This is primarily done by sorting and staining for biochemical parameters, which introduces the basic problem of population overlap even when highly specific markers are used. It is obviously necessary to enrich the rare cells and discriminate them from the vast majority of maternal cells. Unfortunately, because these cells are very rare, single-cell PCR in rare cell analysis faces additional problems when compared with similar applications in pre-implantation genomic diagnosis (PGD). This phenomenon has been linked to autoimmune diseases, but it is also the basis of long-standing efforts to implement non-invasive prenatal diagnosis (NIPD) based on molecular genetic analysis of fetal cells present in the circulation of pregnant women. Examples have been reported of maternal cells present in the tissue of offspring (maternal microchimerism) and, conversely, of fetal cells present in maternal tissues (fetal microchimerism), in which these cells may survive even for years after delivery. Microchimerism constitutes the presence of a small number of cells that are genetically distinct from the host individual.

We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The power of the method was tested using samples containing mixed cells of related and non-related individuals. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. Under these conditions, even highly specific biochemical markers are not perfectly reliable.

This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. The analysis of rare cells is not an easy task.

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