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Drug Discovery combed through patient records from the hospital's cytogenetic department database collected between 2011 and 2012. Characteristic features of myelodysplastic syndromes are a reduction in the number of red or white blood cells or platelets, called cytopenia, as well as morphological abnormalities of cellular maturation and differentiation, or dysplasia. Dr. Vawda sought to investigate the utility of cytogenetics in cases where there was a clinical suspicion of myelodysplastic syndrome because of a reduction in one, two, or all three blood cell types, but there was no defnitive evidence of dysplasia based on a bone marrow biopsy. Could cytogenetics provide a clear indication of myelodysplastic syndrome in these cases? The laboratory performed cytogenetics if a patient's bone marrow report had inconclusive results to warrant this additional test. Dr. Vawda and her collaborators analyzed hospital records of cytogenetics that were successfully performed in 37 patient cases. Karyotyping identifed unique banding patterns of metaphase chromosomes. The researchers saw that abnormalities were only identifed in two of the 37 karyotypic analyses: A 5q deletion in two of 75 metaphases from a unicytopenic patient with a reduction in one blood cell type, and a 9;19 translocation in all 20 metaphases in a pancytopenic patient with a reduction in all three blood cells. Although patient management was ultimately not altered by these results, the researchers used fuorescence in situ hybridization (FISH) to confrm the presence of a deletion in the q arm of chromosome 5 for the unicytopenic patient. A locus-specifc FISH probe for 5q31 (EGR1 gene) was used, along with a control probe specifc to the p arm of chromosome 5. It did not appear that the 9;19 translocation was confrmed via FISH in the pancytopenic patient, but Dr. Vawda explains that FISH could also be performed based on this karyotypic result, as well. As opposed to a deletion that is missing a fuorescent probe on the q but not p chromosome arm, which would appear as a loss of probe fusion because the two probes are very close to each other, a translocation would be identifed by fusion of two probes that originated from different chromosomes. "As only two abnormal karyotypes were reported, it appeared that cytogenetics is noncontributory in diagnosis and management of myelodysplastic syndrome in cases with no morphological dysplasia, despite the presence of cytopenia," Dr. Vawda concludes. The team now hopes to validate these results in a larger cohort. Dr. Vawda explains that while routine practices for cytogenetics may differ at other hospitals, Vancouver General Hospital is part of the public healthcare system, and thus deciding to perform cytogenetic testing is based largely on workload, turn-around time, and cost effciency. "Ideally, we would perform cytogenetics for all the patients who come through, but in a system with fnite resources, we really need to decide if there is high yield or not for these procedures," she says. Genetic Screening via Flow Cytometry Lisa Filipovich, M.D., studies hemophagocytic lymphohistiocytosis (HLH) and related immune disorders at Cincinnati Children's Hospital Medical Center. HLH is commonly identifed by phagocytosis of red and white blood cells, platelets, and their precursors. Dr. Filipovich runs a diagnostic lab for primary immune defciency disorders, and she and her colleagues have developed specifc assays for screening the genetic forms of HLH diagnosis, as well as to study the functional defects of these disorders. The fve subtypes of familial HLH are each associated with a specifc gene, and Dr. Filipovich's team looks for evidence of specifc intracellular, cytotoxic proteins as a screen for the genetic diagnosis of a patient. For example, one subtype of HLH is caused by a defciency in perforin, a protein that aids other cytotoxic proteins like granzyme B in entering a target cell and setting off an apoptotic cascade. Dr. Filipovich's diagnostic lab developed an assay to quantitate expression of intracellular perforin. This fow cytometry test for detecting perforin proved useful not only for rapid diagnosis, but also for screening potential family members to be donors for a bone marrow transplant, by separating carriers from unaffected individuals. The researchers have since developed similar assays for two X-linked subtypes of HLH by quantifying levels of XLP1 and XLP2, respectively. "Rather than waiting eight weeks for a genetic test, you will know in hours what the likelihood is that the patient is affected with HLH by using this fow cytometry assay," Dr. Filipovich says. "We have gone on to develop more tests of this kind so that we can focus on what are the likely genetic defects in our patients." Another fow cytometry-based assay the lab has used is a degranulation assay. Natural killer (NK) cells contain cytotoxic granules within their cytoplasm as they develop, and these granules contain "killer" proteins like perforin and granzyme B. When NK cells are prepared to kill target cells, these granules are dragged to where the immunologic synapse is forming, so that the cytotoxic cell is in direct contact with the target cell. This then stimulates the release of granular components, and researchers have identifed at least four proteins that are sequentially involved in opening the granules onto the external surface of the NK cell. The degranulation assay indicates if any of these proteins are defective, by looking on the surface of NK cells for proteins that are normally found only on the inside of the granular membrane. When the granules open, proteins like LAMP1 (CD107a), which are normally on the inner surface of granular membranes are now visible on the outside surface of the NK cells. The degranulation assay uses a monoclonal antibody to quantitate LAMP1 via fow cytometry. If there is an abnormal result, Dr. Filipovich and her colleagues will then sequence the four known genes involved in the degranulation pathway to identify which is the culprit. Therefore, this assay can point the researchers in the right direction by identifying that the patient's HLH disorder is related to defciencies in the degranulation pathway, and not X-linked genes or perforin. Dr. Filipovich explained that an alternative NK cytotoxic killing assay, which assesses the ability of cytotoxic cells to lyse chromium-labeled target cells, has many pitfalls: "Our lab has shifted to primarily using the degranulation assay because fow cytometry is much more quantitative and will lead you to the likely genetic defect. Unlike cytotoxic killing assays, the degranulation assay is not dependent on the number of NK cells in the sample," she says. "Moreover, the cytotoxic killing assay is susceptible to immunosuppressive drugs, which many patients are already receiving by the time the testing is planned." Aside from using fow cytometry as a screening tool, Dr. Filipovich and her collaborators are developing an HLH-specifc microarray chip to identify which of the seven known genetic defects might exist in a patient. On a global scale, gene microarray studies of HLH patients have also revealed that many genes involved with key pathways of innate, B cell and cytotoxic immunity are highly downregulated. REPORTS Eye on Single-Cell Analysis A thorough look into the expanding feld of single-cell analysis. Check it out and download now for FREE. This essential report, the frst of a regular series, focuses on circulating tumor cells and covers qualitative and quantitative trends in the marketplace. GENengnews.com Genetic Engineering & Biotechnology News | GENengnews.com | August 2013 | 19