Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D; community with critical information on the tools, technologies, and trends that drive the biotech industry.
Issue link: http://gen.epubxp.com/i/145822
Drug Discovery Hematologic Dx Continued from page 16 clearly two different malignant populations. From 2,600 samples of blood, bone marrow, fne needle aspiration, and tumor cell suspensions, the researchers identifed 43 samples showing two abnormal lymphoid populations—40 of which had two aberrant B-cell populations, the remaining three showing one B- and one T-cell aberrant population. "Previously, we were using a fve-color fow cytometer, but switching to the 10-color panel has allowed for increasingly more accurate diagnostics, as these lymphoma cases with two aberrant cell populations were easier to detect by identifying many more parameters for each cell," Dr. Porwit says. "Additionally, this improves the accuracy of our data: Instead of doing several different assays in multiple tubes and attempting to compare the tubes to each other, now we have everything in one assay." Dr. Porwit explains that this 10-color fow cytometry method is an important advancement in cellular analysis, not only because of the machinery, but also the types of software for analyzing the data. The Kaluza® software provides easy protocols for analyzing this complex data of 12 parameters per cell: 10 antibodies, cell size, and granularity. She also notes, though, that it is critical to monitor fuorescence compensation during data analysis, by calculating the amount of interference of a fuorochrome's emission profle in a different channel. Ideally, fuorochromes at different ends of the spectrum should be used so that there is no spillover, but this is not always practical with analysis of increasing numbers of antibodies. Dr. Porwit will continue to use 10-color flow cytometry for hematopathological diagnosis, especially myelodysplastic syndrome. She tells GEN that a research initiative through the International Council for Standardization in Hematology is currently establishing guidelines for identifying malignancies in acute myeloid leukemia using fow cytometry methodologies. after therapy initiation—in order to quantify residual tumor cells. He noted that minimal manipulation of the sample is ideal, as certain preparation methods may in fact lead to a loss of the abnormal cell population in the sample. Samples should be prepared at a higher concentration than what is normally used for fow cytometry; if a sensitivity of 0.01% is target, at least 1 x 106 cells should be collected. Dr. Keeney and his colleagues are working with a quality assurance group from the U.K. called NEQAS, which provides blood samples containing small numbers of tumor cells to participating labs, so that researchers can test their samples against these controls. Additionally, Dr. Keeney is collaborating with the NIH on a recent initiative to investigate standardization methods for detecting these small numbers of residual tumor cells. As a form of Flow Techniques for Counting Cells intralab quality control, he has also sent many fow cytometry reference images to new labs utilizing these procedures, so that other researchers can understand the differences between normal and abnormal cell populations. Dr. Keeney indicates that rare event detection has its limitations—both statistical and bench work expertise are required to effectively identify these cell populations. However, fow cytometry still has many advantages, he notes. "Flow cytometry is well-suited for rare event analysis in different hematological diseases. Compared to PCR or genome sequencing, it remains the least expensive method for identifying minimal residual disease, and it is a speedy process—results are obtained in the same day as running the sample," he says. Rare event detection has mostly been used in acute lymphoblatic leukemias, but the methodology is now being applied to adults with acute myeloid leukemias. Similar to Dr. Porwit's studies, Dr. Keeney has also utilized 10-color fow cytometry to study blood samples from these leukemia patients, as it is much harder to clearly identify abnor- Michael Keeney, M.D., from the London Health Sciences Centre is working to improve fow cytometry methods in cases where cell counts are particularly low. Dr. Keeney has collaborated with breast cancer researchers to detect circulating tumor cells in peripheral blood at 1 cell/mL. He has also worked on the detection of minimal residual disease in pediatric patients with acute lymphoblastic leukemia after induction therapy. Dr. Keeney tells GEN that rare event detection of acute lymphoblastic leukemia is critical for re-stratifying a patient group based on their response to a particular therapy. These small populations of residual tumor cells, which may be on the order of one in 10,000 cells, establish the patient's prognosis and will inform the clinician regarding whether or not to alter the treatment. Leukemic patients may have up to 1012 leukemic cells, and