Rapid quantitative detection of BCR-ABL transcripts in chronic myeloid leukemia patients by real-time reverse transcriptase polymerase-chain reaction using fluorescently labeled probes.
OBJECTIVE
The limited value of qualitative reverse transcription polymerase chain-reaction (RT-PCR) for monitoring chronic myeloid leukemia (CML) patients has prompted the development of quantitative assays. We have developed a quantitative real-time PCR (QC-PCR) method in the LightCycler, based on the use of fluorescently labeled probes (HybProbes), to estimate BCR-ABL fusion gene transcripts in samples from CML patients.


METHODS
Fifty-two samples (45 peripheral blood, five bone marrow, and two apheresis product samples) from nine patients with CML were analyzed. Seven patients were studied at diagnosis and during follow-up after hematopoietic stem cell transplantation (HSCT), whereas two were evaluated only after HSCT. The PCR reaction was carried out in capillary tubes in a final volume of 10 microL, using 2 microL cDNA, the Mensik et al. primers, and two HybProbes. The results for BCR-ABL were normalized with reference to ABL. The PCR program is completed in only 45 min.


RESULTS
The sensitivity attained allowed the detection of rearrangements at dilutions of between 5-10(-4) and 10(-5) K562 cDNA. The within-assay coefficient of variation was 11% for BCR-ABL, and 9% for ABL. A greater than 2 log reduction in the BCR-ABL/ABL ratio was evident shortly after transplantation in all allografted patients.


CONCLUSIONS
We may conclude that the TaqMan probe technology can be easily adapted to HybProbes with equivalent results. Besides, the results of BCR-ABL quantification in the follow-up of patients clearly confirm that real-time PCR with HybProbes is a reliable and sensitive method for monitoring minimal residual leukemia after HSCT in CML patients.
Bolufer
P
P
Department of Clinical Pathology, Hospital Universitario La Fe, Valencia, Spain. bolufer_pas@gva.es
Sanz
G F
GF
Barragán
E
E
Sanz
M A
MA
Cervera
J
J
Lerma
E
E
Senent
L
L
Moreno
I
I
Planelles
M D
MD
D000293:Adolescent; D000328:Adult; D001853:Bone Marrow / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D005500:Follow-Up Studies; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000097:blood / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl