Evaluation of PML immunofluorescence, flow cytometric immunophenotypic analysis, and reverse transcriptase polymerase chain reaction for PML/RARa for rapid diagnosis of acute promyelocytic leukemia


We read with great interest the article entitled “Rapid and reliable confirmation of acute promyelocytic leukemia by immunofluorescence staining with an antipromyelocytic leukemia antibody: the M.D. Anderson Cancer Center experience of 349 patients” by Dimov et al.1

We herein share our experience with PML immunofluorescence (IF), reverse transcriptase polymerase chain reaction (RT-PCR), and flow cytometric (FCM) immunophenotyping in the diagnosis of acute promyelocytic leukemia (APL). Between February 2008 and November 2009, 103 acute nonlymphocytic leukemia cases were analyzed, after obtaining informed consent. We performed RT-PCR for t(15;17), FCM immunophenotyping using a panel of 24 monoclonal antibodies, and PML immunofluorescence using monoclonal anti-PML antibody (PG-M3 clone). RT-PCR for t(15;17) was positive in 28 of 103 cases (long form in 13 and short form in 15). Twenty-seven cases had classical APL morphology. PML immunofluorescence staining revealed a nuclear microgranular staining pattern in 27 of 28 cases. Flow cytometry, performed in 16 of 28 cases, showed typical APL phenotype (CD34−, HLA-DR−, CD13+, CD33+) in 13.

In 75 cases, RT-PCR was negative for t(15;17) and showed a speckled staining pattern. Morphologic diagnoses ranged from M0 to M7. FCM immunophenotyping, performed in 41 of 75 cases, showed an immunophenotype suggestive of non-APL in 36. Considering RT-PCR as the gold standard, PML immunofluorescence had 96.4% sensitivity and 100% specificity; FCM immunophenotyping had 81.25% sensitivity and 87.8% specificity. This is in close agreement with the sensitivity and specificity of PML immunofluorescence testing of 98.9% and 98.7%, respectively, reported by Dimov et al1 using a polyclonal anti-PML antibody.

In our experience, time required was 2 to 4 hours for the immunostaining procedure, 12 to 24 hours for RT-PCR, and 3 to 5 hours for FCM immunophenotyping.

Dimov et al studied a much larger number of APL cases.1 However, ours is perhaps the first study conducted in a resource-constrained setup to evaluate the role of PML immunofluorescence, RT-PCR, and FCM immunophenotyping for rapid diagnosis of APL. These results have been briefly communicated previously.2

PML immunofluorescence was found to be a simple and rapid technique to detect t(15;17), having good concordance with the gold standard RT-PCR. It should be applied as a first-line tool to detect APL cases, especially in under-resourced countries.

Neelam Varma MD*, Chetna Agarwal MD*, Subhash Varma MD†, * Department of Hematology, Postgraduate Institute of Medical Education & Research, Chandigarh, India, † Department of Internal Medicine, Postgraduate Institute of Medical Education & Research, Chandigarh, India.