Semiquantitative reverse transcription polymerase chain reaction analysis for detection of bcr/abl rearrangement using RNA extracts from bone marrow aspirates compared with glass slide smears after 0, 2 and 4 d of storage.
The polymerase chain reaction (PCR) is an established tool for the detection of specific chromosomal aberrations in different haematological malignancies. Owing to fast degradation of RNA, the immediate processing of samples is thought to have a major influence on the reliability of results. Any delay caused by transport may be an obstacle to reverse transcription PCR (RT-PCR)-based methods in multicentre studies. However, as air-dried bone marrow smears are usually available, we have improved a method to use smears as a source for routine RT-PCR investigations. We studied whether this source of RNA could overcome problems caused by delayed transport of samples. The aim of the present study was (i) to investigate the influence of a storage period of up to 4 d before processing of a specimen by nested bcr/abl RT-PCR, and (ii) to compare bone marrow aspirates with bone marrow smears stored at room temperature in parallel. Bone marrow aspirates and smears were taken from 11 patients with Ph-positive chronic myeloid leukaemia (CML). PCR results were semiquantified using a limiting dilution assay. We observed a loss of sensitivity < 1 log in stored bone marrow aspirates, even after 96 h. Results obtained from air-dried unstained glass slide smears were similar to investigations performed on approximately 1 x 10(5) cells of a bone marrow aspirate. We conclude that a storage period of up to 96 h has little influence on the detection of a bcr/abl fusion transcript in CML at diagnosis. Glass slide smears were equivalent to bone marrow aspirates in 8 out of 11 cases as a source for RT-PCR analysis when nested PCR was performed.
Schoch
R
R
Department of Internal Medicine II, Christian-Albrechts-University of Kiel, Germany. r.schoch@med2.uni-kiel.de
Pitako
J
J
Schafhausen
P
P
Jenisch
S
S
Haferlach
T
T
Kneba
M
M
Suttorp
M
M
D001854:Bone Marrow Cells; D002892:Chromosomes, Human, Pair 22; D002899:Chromosomes, Human, Pair 9; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D015321:Gene Rearrangement; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013997:Time Factors; D014158:Transcription, Genetic
D016044:Fusion Proteins, bcr-abl