medium: human recom-binant erythropoietin was biotinylated usingthe method of Wojchowski and Caslake(1989), since it was not available commer-cially. Labelling reactions and stability duringstorage were verified by electrotransferringeach SDS-PAGE gel onto a Hybond-C mem-brane and performing a Western blot with ahorse-radish peroxidase (HRP)-streptavidinconjugate. Following the published protocolof Valerio et al. (1996), isolated mononuclearcells were incubated with biotin-(sialyl)-erythropoietin at 4)C for either 6 h or over-night under sterile conditions. Cells were thenseparated by MiniMacs cell sorting usingstreptavidin-conjugated microbeads (MiltenyiBiotec, Germany). Positive fractions were col-lected and plated in duplicate in 35 mm Petridishes with 3 ml of semi-solid medium con-taining 50 ng/ml stem cell factor, 10 ng/mlGM-CSF, 10 ng/ml IL3 and 3 U/ml erythro-poietin (Stemcell Technologies Inc, Canada).Cells were incubated at 37)C, 100 per centhumidity and 5 per cent CO2 atmosphere.Negative fractions, which were used asthe control samples of maternal cells, werecentrifuged and resuspended into H2O, thenstored at "20)C until required. Colonieswere counted on days 10 and 14.

Harvesting cells

In order to diagnose small numbers of cells and
leave the colony intact for further (e.g., cyto-
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885

genetic) analyses we used a Nikon Axiovertmicroscope equipped with micromanipulator. Amicropipette loaded with sterile PBS was plungedinto the centre of the colony and approximately200 cells aspirated. These were then transferred toa thin-walled 0·5 ml eppendorf tube containing10 ìl of ice-cold sterile water prior to fluorescencePCR analysis. For conventional PCR detectionand cytogenetic analyses, single colonies werepicked with sterile Pasteur pipettes and transferredto separate eppendorf tubes containing 300 ìlPBS. Cells in PBS were thoroughly dispersed bypipetting up and down and divided into twoaliquots. One aliquot of cells was immediatelycentrifuged onto clean microscope slides using acytospin. The other aliquot was centrifuged andresuspended into H2O. All samples were thenstored at "20)C until required.

PCR

(1) Conventional PCR: both the ZFY/ZFX gene(Lau and Chan, 1989) and the D9S154 micro-satellite locus (Gyapay et al., 1994) weretargeted for DNA amplification and detec-tion. Cells were boiled for 5 min and centri-fuged at 13 000 rpm for 10 min. Each reactioncomprised of a 3 ìl lysed sample, 100 ng ofeach primer, 1·5 mM MgCl2, 200 ìM dNTPand 0·6 units Taq DNA polymerase (HTBiotechnology Ltd, UK) in a total PCR reac-tion volume of 25 ìl. Taq DNA polymerasewas not added to each reaction mixture untilafter the first 3 min of denaturation at 94)C.35 cycles of PCR amplification were per-formed on the targeted gene loci. Each ampli-fication cycle consisted of 30 s at 94)C, 30 sat 55)C and 1 min at 72)C. A final 10 minextension was conducted at 72)C.

PCR products from the ZFY/ZFX geneamplification were separated on 1·4 per centagarose gels. PCR products using the D9S154microsatellite marker were fractionated on a6 per cent polyacrylamide gel containing 7Murea and developed by silver staining.

(2) Fluorescence PCR: in order to perform sen-sitive diagnosis on a small number of cells,fluorescence-labelled primers were used. Inthe initial set of experiments, primers forthe amelogenin gene (Sullivan et al., 1993)and the cystic fibrosis locus ÄF508 (Wuet al., 1993) were used in a duplex PCRreaction, both were HPLC purified (Genosys,


