Zebrafish embryo development in a microfluidic flow-through system.

The zebrafish embryo is a small, cheap, whole-animal model which may replace rodents in some areas of research. Unfortunately, zebrafish embryos are commonly cultured in microtitre plates using cell-culture protocols with static buffer replacement. Such protocols are highly invasive, consume large quantities of reagents and do not readily permit high-quality imaging. Zebrafish and rodent embryos have previously been cultured in static microfluidic drops, and zebrafish embryos have also been raised in a prototype polydimethylsiloxane setup in a Petri dish. Other than this, no animal embryo has ever been shown to undergo embryonic development in a microfluidic flow-through system. We have developed and prototyped a specialized lab-on-a-chip made from bonded layers of borosilicate glass. We find that zebrafish embryos can develop in the chip for 5 days, with continuous buffer flow at pressures of 0.005-0.04 MPa. Phenotypic effects were seen, but these were scored subjectively as 'minor'. Survival rates of 100% could be reached with buffer flows of 2 µL per well per min. High-quality imaging was possible. An acute ethanol exposure test in the chip replicated the same assay performed in microtitre plates. More than 100 embryos could be cultured in an area, excluding infrastructure, smaller than a credit card. We discuss how biochip technology, coupled with zebrafish larvae, could allow biological research to be conducted in massive, parallel experiments, at high speed and low cost.

Automated red blood cell differential analysis on a multi-angle light scatter/fluorescence hematology analyzer.

BACKGROUND: Most hematology analyzers today are capable of performing white blood cell differential analysis but only limited red blood cell (RBC) differential parameters are available. Because of incomplete morphological information on RBC abnormalities, 5-10% of samples in hematology laboratories routinely undergo smear review. A more complete automated RBC differential capability is desired. METHODS: Abbott CELL DYN 4000 analyzer was modified to perform three-dimensional (3D) RBC differential analysis (RBC/diff) on cell-by-cell basis at 488 nm as well as at 633 nm. Immature RBCs and all nucleated cells are labeled with a fluorescent nuclear stain, prior to RBC/diff using light loss and forward scatter (FSC) signal or two FSC signals and a third side scatter signal, projecting the cytogram onto a precalibrated 3D surface containing grid lines of volume (V) and hemoglobin concentration (HC), to determine the V and HC of a cell. Abnormally shaped RBCs (AbnRBC) are quantitated by the distance of each event to the 3D surface. A total of 154 normal and 484 clinical samples with various hemoglobinopathies have been analyzed. RESULTS: The 3D cytogram and the bivariate distribution of V and HC of individual event provided information to extract quantitative data on V, HC, schistocytes, and AbnRBC of mature and immature RBCs. The accuracy level of the 3D data is difficult to assess because of the semiquantitative nature of smear review, the only available reference method. CONCLUSIONS: The 3D RBC cytograms provide a wealth of morphologic information useful for diagnosis and treatment of patients. With powerful computer programs available today, it is evident that the rate of smear review can be significantly reduced as a result.