Ongoing
ResearchOngoing
Research
BASIC RESEARCH PROGRAM
Molecular Phenotyping by Flow Cytometry: Flow cytometry has been the method of choice for identifying and quantifying the binding of fluorochrome labeled antibodies to specific protein antigens on the membrane or inside of cells. The methodology to do this is well established, reliable and has important implications in both the research and clinical environments.
The polymerase chain reaction (PCR) has become an indispensable tool in the field of molecular biology. Starting with a minute amount of DNA or RNA, the PCR can amplify a specific nucleic acid sequence to the point where it can be easily detected on an ethidium bromide stained agarose gel. This completely eliminates the need for radioactive probes for detection. Many variations of this procedure have been developed such as inverse PCR, anchored PCR and asymmetric PCR to accommodate the needs of individual researchers. For evaluating gene expression, reverse transcription-PCR (RT-PCR) is used. Gene amplification or expression can then be quantified using competitive PCR or the new automated from . The major disadvantage, however, of all of these technologies is that once the RNA or DNA is extracted from a heterogeneous cell population, it becomes extremely difficult to relate the data obtained by PCR to the specific cells from which the product was derived.
Our strategy is to use multiparameter flow cytometry to identify and sort specific populations of cells out of a heterogeneous cell population for further analysis by PCR. The use of PCR to amplify the DNA or RNA of interest is highly advantageous since very few cells are required for analysis. Thus, by combining immunophenotyping with molecular biology, quantitative analysis of gene amplification and expression can be achieved in a specific cell population. Even though further development is necessary, it also seems reasonable that direct molecular phenotyping on a cell by cell basis utilizing fluorescence in situ hybridization (FISHES) or in situ PCR (FLIP) can be accomplished with high sensitivity using fluorochrome labeled probes.
Cytokine Receptor Expression on Hematopoietic Progenitor Cells: Hematopoietic cells are derived from a small pool of primitive progenitor cells and differentiate from multipotential stem cells into committed progenitor cells that expand to functional end cells. Antibodies have been produced to some of the proteins on the membrane of hematopoietic cells. Since a unique repertoire of proteins is displayed for each subset along the differentiation pathway, we are able to separate and identify specific cell populations using Flow Cytometry and sorting.
Hematopoietic cells are responsive to a number of cytokines. We hypothesize that hematopoietic cell subsets express a unique repertoire of cytokine receptors whose engagement by ligand regulates both proliferation and differentiation. This regulation can be directed by altering the combination of cytokines within the cell's immediate microenvironment. We have developed a process to simultaneously immunophenotype and molecular phenotype cells to evaluate cytokine receptor and ligand expression.
INSTRUMENTATION RESEARCH AND DEVELOPMENT
We have an active R & D program in collaboration with the research service component of BDIS. Our main goal is to improve instrument performance.
Sorted Cell Recovery: In the past, the emphasis of flow cytometry sorting has been on purity. There was little concern for recovery, which we define as the number of desired cells found in the sort tube divided by the number present in the sample tube. Our experience has been poor, as recovery is consistently lower than 30%. Based on the realization that recovery may be the most important endpoint, new high speed pulse processing electronics have been built. Recovery is consistently better than 80% and accountability is better than 95%. Accountability means knowing where every cell has gone, i.e. not detected, detected but aborted, detected and interrogated. One problem with stream in air detection is loss of sensitivity. Up to now this loss has been balanced by the advantage of easier sorting. Over the years, improvements in flow cell design and droplet generation have provided us the opportunity to develop a liquid in liquid flow cell for high speed sorting and enhanced sensitivity.
We are also developing a monitoring
system for droplet breakoff. This has two important features: it will terminate
the sort if the flow cell becomes obstructed and it will automatically
maintain the droplet breakoff at ± 0.1 drops. This will allow for
unattended sorting.
