FLOW CYTOMETRY-BASED ASSAY FOR DETECTION OF BCR-ABL FUSION PROTEIN IN BLOOD CELLS FROM CML PATIENTS
(Abstract release date: 05/21/15)
EHA Library. Löf L. 06/12/15; 102807; PB1752
Disclosure(s): Uppsala UniversityDepartement of Immunolgy, Genetics and Pathology
Liza Löf
Contributions
Contributions
Abstract
Abstract: PB1752
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is currently diagnosed using RT-PCR and/or FISH to reveal the presence of the fusion mRNA transcripts for BCR-ABL, or of the characteristic Philadelphia chromosome. RT-PCR is also used to monitor the effects of treatment by sensitively measuring transcripts representing minimal residual disease (MRD). It has not been possibly to use flow cytometry to identify the neoplastic cells but such a method would be helpful in the workflow of a hematopathology lab.
Aims
We have now developed a method that successfully detects and enumerates cells harboring the fusion protein BCR-ABL by flow cytometry in CML patients.
Methods
The method uses the in situ proximity ligation assay (PLA) (Söderberg et al 2006, Leuchowius et al 2009), where two antibodies target the BCR and the ABL part, respectively, of the fusion protein. The antibodies are equipped with DNA oligonucleotides that – when brought in proximity – guide the formation of a circular DNA molecule as a template for localized DNA amplification through rolling circle amplification (RCA). Each RCA-product is then labeled with around 1,500 fluorophore-coupled DNA oligonucleotides, allowing cells to be detected by flow cytometry.
Results
The method has proven very sensitive, able to detect very low number of cells in patient samples, and it allows identification of patients in relapse at a very early state. We have analyzed 25 CML patient samples using PLA-based flow cytometry, and the results were compared to the routine RT-PCR analysis. More than 50% of the analyzed samples were positive for both methods, ranging from 75 IS% down to 0.03 IS% for the RT-PCR. Five of the samples were positive only in RT-PCR, while three were positive only in the PLA method.
Summary
Since the method is developed for readout with flow cytometry all the advantages with multicolor fluorescence analysis can be achieved simultaneously. In addation, the method allows for standard cytological markers to further characterize the malignant cells.
Session topic: Publication Only
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is currently diagnosed using RT-PCR and/or FISH to reveal the presence of the fusion mRNA transcripts for BCR-ABL, or of the characteristic Philadelphia chromosome. RT-PCR is also used to monitor the effects of treatment by sensitively measuring transcripts representing minimal residual disease (MRD). It has not been possibly to use flow cytometry to identify the neoplastic cells but such a method would be helpful in the workflow of a hematopathology lab.
Aims
We have now developed a method that successfully detects and enumerates cells harboring the fusion protein BCR-ABL by flow cytometry in CML patients.
Methods
The method uses the in situ proximity ligation assay (PLA) (Söderberg et al 2006, Leuchowius et al 2009), where two antibodies target the BCR and the ABL part, respectively, of the fusion protein. The antibodies are equipped with DNA oligonucleotides that – when brought in proximity – guide the formation of a circular DNA molecule as a template for localized DNA amplification through rolling circle amplification (RCA). Each RCA-product is then labeled with around 1,500 fluorophore-coupled DNA oligonucleotides, allowing cells to be detected by flow cytometry.
Results
The method has proven very sensitive, able to detect very low number of cells in patient samples, and it allows identification of patients in relapse at a very early state. We have analyzed 25 CML patient samples using PLA-based flow cytometry, and the results were compared to the routine RT-PCR analysis. More than 50% of the analyzed samples were positive for both methods, ranging from 75 IS% down to 0.03 IS% for the RT-PCR. Five of the samples were positive only in RT-PCR, while three were positive only in the PLA method.
Summary
Since the method is developed for readout with flow cytometry all the advantages with multicolor fluorescence analysis can be achieved simultaneously. In addation, the method allows for standard cytological markers to further characterize the malignant cells.
Session topic: Publication Only
Abstract: PB1752
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is currently diagnosed using RT-PCR and/or FISH to reveal the presence of the fusion mRNA transcripts for BCR-ABL, or of the characteristic Philadelphia chromosome. RT-PCR is also used to monitor the effects of treatment by sensitively measuring transcripts representing minimal residual disease (MRD). It has not been possibly to use flow cytometry to identify the neoplastic cells but such a method would be helpful in the workflow of a hematopathology lab.
Aims
We have now developed a method that successfully detects and enumerates cells harboring the fusion protein BCR-ABL by flow cytometry in CML patients.
Methods
The method uses the in situ proximity ligation assay (PLA) (Söderberg et al 2006, Leuchowius et al 2009), where two antibodies target the BCR and the ABL part, respectively, of the fusion protein. The antibodies are equipped with DNA oligonucleotides that – when brought in proximity – guide the formation of a circular DNA molecule as a template for localized DNA amplification through rolling circle amplification (RCA). Each RCA-product is then labeled with around 1,500 fluorophore-coupled DNA oligonucleotides, allowing cells to be detected by flow cytometry.
Results
The method has proven very sensitive, able to detect very low number of cells in patient samples, and it allows identification of patients in relapse at a very early state. We have analyzed 25 CML patient samples using PLA-based flow cytometry, and the results were compared to the routine RT-PCR analysis. More than 50% of the analyzed samples were positive for both methods, ranging from 75 IS% down to 0.03 IS% for the RT-PCR. Five of the samples were positive only in RT-PCR, while three were positive only in the PLA method.
Summary
Since the method is developed for readout with flow cytometry all the advantages with multicolor fluorescence analysis can be achieved simultaneously. In addation, the method allows for standard cytological markers to further characterize the malignant cells.
Session topic: Publication Only
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is currently diagnosed using RT-PCR and/or FISH to reveal the presence of the fusion mRNA transcripts for BCR-ABL, or of the characteristic Philadelphia chromosome. RT-PCR is also used to monitor the effects of treatment by sensitively measuring transcripts representing minimal residual disease (MRD). It has not been possibly to use flow cytometry to identify the neoplastic cells but such a method would be helpful in the workflow of a hematopathology lab.
Aims
We have now developed a method that successfully detects and enumerates cells harboring the fusion protein BCR-ABL by flow cytometry in CML patients.
Methods
The method uses the in situ proximity ligation assay (PLA) (Söderberg et al 2006, Leuchowius et al 2009), where two antibodies target the BCR and the ABL part, respectively, of the fusion protein. The antibodies are equipped with DNA oligonucleotides that – when brought in proximity – guide the formation of a circular DNA molecule as a template for localized DNA amplification through rolling circle amplification (RCA). Each RCA-product is then labeled with around 1,500 fluorophore-coupled DNA oligonucleotides, allowing cells to be detected by flow cytometry.
Results
The method has proven very sensitive, able to detect very low number of cells in patient samples, and it allows identification of patients in relapse at a very early state. We have analyzed 25 CML patient samples using PLA-based flow cytometry, and the results were compared to the routine RT-PCR analysis. More than 50% of the analyzed samples were positive for both methods, ranging from 75 IS% down to 0.03 IS% for the RT-PCR. Five of the samples were positive only in RT-PCR, while three were positive only in the PLA method.
Summary
Since the method is developed for readout with flow cytometry all the advantages with multicolor fluorescence analysis can be achieved simultaneously. In addation, the method allows for standard cytological markers to further characterize the malignant cells.
Session topic: Publication Only
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