Cambridge, UK) and labelled with theblue fluorescence dye 6-FAM (AppliedBiosystems). The amelogenin primers wereused at a concentration of 0·1 ìM and CFprimers at 0.05 ìM. HT Biotech ‘Supertaq’and standard PCR buffer (1·5 mM MgCl2)were also used in a 25 ìl reaction. Thermalcycling was for 30 cycles with an annealingtemperature of 61)C. Since the amelogeninprimers were only capable of distinguishingbetween maternal and fetal colonies in 50 percent of the cases (i.e., male pregnancies) weemployed the microsatellite primer setsD18S51, D19S253 and THO (Urquhart et al.,1995). These primers are in common usagefor the purposes of genetic fingerprinting.They were labelled with 6-FAM (blue),TAMRA (yellow) and HEX (green), respect-ively, and used at concentrations of 0·02 ìM,0·032 ìM and 0·06 ìM, respectively, in a tri-plex PCR reaction. All conditions were thesame as for the amel/CF primers set exceptthat the annealing temperature was 62)C. Allreactions were performed on a Biometra Triothermoblock. Analysis was performed on anApplied Biosystems sequencing gel apparatusequipped with Genescan 672 software forsizing of the PCR products in relation to aRox 350 (red) labelled internal size marker(Applied Biosystems). The approach ofmicrodissection followed by fluorescencePCR allowed sensitive genotype analysis ofcells from the colony and left sufficient cells toperform immuno-phenotyping and FISHanalysis.

Immuno-phenotyping

All procedures followed the method of Zhenget al. (1993), with minor modifications. Cytospinslides, stored at "20)C, were fixed in 4 per centparaformaldehyde, 15 mM MgCl2 at room tem-perature for 10 min and then blocked with 15 percent human AB serum in PBS at room temperaturefor 30 min. Fetal haemoglobin (HbF) antibodies(UCHã) were diluted 1:10 in PBS containing 10 percent human AB serum of which 100 ìl was appliedto each slide. Cells were then incubated at roomtemperature for 60 min in a humid chamber.Specific binding was detected by incubating theslides with FITC-labelled goat anti-mouse IgG(1:200 dilution) at room temperature for 30 min.

Prenat. Diagn. 18: 883–892 (1998)

886 . .

After a final wash in PBS, the slides were counter-stained with propidium iodide and mounted in anantifade mountant (Citifluor AF1). Cells stainedwith the secondary antibody only were used ascontrols in each experiment.

FISH

After microscopic examination of immuno-stained cells, slides were washed twice in 2#SSC/0·05 per cent Tween-20 at 50)C for 15 minand dehydrated in an ethanol series. AnX-chromosome specific centromeric repeat probe(pBamX5) and a Y-chromosome specific DYZ1repeat probe (Cy98) were labelled by nick transla-tion with biotin-16-dUTP and digoxigenin-11-dUTP, respectively. A mixture of these X andY-chromosome probes was added to the slides andsimultaneously denatured with chromosomalDNA at 90)C for 10 min. Hybridization proceededovernight at 42)C. The slides were washed twice in50 per cent formamide/2#SSC, twice in 2#SSCand once in 0·1#SSC. All washes were performedat 45)C for 5 min each. The slides were thenblocked in 4#SSC/0·05 per cent Tween-20/3 percent BSA at 37)C for 30 min and incubated withavidin-FITC and anti-digoxigenin-rhodaminediluted (1:400 and 1:200, respectively) in 4#SSC/0·05 per cent Tween-20/1 per cent BSA at 37)C for30 min. After a final wash in 4#SSC/0·05 per centTween-20, the slides were counter-stained in DAPIfor 2 min and mounted in antifade mountant(Citifluor AF1).

All slides were examined using a NikonMicrophot-SA fluorescence microscope equippedwith an automatic filter wheel and a cooled CCDcamera controlled by a Macintosh Quadra 950computer system using Smartcapture software(Digital Scientific, U.K.).

Chromosome analysis

The whole colony was aspirated using a Gilsonpipette and the intact colony placed in 100 ìl ofRPMI medium containing 20 per cent fetal calfserum and colcemid. After a 2–6 hour incubation,900 ìl of 75 mM KCL hypotonic solution wereadded and after a further 12 min incubation, thecells were dispersed, spun down in a microcentri-fuge and fixed gradually in 3:1 methanol–aceticacid. Following a second centrifugation and fixa-


tion, standard chromosome preparations weremade. Fluorescent banding was performed usingDAPI and analysis was done on a Zeiss Axioplanepifluorescence microscope.

RESULTS

Cell cultures

16 of the 27 maternal blood samples containedenough mononuclear cells (i.e., >107 cells) forMACS enrichment and culturing in completemedium with growth factors. The Ep receptor-positive cells were eluted and found to vary innumber from 3#104 to 4·5#105. Culturing thepositive fraction for 7–10 days resulted in theformation of CFU-Es (colony forming unit-erythroid). After a total incubation time of 14days, each CFU-E was replaced by a large colonycharacterized by several subcolonies of haemo-globinized erythroblasts (BFU-E, burst formingunit-erythroid) (Fig. 1(a)). The total number ofcolonies obtained for each case ranged from 2 to60, with a mean of 15·4 (Table I).

For each case, a fraction of the unsorted mono-nuclear cells was also cultured in a semi-solidmedium containing only 1 U/ml Ep. Most eryth-roid colonies which were obtained under theseculture conditions developed into relatively smallcolonies (as compared with those grown in com-plete medium) and were light red in colour(Fig. 1(b)). A few other colony types, presumablyderived from granulocyte or macrophage precur-sors, were also observed under these culture con-ditions. The number of erythroid colonies/caseobtained varied from 3 to 111, with a mean of13·8&11·1 per 105 mononuclear cells (Table I).

The results from five non-pregnant women arealso shown in Table I. In general, blood samplesfrom these adults produced fewer colonies thanmaternal samples.

Fetal sex determination

Fetal sex was determined by amplifyingtwo different loci on both the X and theY-chromosome. Amplification of the ZFX geneyielded a 488 bp fragment whereas amplification ofthe ZFY gene produced a 340 bp fragment. Hence,DNA from XY males produced two resolvablebands and DNA from XX females showed a singleband (Fig. 2(a)). Amplification of the amelogenin

Prenat. Diagn. 18: 883–892 (1998)

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Fig. 1—Erythroid colonies (14 days): (a) a BFU-E colony oncomplete medium (#100); (b) a BFU-E colony on culturemedium containing 1 U/ml Ep (#100)

gene on the X-chromosome using the fluorescencePCR technique produced a 107 bp band andamplification of its homologous gene on theY-chromosome produced a 113 bp fragment(Fig. 2(b)). The results of fetal sex determinationfrom PCR experiments of single colonies are sum-marized in Table II. Although seven colonies fromfour cases showed a Y-specific band on PCRamplification of the ZFY/ZFX locus, this wasinconsistent with results on the same colonies fromD9S154 PCR (Fig. 2(a)).

Microsatellite PCR was used to distinguishbetween maternal and fetal cells of a colony and toassess the overall level of contamination. Ninedifferent alleles have been amplified with theD9S154 primer pair from maternal cells of 16 casesin this study. The identity of the alleles present inthe colonies and the mothers’ genotypes are shownin Table II. A total of 140 colonies were screened


by D9S154 PCR and the results revealed no differ-ences between all harvested cells and the corre-sponding maternal cells. Similarly, when usingmicrosatellite primers for D18S51, D19S253 andTHO by fluorescence PCR in five pregnanciesfrom high-risk populations, no colony was foundwith a genotype which differed from the maternalgenotype (Table II and Fig. 2(c)).

As shown in this table, amniocentesis confirmedthat patient 14 carried a male fetus. From a totalof 23 colonies obtained from culturing erythroidcells of this patient in complete medium and 10colonies picked at random in culture conditionscontaining only 1 U/ml Ep, no Y-specific signalcould be detected using either the amelogenin geneor ZFY/ZFX gene PCRs. Similar results were alsoobtained with a control case which had a numberof cord blood-isolated male cells mixed withmononuclear cells from a non-pregnant woman.For the other four cases, where each mothercarried a female fetus, microsatellite PCRs for alltested colonies showed an identical genotypebetween the mother’s cells and harvested cells. Thissuggests that all colonies tested from each samplewere actually derived from maternal erythroidprogenitor cells.

