Background
- Flow cytometry uses laser techniques to sort the cells.
- The cells are labeled with monoclonal antibodies.
- These monoclonal antibodies are coupled with flourescense molecules.
- There are many types of molecules, e.g. FITC (green), PE (red, strong), PE-Cy5 (PE conjugated) and so on.
- The laser hits the electron on the molecule and when the laser stops, the electrons needs to go back to their original circuit and this process emits light.
- The light has various length (in nm).
- There are also several types of length.
- There is overlap between the fluorescense --> matrix for the correction is needed.
- Main producers of the machines are: Beckton Dickinson and Beckman Coulter.
Sorting cells
- The cells are also sorted because they have difference size and complexity (granules).
- Forward scatter measures the size of the cells (shadow), and the side scatter meas
ures the complexity.
- In this figure: green is lmphocyte, orange is neutrophils.
CD (cluster differentiation) and other markers
- The monoclonal antibodies bind to CD molecules on the surface of the cells, but also on heavy or light chains on plasma cells (remember that plasma cells are cells that secretes immunoglobulines and they differ than virgin B cells; remember also that the free immunoglobulines can be determined using other techniques such as BN II from Siemens).
- Some important markers: CD45 (leukocytes), CD34 (stam cells), CD3 (T lymphocyte), CD 56 (NKcells), CD41 and CD61 (thrombocytes), CD8 (cytotoxic T cells), CD4 (T helper cells), CD19 and CD20 (B cells).
- There are also intracellular markers, such as tDt and MPO and immunoglobuline such as IgG, IgM, IgG and IgA.
Specific markers
- koRSA: associated with Philadelhia chromosome, B-ALL, also used as prognostic markers.
- CD103 and CD25: hairy cell leukemia.
- MPO: acute myeloid leukemia.
- CD19 --> CD10 --> CD20 (from pan B cells --> more riper B cells)
Other aspects
- Positive if >20% cells are positive.
- Cell can also have natural fluorescense.
- A CD marker can positive while cytoplasmic marker can be negative (e.g. myeloma vs. B-cell neoplasia). .
Practical
Work flow
* CLL
- Suspected CLL due to high lymphocytes number during the time.
- Screening panel on T- (CD3, CD4), B- cells (CD19) and NK cells (CD 56).
- B-cell panel, such as IgD, IgM, IgG and IgA, CD10, and CD20.
- CLL panel for the Matutes score (weak CD79, positive CD23 and CD5, weak Smig, negative Fmc7).
* Myeloma
- Screening panel on T- (CD3, CD4), B- cells (CD19) and NK cells (CD 56).
- B-cell panel, such as IgD, IgM, IgG and IgA, CD10, and CD20.
- Myeloma panel, with cytoplasmic IgG, IgM, IgG and IgA (in general, these markers do not present on the surface of B-cell lymphomas except in Waldenstrom).
* AML or ALL
- Blasts on morphology.
- Screening panel on T- (CD3, CD4), B- cells (CD19) and NK cells (CD 56).
- B-cell panel, such as IgD, IgM, IgG and IgA, CD10, and CD20.
- Panels needed to type the malignancy. More information (in Dutch) in the website of the Dutch Union of Cytometry.
Bulk lysis vs. Ficoll gradient
Ficoll to concentrate in MDS and in myeloproliferative diseases (to concentrate stam cell).
- 20 uL antibodies with markers + 50 uL blood sample + lysis buffer --> wash --> measurement.
- Ficoll gradient (hold the mononuclear and stam cells, let erythrocytes pass) --> isolation of the monunuclear layer --> labelling --> measurement.
Abberant markers
- Markers that are unique for the patients, i.e. CD45 and CD19 an CD107.
- These are gated against pool of controls.
- Pool of controls consisted of patients treated with ALL (for AML) and vice versa. This choice is based to see the development of stimulated bone marrow (vs. bone marrow in not diseased).
- There should be maximum 0.001% overlap with the control.
- After all, the aim is to determine minimal residual disease; due to the limitation described, it is very difficult to defined.
Several practical clinical issues
- After solid organ transplantation patients receive immunosuppresion such as anti thymocyte globulin for a short time (<1 year). The screening T cell (CD3 and CD4) is performed at this time. After this time the immunosuppresva is switched and the full panel of T cell is performed (more time available) but in lower frequency.
