Flow cytometric immunobead assay for fast and easy detection of PML–RARA fusion proteins for the diagnosis of acute promyelocytic leukemia

Abstract

The PML–RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML–RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4–5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML–RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML–RARA immunobead assay showed full concordance with the PML–RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.

The CD9+CD11b−HLA‐DR− immunophenotype can be used to diagnose acute promyelocytic leukemia

“…According to differences in SSC, APL can be divided into different types: high SSC, low SSC, and medium SSC. The high SSC type is the common clinical type; in the present study, it accounted for 82% of the total APL in this study group, similar to previous reports . The low SSC type accounted for 15% of APL cases, consistent with previous reports.…”

“…The high SSC type is the common clinical type; in the present study, it accounted for 82% of the total APL in this study group, similar to previous reports. 13 The low SSC type accounted for 15% of APL cases, consistent with previous reports. These promyelocytic cells are often similar in location CD9 is helpful in APL identification aside from CD34 and HLA-DR, and should be included in routine screening panels.…”

“…Most cases of APL are relatively easy to diagnose because of their typical karyotype, fusion gene, and relative morphological features (such as their rod shape). However, some APL cases are not easily diagnosed because they have no typical genetic alteration …”

“…However, some APL cases are not easily diagnosed because they have no typical genetic alteration. 12,13 According to differences in SSC, APL can be divided into different types: high SSC, low SSC, and medium SSC. The high SSC type is the common clinical type; in the present study, it accounted for 82% of the total APL in this study group, similar to previous reports.…”

Immunophenotyping of Acute Leukemias – From Biology to Clinical Application

“…In the field of genotype prediction, a novel application of flow cytometry (FC) based upon an immunobead assay has been developed. This kind of test is able to detect the presence of chimeric proteins coded by relative fusion genes such as PML/RARalfa and BCR/ABL and, obviously, has the advantage to provide earlier results compared to PCR [18,19].…”