Fetal cell detection by immuno-staining and FISH

HbF was detected by immuno-staining cellsfrom a single colony. Control slides subjected toonly the secondary antibody and human AB serumclearly demonstrated the absence of significantamounts of non-specific binding. Colonies derivedfrom the culturing of cells from non-pregnantadults were also tested with HbF immuno-staining. Harvested colonies from maternal bloodsamples displayed 13·4 per cent of cells positive forHbF whereas colonies from four out of the fivenon-pregnant adults found 11·7 per cent of cellspositive for this same antibody (Table III).

After the removal of HbF antibodies, cells werehybridized with chromosome X and Y-specificDNA probes. The purpose of using both probesinstead of a single Y-chromosome specific probewas to distinguish false-positive Y-signals causedby non-specific binding. Indeed, on certain prepa-rations, we observed background fluorescent sig-nals similar to those of the Y-chromosome whichcould only be distinguished with difficulty by thetrained eye and by the fact that two X-signals wereclearly observed on the HbF positive cells from

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Table I—Results from cell cultures

MACS-complete medium Ep 1 U/ml

Valerio Cambridge Pregnant Non-pregnant

Number of cases 8 16 16 5Gestation (weeks) 14–16 a 9–17a a 9–17a

Days of culturing 6–11 10–14 10–14 14Colonies/case: minimum 1 2 3 19

maximum 34 60 111 101Average colonies/case 15·1 15·4Colony/105 MNCc 13·8&11·1 10·9&3·8Total number of colonies b121b 210 669 181

Valerio—Valerio et al. (1996).Cambridge—present study.MNC—mononuclear cells.aNine of the cases were obtained from pregnancies in the second trimester.bTotal number of colonies from Valerio included erythroid colonies and CFU-GEMM (colonyforming unit-granulocyte, erythrocyte, macrophage, megakaryocyte). The present study onlyshows the number of erythroid colonies.cData are presented with a mean&1 standard deviation.

male pregnancies and controls. To further deter-mine the origin of colonies with HbF positive cells,microsatellite PCR was carried out and resulted inthe same alleles for both the mother’s cells andHbF positive colony cells. In addition, we weresuccessful in producing analysable chromosomepreparations from 16 out of 29 colonies. Althoughwe believe that this success rate could be improvedas we become more familiar with the technique, inall cases tested the karyotype of colonies was46,XX regardless of the fetal karyotype.

DISCUSSION

In this study, we have evaluated the feasibility ofusing recently published techniques to culture fetalerythroblasts from maternal blood for non-invasive prenatal diagnosis. The two methodschosen included culturing in complete mediumwith prior enrichment and culturing mononuclearcells in a selective medium. Both culture conditionsacquired a similar average number of colonies aswas found in Alter (1994) and Valerio et al. (1996).However, detailed analysis of large numbers ofsingle colonies failed to confirm that any were offetal origin.

The earlier study by Valerio et al. (1996)involved pooling colonies or collecting PBS wash-
ings from an entire petri dish, thereby increasing

the chance of including the few of fetal originamong a preponderance of maternally-derived col-onies. Furthermore, many of their maternal bloodsamples were taken after an invasive procedure.However, if the procedure is to be useful forprenatal diagnosis, it must be capable of yieldingsufficient fetal colonies uncontaminated withmaternal cells which can be used for genotypingand chromosome analysis. We have chosen toisolate and test cells from single colonies in orderto evaluate this possibility. Our results stronglysuggest that maternal erythroid progenitors so faroutnumber those of fetal origin in maternal blood,as to make the chance of being able to identifyfetal colonies an impractical proposition in a diag-nostic setting until much better techniques forenrichment are available. Our study also revealsthat the culture of erythroid progenitors can yieldsuitable material for accurate genotyping, particu-larly with the fluorescence PCR technology, andfor chromosome analysis both by conventionalmethods and by interphase FISH. Thus, non-invasive prenatal diagnosis will be feasible byculture once the problems of fetal cell isolation andenrichment are overcome.

