Automatic retrieval of single microchimeric cells and verification of identity by on‐chip multiplex PCR

Abstract

The analysis of rare cells is not an easy task. 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. Under these conditions, even highly specific biochemical markers are not perfectly reliable. 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. The power of the method was tested using samples containing mixed cells of related and non-related individuals. 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 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. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.

Prenatal, noninvasive and preimplantation genetic diagnosis of inherited disorders: hemoglobinopathies

“…However, recently, e��orts have been revived to isolate �etal cells using approaches that are based on more automated cell-sorting techniques. To date, technologies that have been investigated include automated microscopy [68], microfluidics [69], light-scattering spectroscopy [70] and a combination o� automatic screening �or enriched target cells (based on immunofluorescence labeling) with isolation o� single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and finally low-volume on-chip multiplex PCR �or DNA fingerprint analysis [71]. Preliminary results with these methods have been encouraging and indicate potential �or NIPD applications, assuming all limitations and technical problems encountered so �ar can be addressed [8,63].…”

Verification of the genomic identity of candidate microchimeric cells

“…We suggest this procedure as a standard for sex-independent identification of fetal cells in the setting of rare cell analysis. 10 Furthermore, we developed a method which avoids exhaustion of the available DNA for target cell identification and which allows for both genomic identification as well as for molecular genetic and cytogenetic analysis of the same cell. For this purpose we implemented a step of ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

Low-Volume On-Chip Single-Cell Whole Genome Amplification for Multiple Subsequent Analyses

“…A fast growing number of techniques are available and reviewed in detail by Panabiers et al [12]. Although enrichment steps are highly efficient, an excess of background cells remain which makes additional isolation procedures like laser capture microdissection or micromanipulation necessary [6,7,13,14]. Here, we describe a protocol that uses LMPC to transfer single candidate cells onto 48-reaction slides and perform on-chip whole genome amplification in less than 2 μL [7].…”