Cultivation of fetal erythroid precursors from maternal blood: isolation and characterization by PCR and FISH.

Cultivation of fetal progenitor cells from maternal blood offers the opportunity to produce sufficient fetal cells for prenatal molecular genetic and cytogenetic analysis. For in vitro cultivation, 10 ml of blood was collected from 22 women carrying a male fetus. After triple-density gradient centrifugation, the mononucleated cells were cultivated for 10 to 14 days in special hematopoietic growth medium. Red and white colored cell colonies were individually collected by micromanipulation. A representative sample from each colony was characterized by chromosome Y-specific polymerase chain reaction (PCR) systems. The remaining cells of the Y-positive colonies were used to perform chromosome preparations and fluorescence in situ hybridization (FISH) to detect XY-positive interphase nuclei and metaphases. Y-positive signals could be detected in 15 (68%) of the 22 analyzed blood samples. With SRY PCR 10.5% (40/379) of the collected red colonies were determined to be of fetal origin and 6.1% (32/522) of the colonies analyzed by amelogenin PCR were Y-positive. All collected white cell colonies were Y-negative. FISH analyses of PCR-positive colonies revealed that less than 30% of the cells within a colony are of fetal origin and reflect more precisely the actual situation within a single colony. Moreover the successful preparation of fetal metaphases in non-invasive prenatal diagnosis is presented.

Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock.

The function of phagocytic and antigen presenting cells is of crucial importance to sustain immune competence against infectious agents as well as malignancies. We here describe a reproducible procedure for the quantification of phagocytosis by leukocytes in whole blood. For this, a pH-sensitive green-fluorescent protein- (GFP) like dye (Eos-FP) is transfected into infectious microroganisms. After UV-irradiation, the transfected bacteria emit green (≈5160 nm) and red (≈581 nm) fluorescent light at 490 nm excitation. Since the red fluorescent light is sensitive to acidic pH, the phagocytosed bacteria stop emitting red fluorescent light as soon as the phagosomes fuse with lysosomes. The green fluorescence is maintained in the phagolysosome until pathogen degradation is completed. Fluorescence emission can be followed by flow cytometry with filter settings documenting fluorescence 1 (FL 1, FITC) and fluorescence 2 (FL 2, phycoerythrin, PE). Eos-FP transfected bacteria can also be traced within phagocytes using microscopical techniques. A standardized assay has been developed which is suitable for clinical studies by providing clinicians with syringes pre-filled with fixed and appropriately UV-irradiated Eos-FP E. coli (TruCulture™). After adding blood or body fluids to these containers and starting the incubation at 37°C, phagocytosis by granulocytes proceeds over time. Cultures can be terminated at a given time by lysing red blood cells followed by flow cytometry. A pilot study demonstrated that Eos-FP E. coli phagocytosis and digestion was up-regulated in the majority of patients with either severe sepsis or septic shock as compared to healthy donors (p < 0.0001 after o/n incubation). Following treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in selected patients with sepsis, phagolysosome fusion appeared to be accelerated.