In the present study, seven colonies were foundto produce a false-positive Y-band by ZFY/ZFXgene PCR. These results were presumably causedby contamination, despite the fact that all PCR
procedures were performed with strict precautions
Prenat. Diagn. 18: 883–892 (1998)

889

Fig. 2—(a) Amplification of the ZFY/ZFX gene: (1) 100 bp marker; (2) PCR product from a control male; (3) PCR product froma control female; (4–10) PCR products from different individual colonies, three of them show a Y-specific band (arrow) and lessdensity than an X-specific band compared with normal male (2); (11) reagent control. The band sometimes observed at <100 bp,corresponds to primer dimers. (b) Duplex fluorescent PCR of amelogenin and ÄF508 loci reveals that colonies recovered frompatient 7 are female and heterozygous for the mutant (3 bp deletion) allele. (c) Triplex fluorescent PCR using microsatellite primersets D18S51, D19S253 and THO

against contamination and all reagent controlswere found to be blank. It is clear that due to thesensitive nature of these experiments, thereremains a risk of PCR contamination. Use of thefluorescent amelogenin primers in conjunctionwith micromanipulation of a few cells, however,seemed to circumvent this problem. Thus, westrongly recommend the use of the second of ourtwo approaches for fetal sex determination. Fluor-escence PCR is also amenable to multiplex reac-tions. Indeed, we simultaneously amplified theamelogenin loci with primers from the ÄF508locus of the cystic fibrosis gene and, in one case,determined that the maternal genome carried amutant allele with a three base pair deletion(Fig. 2(b)). Furthermore, although for practicalreasons in this study, we performed diagnosis ofsex/CF separately from genetic fingerprintingexperiments (Fig. 2(c)), it is possible to use thisapproach to detect simultaneously amelogenin,cystic fibrosis and microsatellites in one singlereaction (Findlay et al., 1995). The absence of aY-chromosome signal in any of the colonies, plus


patterns consistent with the maternal genotype inall colonies tested using microsatellite markers,provides the basis for our assertions that no fetalcolonies were isolated.

Despite establishing genetically that no colonieswere fetally derived, several gave strong signalsusing the HbF antibody. Thus, adult erythroidprogenitor cells were shown to produce higherthan expected levels of HbF during in vitro cultur-ing. This is consistent with the observations ofFujimori et al. (1990). Normally, the synthesis ofHbF is reduced to very low levels of less than 0·6per cent of total haemoglobin in adults (Rochetteet al., 1994). Therefore, although the precisemechanisms for this increased HbF production inculture have not yet been elucidated (Fujimoriet al., 1990), we suggest that HbF antibodies maynot be suitable for fetal cell detection in culture.

The procedure described in this study does notenrich for fetal cells per se, rather it selects foruptake of erythropoetin, which is much moreabundant in fetal than adult cells. Hence, a lowconcentration of Ep was chosen for selective

Prenat. Diagn. 18: 883–892 (1998)

Table II—Sex and genotype determination on cells derived from single colonies

Fluorescence PCR

Fetalckaryotypeor fetal sex

at birth

F Microsatellites

onytypes

Maternalgenotypee

Colonygenotypes

al, 1Bal, 1B

carrierf Femalermal Femaleal, 1B

rmalal, 1B 1/1, 1/2, 1/2 11 same as maternal, 1B 46, XX

rmal 5/5, 3/4, 6/7 25 same as maternal, 5B 46, XXrmal Femalermal 2/2, 5/6, 3/4 7 same as maternal, 15B 46, XYrmal 3/4, 7/8, 5/5 21 same as maternal, 1B 47, XX+21rmal 6/7, 9/10, 8/8 21 same as maternal, 5B 46, XXrmal 46, XYrmal,

rrier, 4B85 same as maternal, 27B

isolated from cord blood mixed with mononuclear cells from a

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883–892(1998)

