Immunostaining for Rapid Diagnosis of Acute Promyelocytic Leukemia with the Tetramethylrhodamine-5-Isothiocyanate–Conjugated Anti–Promyelocytic Leukemia Monoclonal Antibody PG-M3

Abstract

Context.-Anti-promyelocytic leukemia (PML) immunofluorescence staining is a known diagnostic tool for rapid diagnosis of acute promyelocytic leukemia (APL).Objective.-We describe our methods using the recently developed, commercially available, tetramethylrhodamine-5-isothiocyanate-labeled PG-M3 anti-PML antibody for APL testing.Design.-Immunofluorescence staining with the tetramethylrhodamine-5-isothiocyanate-labeled PG-M3 antibody was used to detect PML-RARA in bone marrow aspirate and/or peripheral blood smears from 30 patients with acute leukemia. The results were compared with those of concurrent testing with our in-house polyclonal anti-PML antibody and with established tests.Results.-All APL cases showed a positive (fine/microgranular) immunofluorescence staining pattern, whereas non-APL cases showed a negative (chunky/macrogranular) pattern. These results, which were available within 2 hours, were validated by testing with the polyclonal anti-PML antibody and with established cytogenetic and molecular testing methods.Conclusions.-We validated the utility of the tetramethylrhodamine-5-isothiocyanate-labeled anti-PML antibody PG-M3 for the diagnosis of APL. Our results indicate that immunofluorescence staining with this antibody is a rapid and reliable method for the diagnosis of APL.(Arch Pathol Lab Med. 2013;137:1669-1673; doi: 10.5858/arpa.2012-0565-OA) A cute promyelocytic leukemia (APL) is characterized by fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes, leading to expression of the aberrant PML-RARA protein. This abnormality is fundamentally important in the pathogenesis of this disease.1 Acute promyelocytic leukemia is known for a high frequency of lethal hemorrhagic complications that can be prevented by prompt administration of available effective therapy, making accurate and timely diagnosis of APL essential for a favorable outcome. The morphology of the promyelocytes in APL shows substantial variation, and other subtypes of acute myeloid leukemia (AML) can show morphologic similarities to APL.Flow cytometric immunophenotypic analysis can support the initial morphologic impression, but the immunophenotype is not completely specific and there is also immunophenotypic heterogeneity that can lead to diagnostic difficulties.3 Conventional cytogenetics, fluorescence in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) are excellent methods for detecting t(15;17)(q24.1;q21.2) or the PML-RARA fusion gene, but these methods require relatively long turnaround times and/ or a laboratory with specialized, highly skilled personnel, which are not typically available in the community setting.Another approach for diagnosis of APL is immunofluorescence staining for the PML protein. 4 This protein is crucial for the formation of PML nuclear bodies, also known as PML oncogenic domains, where PML interacts with other proteins such as p53, Daxx, Sp100, and pRb. 5Promyelocytic leukemia oncogenic domains can be visualized with the PML stain as 5 t...

Early treatment of acute promyelocytic leukaemia is accurately guided by the PML protein localisation pattern: real-life experience from a tertiary New Zealand centre

“…In a study involving 30 patients with acute leukaemia, including nine APL patients, TRITC (tetramethylrhodamine-5-isothiocyanate) conjugated PG-M3 antibody was equally effective in determining PML localisation pattern as the indirect procedure we are currently using. 27 Although immunocytochemistry is easier to examine under light microscopy and provides a permanent record, this method was less sensitive than indirect IF in earlier studies. 15,20 Multi-parameter flow cytometry is unlikely to eliminate the need for PML IF but may reduce numbers of tests being required.…”

Using Antigen Expression of Leukemic Cells for a Fast Screening of Acute Promyelocytic Leukemia by Flow Cytometry

“…A rapid diagnostic screening for APL is essential for timely starting an appropriate treatment, thus preventing complications due to bleeding. This screening is usually done by cytology and immunophenotyping, or by immunocytochemistry with anti-PML staining [16] or FISH [17], which depends on the availability of the appropriate reagents.…”

Acute Myeloid Leukemia With Recurrent Cytogenetic Abnormalities

“…a At least one genetic test required for definitive diagnosis of APL. [9][10][11][12][13] Flow cytometry immunophenotyping also provides clues to APL, but multiple features must be assessed, including high side scatter in addition to the overall immunophenotypic profile (Table 3). 11,12 If flow cytometry features prompt consideration for APL, cytospin Wright stain review along with MPO cytochemical staining should be performed ❚Image 2❚.…”

“…[9][10][11][12][13] Note that turnaround times are estimates and vary significantly based on whether the test is performed in house or is sent out. Morphology is still highly relevant in APL diagnosis and is a very rapid modality, particularly in clinical practice situations optimized for stat CBC counts and pathologist review.…”