Variability in the levels of PML-RAR alpha fusion transcripts detected by the laboratories participating in an external quality control program using several reverse transcription polymerase chain reaction protocols.
OBJECTIVE
The detection of PML-RAR by reverse transcription (RT) polymerase chain reaction (PCR) in acute promyelocytic leukemia (APL) patients who are in hematologic remission influences therapeutic decision making in several trials. In the light of this, the Spanish group has recently designed an external quality assessment program (EQAP) of RT-PCR detection of PML-RAR, which includes a study of sensitivity of the participating laboratories.


METHODS
Eighteen laboratories were involved in the program. Ten laboratories followed the method of Biondi et al., 5 employed that of Borrow et al. and the 3 remaining used other protocols. The sensitivity was studied in five rounds of quality control. The first two shipments consisted of dilutions of NB4 RNA into non-APL RNA. The third round consisted of serial dilutions of the NB4 cell line into HL60 cells. The fourth and five rounds consisted of plasmid dilutions containing the bcr1 and bcr3 PML-RAR isoforms.


RESULTS
The results showed that the distinct methods allow detection of the PML-RAR hybrid up to a dilution of 10(-4), and exceptionally, up to 10(-5). The laboratories following the method of Biondi et al. usually detected the 10(-3) dilution and less frequently the 10(-4) one, whereas those using other methods usually detected PML-RAR transcript in the 10(-4) dilution, and less commonly in the 10(-5) dilution. However, each of the PCR methods used by EQAP participating laboratories successfully detected at least 50 copies of PML-RAR alpha fusion transcript in plasmid dilution controls.


CONCLUSIONS
The results point to heterogeneous sensitivity amongst participating laboratories. This may reflect differences in methodology, although variations in sample quality may also account for discrepant findings.
Bolufer
P
P
Laboratory of Molecular Biology, Department of Clinical Pathology, Hospital Universitario La Fe, Valencia, Spain. bolufer_pas@gva.es
Lo Coco
F
F
Grimwade
D
D
Barragán
E
E
Diverio
D
D
Cassinat
B
B
Chomienne
C
C
Gonzalez
M
M
Colomer
D
D
Gomez
M T
MT
Marugan
I
I
Román
J
J
Delgado
M D
MD
García-Marco
J A
JA
Bornstein
R
R
Vizmanos
J L
JL
Martinez
B
B
Jansen
J
J
Villegas
A
A
de Blas
J M
JM
Cabello
P
P
Sanz
M A
MA
D006801:Humans; D007753:Laboratories / Q000592:standards*; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics; D015588:Observer Variation; D015514:Oncogene Proteins, Fusion / Q000032:analysis* / Q000235:genetics; D011786:Quality Control; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein