Sensitivity of different methods for the detection of myeloperoxidase in leukemia cells.
We examined the sensitivity of different myeloperoxidase (MPO) detection methods in leukemia cell lines. To this end the MPO-positive acute promyelocytic leukemia cell line NB-4 was diluted into cell populations of the MPO-negative myeloma cell line MM-1 at different ratios. MPO protein was identified by classical cytochemical staining and by a specific anti-MPO monoclonal antibody in an immunofluorescent reaction. Cytochemical staining detected 1% positive cells among 99% negative cells. Careful, but time-consuming observation enabled the detection of positive cells in even higher dilutions. At least a 10-fold increase in sensitivity was achieved with the immunofluorescent method, as brightly fluorescent cells are more amenable for a screening of slides at lower microscopic magnification than the cytochemically visualized cells. MPO mRNA expression was examined in whole cell populations by Northern blotting (maximal sensitivity 1%), a reverse transcriptase-polymerase chain reaction (RT-PCR) amplification assay (sensitivity 0.1%), and by RT-PCR followed by Southern blotting (sensitivity 0.05%). The high sensitivity of PCR-based techniques is offset by the fact that these methods do not allow for the identification and further characterization of the individual, MPO-positive cells. Thus, methods examining bulk populations require homogeneous cell samples in order to avoid false-positivity stemming from a few residual bystander cells. The five different techniques were used to determine the status of MPO expression in 20 randomly chosen leukemia cell lines of myelomonocytic origin. In 11 cell lines (8 positive and 3 negative) all five tests provided concordant results. Three cell lines were Northern-negative, but RT-PCR-positive and MPO protein-positive suggesting that Northern blot analysis is the least sensitive tool. Six cell lines were devoid of MPO protein, at least according to the methods used here, but trace expression of MPO message was documented by PCR. All five techniques have advantages and drawbacks and must be carefully selected in order to obtain useful data. The detection of MPO is of experimental and clinical importance in the distinction of myeloid from lymphoid leukemias, and in the lineage assignment of apparently biphenotypic or unclassifiable cases.
Ma
W
W
DSM, German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig.
Hu
Z B
ZB
Drexler
H G
HG
D015152:Blotting, Northern; D015139:Blotting, Southern; D005455:Fluorescent Antibody Technique; D006651:Histocytochemistry; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology*; D009101:Multiple Myeloma / Q000201:enzymology; D009195:Peroxidase / Q000032:analysis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000201:enzymology
D012333:RNA, Messenger; D009195:Peroxidase