remission can be identifed as up to 1010 residual leukemic cells. In his study, Dr. Keeney examined antigen expression in the remission state—i.e., 29 days 18 | mal from normal cells in these patients. The testing is also being rolled out to a broader patient set, such as a chronic lymphocytic leukemia group in Europe. According to Dr. Keeney, "We will continue to make improvements to the standardization protocols over the next few years. In the meantime, any labs performing these methods should participate in the quality assurance program." Along with their colleagues at Abbott Laboratories and South Western Area Pathology Service Liverpool Hospital, Dr. Keeney and London Health Sciences Centre's Benjamin Hedley, M.D., are investigating standardization and validation methods for counting reticulated platelets. Reticulated blood cells and platelets are useful tools for differentiating causes of anemia, but fewer laboratories Hemophagocytic lymphohistiocytosis (HLH) is commonly identifed 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 August 2013 | GENengnews.com | Genetic Engineering & Biotechnology News utilize reticulated platelet enumeration. Dr. Hedley explained that a major application for counting reticulated platelets is to monitor the effciency of certain drugs that mimic the body's natural ability to make platelets. "We need to be able to identify early on which patients are responding to these drugs," he says. "This requires determining if the patient is producing more platelets, is not producing more platelets, or is producing platelets that are somehow getting destroyed by another factor. By measuring the number of immature, reticulated platelets, this tells us if the drug is helping the patient produce platelets in the bone marrow, whether or not they may be destroyed later on." However, the problem remained that there was no standard method for enumerating immature platelets newly released from the bone marrow. To tackle this issue, Dr. Hedley and his team utilized a previously published flow cytometric method from 1990 for analyzing reticulated platelets using Thiazole Orange dye, and combined this with a CD41/CD61 (platelet glycoproteinsIX and IIIa) platelet enumeration method that was standardized by the International Society for Laboratory Hematology. "The main advantage of combining the platelet enumeration method with Thiazole Orange is that the number of platelets and percentage of immature platelets is obtained at once from the same experimental tube," Dr. Hedley reprots. The samples were analyzed using Kaluza fow cytometry software from Beckman Coulter. Dr. Hedley explains that Thiazole Orange, which is a fuorochrome that binds nucleic acids, distinguishes reticulated platelets because mature platelets have lost their RNA content. He also notes that using an eight- or 10-color fow cytometer is not necessary for counting platelets; rather, one fuorochrome to identify platelets and another to identify if they are immature or mature platelets is suffcient. He and his collaborators will be comparing their methodology using different instruments, such as two-color Beckman Coulter and BD fow cytometers. Dr. Hedley and his team determined that a fnal concentration of 10% of the initial Thiazole Orange used was suffcient for staining reticulocytes for 30 minutes, could be quenched with formaldehyde, and was stable for one hour. "Stability is key. Clinically, we cannot expect to run our samples within minutes of being prepped. The samples are prepared all at once but need to be stable until each has been run through the fow cytometer," he says. The researchers used normal donor samples to establish a normal range of reticulated platelets for each assay. However, Dr. Hedley notes that they proceeded with caution when evaluating blood work from donors not subject to any other treatments or medication. For example, some donors had normal blood work, but upon closer investigation, their immature platelet fraction was not normal because the donors had undergone chemotherapy a few weeks prior. Hematology counters that can screen complete blood counts do not accurately measure low numbers of platelets, and immature platelets are also larger than mature platelets and may fall outside of the set size or volume dimensions recognized by hematology analyzers. Instead, fow cytometric methods rely on antibodies and thus can still accurately count larger, immature platelets in low quantities. Identifying Chromosomal Abnormalities In addition to fow cytometry, hematopathological diagnostics often involve analysis of cytogenetics—the study of chromosome structure and function. Half of all patients with myelodysplastic syndrome have abnormal karyotypes, and the International Prognostic Scoring System includes monitoring chromosome changes in bone marrow cells to assess the severity of a patient's disease state. To investigate the effectiveness of this technique, Ayesha Vawda, M.D., at the University of British Columbia and her colleagues at Vancouver General Hospital