Major Interference from Leukocytes in Reverse Transcription-PCR Identified as Neurotoxin Ribonuclease from Eosinophils: Detection of Residual Chronic Myelogenous Leukemia from Cell Lysates by Use of an Eosinophil-depleted Cell Preparation

Abstract

AbstractBackground: The extraction of RNA from leukocytes for reverse transcription-PCR (RT-PCR) is time-consuming and contributes to variation in analysis of the Philadelphia (Ph1) chromosome of chronic myelogenous leukemia (CML) by RT-PCR. To detect residual CML after bone marrow transplantation, mRNA from at least 105 leukocytes should be analyzed, but the RNase activity of the cells precludes simple leukocytes lysis as an alternative to RNA extraction. We sought to identify the main source of RNase activity of leukocytes.Methods: We used a three-step chromatographic process and amino acid sequence analysis. We selected eosinophil-free granulocytes by using a biotinylated CD16 antibody and selected mononuclear cells by fractionating the leukocytes with a Ficoll-Paque® density gradient.Results: Chromatography and amino acid sequencing identified eosinophil-derived neurotoxin (EDN) as the main source of leukocyte RNase. Depletion of eosinophils reduced the EDN content of cell lysates by ∼90%, allowing a signal from a lysate of 50 K562 Ph1-positive cells mixed with 105 CD16+ granulocytes that was equivalent to 77% of the signal in the absence of leukocytes. A similar lysate with mononuclear cells gave a signal equivalent to 53% of that without mononuclear cells. RNA extraction gave a signal equivalent to only 24% of the leukocyte-free control.Conclusion: The depletion of eosinophils during the preparation of leukocyte samples for RT-PCR efficiently reduces the risk of mRNA degradation by ribonucleases, enabling RT-PCR analysis directly from cell lysates with a better signal than can be obtained by RNA extraction.