Contributions
Type: Publication Only
Background
Emerging data strongly indicate that quantification of Minimal Residual Disease (MRD) in Chronic Lymphocytic Leukemia (CLL) appears to be a powerful predictor of progression-free and overall survival, regardless of the clinical response, type of therapy and known biological markers. Two methods are currently available for assessing complete remission according to the international consensus and IWCLL guidelines, i.e. allowing to detect one malignant cell in 10.000 leukocytes. These approaches are either based on specific immunoglobulin heavy chain rearrangement in each clone (RQ-ASO IgH-PCR) or on the unique immunophenotype profile of CLL cells using multiparameter flow cytometry.
Aims
Our aim was to assess complete remission in CLL on Reunion Island. The assay had to be not as complex, expensive and time-consuming as previously published techniques. This study describes the different steps in implementing and optimising an eight-colour flow cytometry MRD assay in peripheral blood.
Methods
Our work is based on an antibody panel designed by the ERIC expert group (www.ericll.org) including the following combination: CD81/CD79b/CD22/CD19/CD43/CD20/CD5/CD3. The malignant clone is identified among normal B cells thanks to 15 scattergrammes combining six CLL highly characteristic markers. Tumor cells typically express CD5, dim CD22, dim CD20, dim or negative CD79b, CD43, dim or negative CD81. In order to reach a higher specificity and sensibility for detection of CLL cells, we have been working on three different approaches: modification of sample preparation procedure, introduction of a new brilliant fluorochrome for the violet laser line and optimisation of data analysis strategy. Once the implementation of the assay had been carried out, we studied performance criteria according to learned societies, considering clinical relevance and practical feasibility.
Results
After modifying the eight-colour protocol, we achieved a precise estimation of the number of total leukocytes events by excluding unlysed cells (SSC/CD43 dot plot), erythrocytes debris (CD81/CD19 dot plot) and doublets (FSC-A/FSC-H dot plot). This new gating algorithm allows us to calculate the number of CLL cells events as a percentage of total leukocytes, according to international guidelines. The intersection of 16 regions of interest contribute further to clearly define the CLL population by minimizing the error of inclusion risk. Analysis of ten healthy donor blood has been carried on to assess the limit of detection, which is 5.3. The application of this new data analysis strategy achieves a sensitivity of 5x10-6 after acquiring one million leukocytes. Performance criteria proved mostly satisfying: imprecision, accuracy, carryover and specimen stability meet the standards we set.
Summary
We have developed on Reunion Island a flow cytometric technique reliable and sensitive enough to detect the presence of less than one CLL cell in 100.000 leukocyte. This 8-colour assay reduces subjectivity, sample requirements and time taken for acquisition and analysis, compared with 4 and 6-color assays. It also presents the advantage of requiring less specialized equipment and being not as expensive as the sensitive PCR methods.
Keyword(s): Chronic lymphocytic leukemia, Cytometry, Minimal residual disease (MRD)
Session topic: Publication Only
Type: Publication Only
Background
Emerging data strongly indicate that quantification of Minimal Residual Disease (MRD) in Chronic Lymphocytic Leukemia (CLL) appears to be a powerful predictor of progression-free and overall survival, regardless of the clinical response, type of therapy and known biological markers. Two methods are currently available for assessing complete remission according to the international consensus and IWCLL guidelines, i.e. allowing to detect one malignant cell in 10.000 leukocytes. These approaches are either based on specific immunoglobulin heavy chain rearrangement in each clone (RQ-ASO IgH-PCR) or on the unique immunophenotype profile of CLL cells using multiparameter flow cytometry.
Aims
Our aim was to assess complete remission in CLL on Reunion Island. The assay had to be not as complex, expensive and time-consuming as previously published techniques. This study describes the different steps in implementing and optimising an eight-colour flow cytometry MRD assay in peripheral blood.
Methods
Our work is based on an antibody panel designed by the ERIC expert group (www.ericll.org) including the following combination: CD81/CD79b/CD22/CD19/CD43/CD20/CD5/CD3. The malignant clone is identified among normal B cells thanks to 15 scattergrammes combining six CLL highly characteristic markers. Tumor cells typically express CD5, dim CD22, dim CD20, dim or negative CD79b, CD43, dim or negative CD81. In order to reach a higher specificity and sensibility for detection of CLL cells, we have been working on three different approaches: modification of sample preparation procedure, introduction of a new brilliant fluorochrome for the violet laser line and optimisation of data analysis strategy. Once the implementation of the assay had been carried out, we studied performance criteria according to learned societies, considering clinical relevance and practical feasibility.
Results
After modifying the eight-colour protocol, we achieved a precise estimation of the number of total leukocytes events by excluding unlysed cells (SSC/CD43 dot plot), erythrocytes debris (CD81/CD19 dot plot) and doublets (FSC-A/FSC-H dot plot). This new gating algorithm allows us to calculate the number of CLL cells events as a percentage of total leukocytes, according to international guidelines. The intersection of 16 regions of interest contribute further to clearly define the CLL population by minimizing the error of inclusion risk. Analysis of ten healthy donor blood has been carried on to assess the limit of detection, which is 5.3. The application of this new data analysis strategy achieves a sensitivity of 5x10-6 after acquiring one million leukocytes. Performance criteria proved mostly satisfying: imprecision, accuracy, carryover and specimen stability meet the standards we set.
Summary
We have developed on Reunion Island a flow cytometric technique reliable and sensitive enough to detect the presence of less than one CLL cell in 100.000 leukocyte. This 8-colour assay reduces subjectivity, sample requirements and time taken for acquisition and analysis, compared with 4 and 6-color assays. It also presents the advantage of requiring less specialized equipment and being not as expensive as the sensitive PCR methods.
Keyword(s): Chronic lymphocytic leukemia, Cytometry, Minimal residual disease (MRD)
Session topic: Publication Only