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 identified by phagocytosis of red and white blood cells, platelets, and their precursors. Dr. Filipovich runs a diagnostic lab for primary immune deficiency disorders, and she and her colleagues have developed specific assays for screening the genetic forms of HLH diagnosis, as well as to study the functional defects of these disorders.
The five subtypes of familial HLH are each associated with a specific gene, and Dr. Filipovich’s team looks for evidence of specific intracellular, cytotoxic proteins as a screen for the genetic diagnosis of a patient. For example, one subtype of HLH is caused by a deficiency 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 flow 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 flow 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 flow 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 identified 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 flow 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 deficiencies 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 flow 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 flow cytometry as a screening tool, Dr. Filipovich and her collaborators are developing an HLH-specific 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.