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To design a protocol for non-invasive prenatal diagnosis of fetal Rhesus D (RhD) status.A total of 112 single lymphocytes were used to test the efficiency of the assay. The protocol was validated using blood samples from 84 RhD-negative pregnant women at 7-24 weeks of gestation. Cell-free DNA (cfDNA) was enzymatically digested using AciI and analyzed by a polymerase chain reaction (PCR) that allowed simultaneous amplification of RHD exons 7 and 10, SRY, RASFF1A and ACTB.On the one genome-equivalent level, the efficiency of the protocol was ≥ 94.6% for each locus amplified. Conclusive results from the first set of PCRs were obtained for 79 cases with one false-positive. In five cases the analysis was repeated and, subsequently, all cases were accurately diagnosed.The proposed protocol is rapid, applicable in most molecular diagnostic laboratories and provides the basis for non-invasive examination of fetal RhD with 96.7% specificity and 100% sensitivity.

The human osteosarcoma U2OS cell line is one of the first generated cell lines and is used in various areas of biomedical research. Knowledge of its protein expression is limited and no comprehensive study on the proteome of this cell line has been reported to date. Proteomics technology was used in order to analyse the proteins of the U2OS cell line. Total protein extracts were separated by two-dimensional gel electrophoresis (2-DE) and analysed by matrix-assisted laser desorption ionisation-mass spectrometry (MALDI-MS) and MALDI--MS-MS following in-gel digestion with trypsin and, finally, protein identification was carried out by peptide mass fingerprint (PMF) and post source decay (PSD), respectively. Approximately 3,000 spots were excised from two 2-DE gels and were analysed, resulting in the identification of 237 different gene products. The majority of the identified proteins were enzymes, regulatory proteins and RNA-associated proteins, while leukocyte markers and oncogenes were also present. Our findings include 11 protooncogenes (FKBP4, SRC8, PSD10, FUBP1, PARK7, NPM, PDIA1, OXRP, SET, TCTP and GRP75) related to the cancerous state of the U2OS cell line. The U2OS 2-DE database provides the basis for future protein studies.

Proteomic analysis combining two-dimentional electrophoresis (2DE) and mass spectrometry (MS) has the potential for a wide range of applications in biological and medical sciences, as protein screening in tissues obtained from healthy and diseased conditions can determine drug targets and diagnostic markers. Conventionally, amniotic fluid (AF) samples are routinely used for prenatal diagnosis of a wide range of fetal abnormalities. Proteomics have already been applied in the analysis of tissues from fetuses with Down's syndrome, in order to detect differences in their protein profile as compared to the normal profiles and to determine possible diagnostic tools. A detailed protein 2DE for the normal human AF has not been reported. In the present study, the 2D protein database of the normal human AF supernatant (AFS) was constructed. Ten AFS samples from women carrying normal fetuses were analysed by 2DE. A mean of 412 spots per gel were analyzed and protein identification was carried out by MALDI-MS and MALDI-MS-MS. A 2D protein map comprising of 136 different gene products was constructed. The majority of the identified proteins are regulatory proteins, enzymes, secreted proteins, carriers and immunoglobulins. Twelve hypothetical proteins were also included. The normal AFS proteome map is a valuable tool for the study of aberrant protein expression and the search for proteins as possible markers for the prediction of abnormal fetuses.