Applicability of direct In Situ reverse transcription-polymerase chain reaction on bone marrow smears

In situ reverse transciption (RT)-polymerase chain reaction (PCR) is a promising laboratory tool for biomedical investigation at the molecular level in tissues. Direct in-cell amplification of the breakpoint cluster region (BCR)-Abelson (ABL) fusion transcript of chronic myeloid leukemia (CML) has recently been accomplished in Italy using bone marrow mononuclear cell suspensions. The goals of this study are to determine if in situ RT-PCR amplification is possible on bone marrow spirate smears and to demonstrate any unique factors in this procedure. A commercially available method was used because of the existence of published protocols for adaptation. Bone marrow (BM) aspirate smears (n = 17) from patients with CML in blast crisis (positive case material) or other hematological malignancies (negative case material) were evaluated. Satisfactory amplification of the BCR-ABL fusion transcript occurred, and distinct blue cytoplasmic granules that varied in intensity were found in most CML blasts. The negative case materials lacked the specifically amplified granular signals. Overall signal strength and backgrounds were readily affected by the quality of the specimen as well as by changes in assay parameters. In conclusion, the direct in situ RT-PCR technique is applicable for bone marrow aspirate smear evaluation. However, it remains an investigative tool until optimization for sensitivity, specificity, and accuracy can be achieved.