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Aug 1, 2013 (Vol. 33, No. 14)

Hematologic Diagnostics Go with the Flow

  • Identifying Chromosomal Abnormalities

    In addition to flow cytometry, hematopathological diagnostics often involve analysis of cytogenetics—the study of chromosome structure and function. Half of all patients with myelodysplastic syndromes have clonal cytogenetic abnormalities, and the International Prognostic Scoring System includes monitoring chromosome changes in bone marrow cells to assess patient prognosis.

    To investigate the effectiveness of this technique, Ayesha Vawda, M.D., at the University of British Columbia and her colleagues at Vancouver General Hospital 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 blood cells, white blood cells, or platelets, called cytopenia, as well as abnormalities of cellular maturation and morphological abnormalities, called 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 definitive evidence of dysplasia based on a bone marrow biopsy.  Is performing cytogenetics of value in these cases?

    The laboratory had performed cytogenetics in cases where the patient's bone marrow biopsy report warranted this additional test. Dr. Vawda and her collaborators analyzed 100 hospital records of suspected myelodysplastic syndrome cases, of which cytogenetics was successfully performed in 43 patient cases. Karyotyping identified unique banding patterns of metaphase chromosomes.

    The researchers saw that abnormalities were only identified in two of the 43 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 cell lines.

    Although patient management was ultimately not altered by these results, fluorescence in situ hybridization (FISH) had been performed to confirm the presence of the deletion in the q arm of chromosome 5 for the unicytopenic patient. A locus-specific DNA probe for 5q31 (EGR1 gene) was used, along with a control probe specific to the p arm of chromosome 5.

    FISH was not performed for the confirmation of the 9:19 translocation in the pancytopenic patient, as the abnormality was clearly evident cytogenetically. Dr. Vawda explains that FISH could also be performed based on this karyotype result if the specific probes were available. As opposed to looking for loss of a fluorescent signal in the setting of a chromosomal deletion, a translocation would be identified by a fusion signal of two probes that originated from different chromosomes.

    “As only two abnormal karyotypes were reported, it appears that cytogenetics may be noncontributory to patient diagnosis and management in cases of suspected myelodysplastic syndrome 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 the findings of this study will ultimately improve workload, turn-around time, and cost efficiency.

    “Ideally, we would perform cytogenetics for all the patients who come through, but in a system with finite resources, we really need to decide if there is high yield or not for these procedures,” she says.

  • Genetic Screening via Flow Cytometry

    Click Image To Enlarge +
    Hemophagocytic lymphohistiocytosis (HLH) is commonly identified by phagocytosis of red and white blood cells, platelets, and their precursors. Hemophagocytosis is the hallmark of histiocyte (macrophages and dendritic cells) activation, as shown in these images. HLH, while rare, is one of the more common histiocyte disorders. [Cincinnati Children’s Hospital]

    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.


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