Contributions
Abstract: PB1792
Type: Publication Only
Background
Matching the morphology with immunophenotype for individual leukocytes is a major issue in diagnostics of leukemia and lymphoma due to the absence of a method for simultaneous cluster of differentiation surface antigen detection and full leukocyte morphology analysis. This problem can be solved by using a leukocyte-binding antibody microarray.
Aims
We describe an anti-CD antibody microarray on a transparent support for leukocyte sorting and a method for preparation of the microarray-bound cells for high-resolution morphology analysis. The aim of the work was to demonstrate, that the leukocyte binding is highly specific and that the microarray-bound peripheral blood mononuclear cells both from healthy donors and patients with B-cell leukemias and lymphomas are morphologically identical to the same cells in blood smears.
Methods
Anti-CD antibodies were immobilised on plastic coverslips in spots 2 mm in diametre. In order to study the peripheral blood mononuclear cells (PBMC) the mononuclear fraction separated by density gradient from peripheral blood are incubated with the microarray in non-mixing conditions at 4 0C. After the unbound cells are washed away the microarray-bound cells are dried in a cytocentrifuge and stained after May-Grünwald-Giemsa for morphology examination. Using this technique we have studied the PBMC from 55 healthy donors and 77 patients with different leukemias and lymphomas: chronic lymphocytic leukemia (CLL, 37 patients), hairy cell leukemia (HCL, 22 patients), splenic marginal zone lymphoma (SMZL, 7 patients), mantle cell lymphoma (MCL, 2 patients), follicular lymphoma (FL, 1 patient), 5 patients with multiple myeloma (MM), 2 patients with large granular lymphocytic (LGL) leukemia and one patient with acute myeloid leukemia (AML M2).
Results
Nonspecific cell binding both inside and outside the spots is below 5%. Due to the non-mixing incubation the density of the cells bound to an anti-CD antibody permits to determine the proportions of cells positive for the corresponding CD antigen with high correlation with flow cytometry. The patterns of the binding densities of the anti-CD-captured PBMC for CLL, HCL and SMZL patients clearly differ both from those for normal PBMC and from each other and agree well with the reported immunophenotypes of corresponding neoplastic cells. Both normal and pathologic microarray-bound PBMC after the proprietary drying procedure are morphologically identical to the same cells in a smear. In cases when pathologic cells are morphologically and/or cytochemically distinct, the anti-CD antibody microarray permits to determine their percentage and immunophenotype by analysing the relative amount of these cells captured by the antibodies against all the CD antigens. The results of such analysis of neoplastic PBMC for the patients with leukemias and lymphomas agree with flow cytometry results for the same patients including expression of CD2 in HCL, CD2 and CD11c in CLL, CD56 in MM. The amount of hairy cells determined morphologically on the microarray varied from 20 to 97 of all anti-CD19-captured cells and 2 to 80% of all lymphocytes and was in good agreement with the percentages of cells with CD19/CD103 and CD19/CD11c coexpression determined in the peripheral blood of the same patients by flow cytometry.
Conclusion
The microarray works as a “sorted smear” with cells positive for certain surface CD antigens localised in a predetermined area and permitting to apply any standard smear-oriented technique to the microarray-captured cells. Combined analysis of the pathologic cells’ immunophenotype, morphology and cytochemistry on the microarray permits to arrive at preliminary diagnosis and can be used in cases of any controversies between morphology, cytochemistry and immunophenotyping. The work is partially supported by 16-34-01030 and 16-04-00282 grants from RFBR.
Session topic: 6. Chronic lymphocytic leukemia and related disorders - Clinical
Keyword(s): Microarray analysis, Leukemia cells, Immunophenotype
Abstract: PB1792
Type: Publication Only
Background
Matching the morphology with immunophenotype for individual leukocytes is a major issue in diagnostics of leukemia and lymphoma due to the absence of a method for simultaneous cluster of differentiation surface antigen detection and full leukocyte morphology analysis. This problem can be solved by using a leukocyte-binding antibody microarray.
Aims
We describe an anti-CD antibody microarray on a transparent support for leukocyte sorting and a method for preparation of the microarray-bound cells for high-resolution morphology analysis. The aim of the work was to demonstrate, that the leukocyte binding is highly specific and that the microarray-bound peripheral blood mononuclear cells both from healthy donors and patients with B-cell leukemias and lymphomas are morphologically identical to the same cells in blood smears.
Methods
Anti-CD antibodies were immobilised on plastic coverslips in spots 2 mm in diametre. In order to study the peripheral blood mononuclear cells (PBMC) the mononuclear fraction separated by density gradient from peripheral blood are incubated with the microarray in non-mixing conditions at 4 0C. After the unbound cells are washed away the microarray-bound cells are dried in a cytocentrifuge and stained after May-Grünwald-Giemsa for morphology examination. Using this technique we have studied the PBMC from 55 healthy donors and 77 patients with different leukemias and lymphomas: chronic lymphocytic leukemia (CLL, 37 patients), hairy cell leukemia (HCL, 22 patients), splenic marginal zone lymphoma (SMZL, 7 patients), mantle cell lymphoma (MCL, 2 patients), follicular lymphoma (FL, 1 patient), 5 patients with multiple myeloma (MM), 2 patients with large granular lymphocytic (LGL) leukemia and one patient with acute myeloid leukemia (AML M2).
Results
Nonspecific cell binding both inside and outside the spots is below 5%. Due to the non-mixing incubation the density of the cells bound to an anti-CD antibody permits to determine the proportions of cells positive for the corresponding CD antigen with high correlation with flow cytometry. The patterns of the binding densities of the anti-CD-captured PBMC for CLL, HCL and SMZL patients clearly differ both from those for normal PBMC and from each other and agree well with the reported immunophenotypes of corresponding neoplastic cells. Both normal and pathologic microarray-bound PBMC after the proprietary drying procedure are morphologically identical to the same cells in a smear. In cases when pathologic cells are morphologically and/or cytochemically distinct, the anti-CD antibody microarray permits to determine their percentage and immunophenotype by analysing the relative amount of these cells captured by the antibodies against all the CD antigens. The results of such analysis of neoplastic PBMC for the patients with leukemias and lymphomas agree with flow cytometry results for the same patients including expression of CD2 in HCL, CD2 and CD11c in CLL, CD56 in MM. The amount of hairy cells determined morphologically on the microarray varied from 20 to 97 of all anti-CD19-captured cells and 2 to 80% of all lymphocytes and was in good agreement with the percentages of cells with CD19/CD103 and CD19/CD11c coexpression determined in the peripheral blood of the same patients by flow cytometry.
Conclusion
The microarray works as a “sorted smear” with cells positive for certain surface CD antigens localised in a predetermined area and permitting to apply any standard smear-oriented technique to the microarray-captured cells. Combined analysis of the pathologic cells’ immunophenotype, morphology and cytochemistry on the microarray permits to arrive at preliminary diagnosis and can be used in cases of any controversies between morphology, cytochemistry and immunophenotyping. The work is partially supported by 16-34-01030 and 16-04-00282 grants from RFBR.
Session topic: 6. Chronic lymphocytic leukemia and related disorders - Clinical
Keyword(s): Microarray analysis, Leukemia cells, Immunophenotype