Development of a Lateral Flow Assay for Prompt, Point of Care Diagnosis of Rapidly-Fatal Acute Promyelocytic Leukemia (APL)
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 project, we demonstrate the sensitive detection of the PML-RARα fusion nuclear protein in blood using a fluorescent lateral flow immunoassay (LFA) after simple leukocyte isolation and lysis.
Methods
Carboxylated polystyrene europium chelate nanoparticles were covalently conjugated with antibodies and used as LFA reporters to detect PML-RARα fusion nuclear oncoprotein in human NB4 APL cells spiked into whole blood. Since PML-RARα oncoprotein has a nuclear localization in the promyelocytes, cell lysis is required to access the protein and whole-cell lysates were prepared in RIPA lysis reagent, which is compatible with the downstream immunoassay. A sample pad, a conjugate pad containing immobilized detection particles, a nitrocellulose membrane containing antibody test and control lines, and an absorbent pad were assembled on an adhesive backing card. The card was dried and cut into LFA strips using a guillotine cutter and strips were stored at low humidity (<20%) until ready to use.
Results
We evaluated the performance of 86 pairs of commercially-available antibodies raised against the amino acid sequence of the fusion junction of the PML-RARα protein or alternatively against different epitopes on the N-terminus of the PML or C-terminus of the RARα protein that are expected to exist in the fusion protein. We confirmed the clinically-actionable detection of 20% leukemic cells in normal peripheral blood mononuclear cell background or whole blood using an affordable, portable fluorescence LFA reader. We confirmed assay specificity with cell lines negative for PML-RARα fusion protein, including the human AML cell line HL-60.
Conclusion
We demonstrated detection of the oncogenic PML-RARα fusion protein in blood using a rapid lateral flow immunoassay (LFA) after simple POC-compatible leukocyte isolation and lysis. 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 utilization of LFA technology in cancer diagnostics and routine patient workup.