Korean J Clin Pathol.  1997 Aug;17(4):668-675.

Detection of Major bcr/abl mRNA from Stored Bone Marrow Aspirate Smears Using the Reverse Transcriptase Polymerase Chain Reaction

• Lee EY
• Shin JH
• Jun ES

Abstract

BACKGROUND: The verse transcriptase polymerase chain reaction (RT-PCR) has been widely used to analyze the bcr/abl fusion mRNA in chronic myelogenous leukemia (CML). Fresh or cryopreserved cells may not always be available for molecular diagnosis. So we investigated the value of stored bone marrow aspirate smears as the sources of material for the detection of bcr/abl mRNA.
METHODS
We extracted RNA using modified Chomczynski method, and amplified bcr/abl mRNA by RT-PCR from the 70 cases of bone marrow smear slides stored from 7 days to 7 years, which were comprised of 49 CML, 11 other chronic myeloproliferative disorders (CMPD) and 10 acute lymphoblastic leukemia (ALL). Sensitivity of RT-PGR was tested using the slide smears prepared with 10(0)-10(6) K562 cells, and RT-PCR results losing each fresh bone marrow cellular suspension and slide smears in 24 patients were compacted. For major bcr/abl rearrangement, RT-PCR was performed by nested PGR afters GDNA synthesis losing downward primer and beta2-microglobulin was used as RNA controls.
RESULTS
The sensitivity of RT-PCR for detecting bcr/abl mRNA was l02 cells per slide. Sixty one cases (86%) of 70 bone marrow aspirate smears showed positive results of beta2-micyoglobulin cDNA as an indicator of intact RNA. Thirty nine cases of 42 beta2-microglobulin cDNA positive CML bone marrow aspirate smears showed 29 b3a2 type mRNA and 10 b2a2 type mRNA. Nine cases of 11 bone marrow aspirate smear with other CMPD showed negative results of bcr/abl mRNA. Two cases of 10 ALL bone mallow aspirate smears had b2a2 type mRNA and b3a2 type mRNA, respectively. The results for detection of bcr/abl mRNA with fresh cell suspensions of 24 patients were same as the bone marrow aspirate smears storied for 7 days to 1 year.
CONCLUSIONS
These data indicated that RNA obtained from bone marrow smears storied for less than 1 year was valuable as the source of RT-PGR for the detection of bcr/abl mRNA in CML and the bone marrow smears stored for much longer period ould be assailable as the specimens for retrospective analysis of specific gene alter-ation in other hematologic malignancy.

MeSH Terms

Bone Marrow*
Diagnosis
DNA, Complementary
DNA-Directed RNA Polymerases
Hematologic Neoplasms
Humans
K562 Cells
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Myeloproliferative Disorders
Polymerase Chain Reaction
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Retrospective Studies
Reverse Transcriptase Polymerase Chain Reaction*
RNA
RNA, Messenger*
RNA-Directed DNA Polymerase*
Suspensions
DNA, Complementary
DNA-Directed RNA Polymerases
RNA
RNA, Messenger
RNA-Directed DNA Polymerase
Suspensions