For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD45+ and CD14+ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71+ erythroid cells were enriched up to 80 per cent by MACS. This double-MACS' procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40-55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45-/CD71+ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45-/CD14- cells, paternal alleles could be detected. In the CD45-/CD71+ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non-invasive prenatal diagnosis.

Our procedure for prenatal paternity testing combines depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell sorting (MACS)' with PCRbased analysis of DNA.Venous blood samples (20 ml) were collected in a 5% EDTA solution from healthy women with uncomplicated pregnancies. The PBMCs were stained after ficolation (Pharmacia, Upsalla) with CD45 PE (murine IgG1, KC56, Coulter) and anti-IgG1 MACS beads and CD14 MACS beads (both from Miltenyi Biotec). After depletion of CD45' and CD14+ cells (leukocytes and monocytes) on a MACS (A2) column, the negative cells were stained with CD71 FTTC (murine IgG2a, Becton Dickinson) and anti-IgG2 MACS beads. CD71' cells (erythroblasts) were enriched by a second MACS separation on a miniMACS. The enrichment of the CD71' cells could be improved up to 80% by using haptenated CD45 and haptenated CD7 1 (both Miltenyi Biotec) and corresponding MACS beads.The highly polymorphic loci of the gene system HLA-DQA l2 and variable number of tandem repeats (VNTR) of the polymorphic gene system D1S803 were analyzed by PCR with and without preamplification with unspecific primers (PEP): After DNA extraction from the double-MACS sorted cells with phenol-chloroform, the concentration of DNA was quantified photometrically. HLA-DQA1 typing was carried out according to the manufacturer's instructions for the "Ampli Type HLA-DQa" system (Cetus, Emmeryville, CA). Amplification of the DlS80 locus was carried out by using the primers described by Kasai et al.5 The PCR cycles had the following phases: denaturation at 95 "C for 1 min; primer annealing at 65 "C for 1 min; primer extension at 70 "C for 2 min. A total of 30 cycles was carried out in a Perkin-Elmer thermocycler. Electrophoretical separation of amplified D 1 S80 alleles was performed 144

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Splines and subdivision curves are flexible tools in the design and manipulation of curves in Euclidean space. In this paper we study generalizations of interpolating splines and subdivision schemes to the Riemannian manifold of shell surfaces in which the associated metric measures both bending and membrane distortion. The shells under consideration are assumed to be represented by Loop subdivision surfaces. This enables the animation of shells via the smooth interpolation of a given set of key frame control meshes. Using a variational time discretization of geodesics efficient numerical implementations can be derived. These are based on a discrete geodesic interpolation, discrete geometric logarithm, discrete exponential map, and discrete parallel transport. With these building blocks at hand discrete Riemannian cardinal splines and three different types of discrete, interpolatory subdivision schemes are defined. Numerical results for two different subdivision shell models underline the potential of this approach in key frame animation.

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