Abstract 4896: Acoustic cytometry for rare event detection of PNH cells
Authors: Bradford Jolene A. , Suter Michael 
Published: June 2012

Abstract
Rare event analysis is an area of broad interest in pathology. In hematopathology, applications include detection of residual leukemic cells in peripheral blood, bone marrow and other fluids, and levels of circulating CD34+ stem cells in blood. An additional area of recent interest is detection of small populations of neutrophils in the blood which lack surface expression of certain markers due to absence of a glycophosphatidylinositol (GPI)-anchor protein. In classic paroxysmal nocturnal hemoglobinuria (PNH), a clone of hematopoeitic stem cells arises which expands in most cases to &gt;80% of marrow cells. Small PNH-like neutrophil populations compromising 1.0 – 0.01% of neutrophils have been detected in the blood of up to 15% of patients who have myelodysplasia (MDS) and 24% of patients with aplastic anemia. These patients do not progress to classic PNH, and do not have the clinical sequelae of hemolytic anemia or thrombosis of PNH. The finding of these minor PNH-like neutrophil populations is of clinical interest because the finding correlates with increased sensitivity to immunosuppressant therapy. There are now preliminary consensus guidelines recommending routine PNH testing on all aplastic anemia and MDS patients who are candidates for therapy.
Flow cytometry is the method of choice for detection of PNH cells, made possible with technological improvements in instrumentation combined with fluorescent reagents to study GPI-linked antigens on cells. One of the practical issues complicating rare event detection and analysis is the need to acquire a very large number of events in order to obtain statistically meaningful data, which can require excessive acquisition time. The current detection limit with conventional flow cytometry using hydrodynamic focusing for rare event detection is in the range of 0.1-0.01% of nucleated cells, accumulating 1 million total events over a 60 minute interval per sample. A recent advance in cytometry instrumentation utilizes acoustic focusing, in which acoustic energy is used to precisely align cells for more sensitive detection, and sample acquisition time can be reduced by 10-fold or more. The detection limit with acoustic focusing cytometry is in the range of 0.1-0.01% of nucleated cells, accumulating 1 million total events over a 5 minute interval per sample.
We present an approach for the detection of PNH-like neutrophils using acoustic cytometry. Utilizing a panel of fluorescent reagents including CD45, CD15, CD16, CD24 and fluorescent aerolysin (FLAER) surface markers, a gating strategy is employed for high-sensitivity analysis of neutrophils using lineage markers. FLAER binds specifically to GPI anchor-deficient cells and is the most useful reagent for detecting PNH neutrohpils. Results of both normal and abnormal patient samples are shown. Acoustic cytometry provides the ability to detect PNH neutrophils by acquiring statistically significant cells for rare event detection.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4896. doi:10.1158/1538-7445.AM2011-4896