PML protein analysis using imaging flow cytometry

Abstract

Acute promyelocytic leukaemia (APML) can be promptly diagnosed by detecting abnormal diffuse staining patterns of PML bodies in abnormal promyelocytes using immunofluorescence microscopy. However, this technique is subjective, with low sensitivity. Using samples from 18 patients with acute myeloid leukaemia (AML) (including four with APML), the authors investigated whether imaging flow cytometry could be a viable alternative to this current technique and improve sensitivity levels. Bone marrow/peripheral blood cells were stained with an antibody to PML, and data were acquired on an ImageStream (Amnis Corporation, Seattle, Washington, USA). Using the modulation feature for data analysis, the authors demonstrated that this technique could successfully identify cases of APML. Imaging flow cytometry, by analysing greater numbers of cells and with the potential to include disease-specific antigens, increases the sensitivity of the current immunofluorescence technique. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of haematological malignancies, including the potential to integrate modalities.

Protein Analytical Assays for Diagnosing, Monitoring, and Choosing Treatment for Cancer Patients

“…Many hospitals have access to flow cytometers with as many as 8 colors to simultaneously assess multiple biomarkers in samples [15,17]. Flow cytometry is less subjective [27] and requires fewer cells than immunohistochemistry [14]. It can also detect as few as 1/10,000 cancer cells, and can be performed in a few hours and more cheaply than immunohistochemistry [15,27,28].…”

“…Flow cytometry is less subjective [27] and requires fewer cells than immunohistochemistry [14]. It can also detect as few as 1/10,000 cancer cells, and can be performed in a few hours and more cheaply than immunohistochemistry [15,27,28]. It is commonly used to diagnose chronic lymphocytic leukemia [19] and a variety of studies have been performed on its use in other leukemias and lymphomas [15,17,27–29].…”

“…It can also detect as few as 1/10,000 cancer cells, and can be performed in a few hours and more cheaply than immunohistochemistry [15,27,28]. It is commonly used to diagnose chronic lymphocytic leukemia [19] and a variety of studies have been performed on its use in other leukemias and lymphomas [15,17,27–29]. However, flow cytometry is capital equipment intensive, and requires cell singularization, limiting its usefulness for solid tumors [30].…”

“…Patients with a low CD38 + population but high CD38 expression per cell had a similar poor prognosis to patients with a high CD38 + population. Another group used image flow cytometry, in which a digital image is taken of the cells in addition to the standard cell counting, to detect promyelocytic leukemia protein and diagnose acute promyelocytic leukemia [27]. Previously promyelocytic leukemia protein bodies could not be assessed using flow cytometry because of the inability to view cell staining patterns [27].…”

“…Another group used image flow cytometry, in which a digital image is taken of the cells in addition to the standard cell counting, to detect promyelocytic leukemia protein and diagnose acute promyelocytic leukemia [27]. Previously promyelocytic leukemia protein bodies could not be assessed using flow cytometry because of the inability to view cell staining patterns [27]. Another advance in flow cytometric cancer detection has been the identification of sets of biomarkers that predict cancers more efficiently than a single biomarker.…”