Casenumber

Conventional PCR

ZFX/ZFY D9S154 Amelogenin C

Colonynumbera

Colonygenotypes

Colonynumber

Maternalgenotype

Colonygenotypes

Colonynumberb

Colonygenotypes

Colgeno

1 12 12F 12 1/4 Same as maternal2 10 3M, 7F 10 4/7 Same as maternal3 10 10F 9 4/7 Same as maternal 9 9F4 9 9F 6 2/5 Same as maternal 8 6F, 2B5 28 1M, 27F 13 3/3 Same as maternal 15 14F, 1B 14 norm6 8 8F 3 2/3 Same as maternal 7 6F, 1B 6 norm7 6 6F 3 7/8 Same as maternal 6 6F 6 CF8 3 3F 1 4/6 Same as maternal 2 2F 2 no9 16 16F 10 5/6 Same as maternal 18 17F, 1B 17 norm

10 17 17F 15 3/6 Same as maternal 11 11F 11 no11 15 1M 14F 9 4/5 Same as maternal 12 11F, 1B 11 norm12 29 2M, 22F, 5B 11 8/9 Same as maternal 30 30F 30 no13 10 10F 4 2/7 Same as maternal 11 11F 11 no14 10 10F 7 1/3 Same as maternal 23 23F 23 no15 10 10F 8 3/7 Same as maternal 22 22F 22 no16 10 10F 19 2/2 Same as maternal 26 26F 26 noControld 14 12F, 2B 13 13F 13 noTotal 217 7M, 203F, 7B 140 All same as maternal 213 207F, 6B 186 no

6 CF ca

F—female.M—maleB—PCR failed.Same—harvested cells and corresponding maternal cells showed a identical genotype.aMost tested colonies were obtained from culture conditions containing only 1 U/ml Ep.bAll tested colonies were obtained from complete medium cultures.cFetal sex ascertained by cytogenetic analysis of amniotic fluid, or phenotypic sex at birth.dBefore magnetic cell sorting and cell cultures, a control was established using a small number of male cells

non-pregnant woman. The mixture corresponded to one fetal cell per 50 000 maternal cells.eMothers’ genotypes are shown for D18S51, D19S253 and THO, respectively.fMother was also a CF carrier.

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enrichment of fetal erythroid progenitor cells toensure maximal fetal erythroblast sensitivity andgrowth. Despite this, attempts made in this studyto establish fetal erythroid progenitor cells circu-lating in maternal blood by these culture con-ditions were disappointing. In addition to theproblems associated with establishing culturesfrom of a small number of fetal erythroblastscirculating in maternal blood, these negativeresults could suggest that there is little or nostatistical significance between the sensitivity offetal and adult erythroblasts to low Ep concen-trations for in vitro colony formations. Obviously,fetal erythroblast growth would also be hamperedby a rapid increase in maternal erythropoiesisduring normal pregnancy.

In conclusion, this study provides clear evidencethat erythroid colonies produced with both com-plete medium and selective media are mainlyderived from maternal erythroid progenitors. Fetalerythroblasts circulating in maternal blood arelikely to have few opportunities of developing intodetectable colonies in culture, even after priorenrichment using current techniques. Substantialimprovement in fetal cell enrichment and isolationwill be required before cell culture methods can beused for prenatal diagnosis in clinical practice.

We would like to thank Dr Sandy Goodburnand the nursing staff and patients in Adden-brooke’s Hospital who enabled us to obtain bloodsamples. We are grateful to Patricia O’Brien,Charles Lee, Fengtang Yang and Debbie Walshfor their help with various aspects of this study.Professor Peter Beverley kindly provided fetal hae-moglobin antibodies (UCHã). H. Chen is a visiting

Table III—Results of HbF immuno-staining

Pregnantwomen

Non-pregnantwomen

Number of cases 10 5Tested slides 25 16FNRBC/200 cells:

minimum 1 0maximum 108 116

#FNRBC (%) 13·4+12·5 11·7+11·0

#FNRBC—nuclear erythrocytes containing fetal haemo-globin (HbF).


scholar of the National Education Committee ofP.R. China. This work was supported by an MRCProject grant to M. A. Ferguson-Smith.

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Evaluating the culture of fetal erythroblasts from maternal blood for non-invasive prenatal diagnosis