IDSSG

10th FETAL CELL WORKSHOP

Report from Dr Dave Miller
Department of Obstetrics and Gynaecology
D Floor, Clarendon Wing
Belmont Grove
Leeds LS2 9JP
UK

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The workshop was in Amsterdam on Saturday 19 June 1999 and arranged by Jan van Lith and Jan Hoovers under the auspices of the Onze Lieve Vrouwe Gasthuis and the Academic Medical Centre. A large number of delegates ended the day information rich and well fed thanks to the organisors and Applied Imaging Inc who partly sponsored the meeting.

Readers will be intrigued to know that despite my absence from the meeting (due to a technical hitch at Leeds-Bradford Airport), I have prepared this summary based partly on the meeting abstracts and partly on my previous experience of and participation in these workshops. Please forgive any omissions of detail arising from my attempt to fill in the gaps. Having looked through the abstracts, compared the data with that from previous meetings and talked at length to Howard Cuckle, my impression is that while progress is being made, there is still some considerable way to go. The report is not necessarily written in the order of presentation.

Wilma Mesker (Leiden) gave us their latest data on the expression of embryonic Hbs during fetal development and their use in the immunological identification of FNRBCs by automated scanning microscopy. RhD status was also investigated by these techniques with promising results.

Jan Hoovers (Amsterdam) provided an update on one of the now three methods of FNRBC isolation (four if you count Jim Schröders's report below) not to rely on immunological separation. This method relies on the buoyant density of FNBRs to separate them from maternal cells depleted of erythrocytes by differential cell lysis (see previous reports). While promising, it is now recognised that some NRBCs are maternal in origin making reliance on HbF based identification, somewhat problematic. The variability in the yield of recovered fetal cells is also a recognised problem.

The application of Dennis Lo's original report on the detection of acellular fetal DNA in maternal bloodplasm to the determination of fetal RhD status was at the core of Ellen van der Schoot's (Amsterdam) report. Using PCR to amplify D-specific DNA, fetal RhD status can be reliably determined with no false-negative or false-positive data obtained from 2nd trimester pregnancies. Had David Miller been able attend, he would have reported a similar study albeit based on the detection of RhD mRNA. The Leeds data suggests an accuracy rate of approximately 83% in 2nd and 3rd trimester pregnancies, falling to <40% in the 1st trimester when the test would be most useful. So it looks as though DNA in the blood plasma wins through although the results of 1st trimester testing are eagerly awaited. There were a number of reports with updates on the traditional density gradient/MACS/FACS/FISH/PCR FNRBS isolation and detection protocols. Sinuhe Hahn (Basel, Switzerland) which is taking part in the NIFTY trial (see below), indicated that a single-density Percoll or Ficoll step followed by anti-GPA immunosegregation was the most efficient method for isolating FNRBCs. Laird Jackson (Philadelphia, USA) found Y-signals in under half of male-bearing pregnancies (292/688) using a similar procedure (targeting the transferrin receptor). A novel variation on the transferrin theme was reported by Martina Serlachus (Helsinki, Finland) who substitutes anti-CD71 MAbs with the transferrin ligand in the immunosegregation step (following a triple density gradient). They detected Y-signals in 3/4 male-bearing pregnancies with this method and no Y-signals in 6 female-bearing pregnancies.

The serious problems all of us have encountered with simultaneous FISH/immunocytochemical procedures was the main topic for Michael Zoccoli (Santa Clara, USA). In particular, the commonly used HbF marker is often incompatible with reliable FISHing for chromosomes X, Y, and 21. Our laboratory gave up trying! The use of new fixation protocols alongside the search and locate functions of the Applied Imaging WinScan system was considered.

There was also a number of interesting alternative systems reported at the workshop. One of them, dielectrophoretic sorting of fetal trophoblast was the subject of Margot Thomas's (Glasgow) talk. The novel use of electric fields in cell sorting tools is not new to us. Both FACS and Steve Wachtel's (Memphis, USA) charge flow (CFS) device use them: Steve presented an overview of fetal cell techniques in Amsterdam. However, Margot's is the first demonstration of a method developed originally for monitoring and isolating potential pathogens from public water supplies! It shares the CFS and Applied Imaging advantage of being wholly non-reliant on immunological reagents and we eagerly await more details in due course.

The use of a laser microbeam to isolate cytotrophoblast from trans-cervical aspirates was presented by Renate Burgemeister (Basel, Switzerland). This instrument is used to isolate single cells, free of maternal cell contamination (a major problem with MACS based procedures on these highly sticky samples) making multiple single-cell diagnostics a real possibility. If the technology proves itself, then this minimally invasive sampling procedure might become more acceptable to its many detractors.

Two reports described attempts to do the same thing but by quite different routes deserve attention. Both described attempts to identify novel markers for fetal cell isolation. The more traditional (Outï Hallikas, Helsinki, Finland)) involves the identification of new MAbs for this purpose using placental antigens. Kit Boye (Copenhagen) is hoping to achieve the same thing by Differential Display (DD-RTPCR) analysis of cellular RNA. Using this method on a FNRBC-like cell line, 1st trimester trophoblasts and female mononuclear cells, she can visualize the differences in gene expression profiles between these cell types. Genes only expressed by one or other can be used as a marker of that cell. It is then a logical step to use the derived sequence information to manufacture synthetic peptides which can behave like natural antigens for MAb production (or perhaps for phage display?). Cees Oudejans (Amsterdam) described his work on circulating trophoblasts in medical conditions as well an aneuploidy. His group have started a study to evaluate the relationship betweeen circulating trophoblast in early pregnancy and the development of pre-eclampsia.

I thought I would leave the interim data from the 5 year multi-center NIH, NIFTY trial of FNRBC isolation protocols until last because it seems to highlight many of the problems associated with the difficult task of delivering a fully working system. Diana Bianchi (Boston, USA) gave details of these problems (hills and valleys as Laird Jackson puts it). To my mind. the trial has generated problems of its own. Being multi-center is laudable, but it could be argued that it has come far too early in assay development to provide any meaningful conclusions. A good example is in the Lessons Learned (so far) which include a need for consistent lab personnel to perform analysis (lab continuity); internal quality control (inter-operator variability); poor reproducibility (the previous two plus the use of different reagents and protocols at each site). Anyway the detection rates achieved in NIFTY were disappointingly low and NIFTY II is now planned to focus on a number of issues and take thinks further.

Having come to the end of my report, readers may still agree with my earlier comments that we have a long way to go. Hence the Workshops will continue for some time to come with the 11th set to take place in Basel, early in the new Year (or Millenium unless you are an advocate for the Julian calendar).

Email: miller@bmb.leeds.ac.uk