(G-253) Prompt and Sensitive Point-of-Care Diagnosis of Acute Promyelocytic Leukemia (APL) Using a Europium Lateral Flow Assay

Introduction:: Acute Promyelocytic Leukemia (APL) is a subtype of Acute Myelocytic Leukemia (AML) characterized by rapid progression and early mortality (sometimes in days), often due to coagulopathy. The hallmark of APL is a chromosomal translocation involving the Promyelocytic Leukemia (PML) and Retinoic Acid Receptor alpha (RARα) genes, creating a PML-RARα fusion protein that inhibits the differentiation of myeloid cells and leads to accumulation of immature cells in the bone marrow. Immediate treatment with all-trans retinoic acid can drive the differentiation of immature promyelocytes and cure APL with a high survival rate (80-90%). Timely detection of APL is crucial, however, due to its rapid progression, standard molecular diagnostics such as FISH and cytogenetics have too-long turnaround times, even in major medical centers. In this study, we demonstrate rapid and sensitive detection of the oncogenic PML-RARα fusion nuclear protein in blood using a fluorescent lateral flow immunoassay (LFA) after simple leukocyte isolation and lysis.

Materials and Methods:: Carboxylated polystyrene europium chelate nanoparticles were conjugated with antibodies and used to detect PML-RARα fusion nuclear oncoprotein in human NB4 APL cells spiked into whole blood. Whole-cell lysates were prepared in RIPA lysis reagent, which is compatible with the downstream immunoassay. The LFA strips were analyzed with a portable time-resolved fluorescence LFA analyzer. A sample pad, a conjugate pad containing immobilized detection particles, a nitrocellulose membrane containing striped test and control lines, and an absorbent pad are assembled on an adhesive backing card. The card is dried and cut into strips using a guillotine cutter and strips are stored at low humidity (< 20%) until ready to use.


Results, Conclusions, and Discussions:: We evaluated the performance of 86 pairs of commercially-available antibodies recognizing different epitopes of the PML-RARα fusion protein, and confirmed the clinically-actionable detection of 20% leukemic cells in normal peripheral blood mononuclear cell background or whole blood using an affordable, portable fluorescent LFA reader. We confirmed assay specificity with cell lines negative for PML-RARα fusion protein, including human AML cell line HL-60. The assay is currently being validated with human clinical samples. Further integration with a point-of-care sample pretreatment workflow will pave the way for the utilization of LFA technology in cancer diagnostics and routine patient workup.


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