- Flow cytometry uses laser techniques to sort the cells.
- The cells are labeled with monoclonal antibodies.
- These monoclonal antibodies are coupled with flourescense molecules.
- There are many types of molecules, e.g. FITC (green), PE (red, strong), PE-Cy5 (PE conjugated) and so on.
- The laser hits the electron on the molecule and when the laser stops, the electrons needs to go back to their original circuit and this process emits light.
- The light has various length (in nm).
- There are also several types of length.
- There is overlap between the fluorescense --> matrix for the correction is needed.
- Main producers of the machines are: Beckton Dickinson and Beckman Coulter.
Sorting cells- The cells are also sorted because they have difference size and complexity (granules).
- Forward scatter measures the size of the cells (shadow), and the side scatter meas
ures the complexity.
- In this figure: green is lmphocyte, orange is neutrophils.
CD (cluster differentiation) and other markers- The monoclonal antibodies bind to CD molecules on the surface of the cells, but also on heavy or light chains on plasma cells (remember that plasma cells are cells that secretes immunoglobulines and they differ than virgin B cells; remember also that the free immunoglobulines can be determined using other techniques such as BN II from Siemens).
- Some important markers: CD45 (leukocytes), CD34 (stam cells), CD3 (T lymphocyte), CD 56 (NKcells), CD41 and CD61 (thrombocytes), CD8 (cytotoxic T cells), CD4 (T helper cells), CD19 and CD20 (B cells).
- There are also intracellular markers, such as tDt and MPO and immunoglobuline such as IgG, IgM, IgG and IgA.
Specific markers
- koRSA: associated with Philadelhia chromosome, B-ALL, also used as prognostic markers.
- CD103 and CD25: hairy cell leukemia.
- MPO: acute myeloid leukemia.
- CD19 --> CD10 --> CD20 (from pan B cells --> more riper B cells)
Other aspects
- Positive if >20% cells are positive.
- Cell can also have natural fluorescense.
- A CD marker can positive while cytoplasmic marker can be negative (e.g. myeloma vs. B-cell neoplasia). .
Practical
Work flow
* CLL
- Suspected CLL due to high lymphocytes number during the time.
- Screening panel on T- (CD3, CD4), B- cells (CD19) and NK cells (CD 56).
- B-cell panel, such as IgD, IgM, IgG and IgA, CD10, and CD20.
- CLL panel for the Matutes score (weak CD79, positive CD23 and CD5, weak Smig, negative Fmc7).
* Myeloma
- Screening panel on T- (CD3, CD4), B- cells (CD19) and NK cells (CD 56).
- B-cell panel, such as IgD, IgM, IgG and IgA, CD10, and CD20.
- Myeloma panel, with cytoplasmic IgG, IgM, IgG and IgA (in general, these markers do not present on the surface of B-cell lymphomas except in Waldenstrom).
* AML or ALL
- Blasts on morphology.
- Screening panel on T- (CD3, CD4), B- cells (CD19) and NK cells (CD 56).
- B-cell panel, such as IgD, IgM, IgG and IgA, CD10, and CD20.
- Panels needed to type the malignancy. More information (in Dutch) in the website of the Dutch Union of Cytometry.
Bulk lysis vs. Ficoll gradient
Ficoll to concentrate in MDS and in myeloproliferative diseases (to concentrate stam cell).
- 20 uL antibodies with markers + 50 uL blood sample + lysis buffer --> wash --> measurement.
- Ficoll gradient (hold the mononuclear and stam cells, let erythrocytes pass) --> isolation of the monunuclear layer --> labelling --> measurement.
Abberant markers
- Markers that are unique for the patients, i.e. CD45 and CD19 an CD107.
- These are gated against pool of controls.
- Pool of controls consisted of patients treated with ALL (for AML) and vice versa. This choice is based to see the development of stimulated bone marrow (vs. bone marrow in not diseased).
- There should be maximum 0.001% overlap with the control.
- After all, the aim is to determine minimal residual disease; due to the limitation described, it is very difficult to defined.
Several practical clinical issues
- After solid organ transplantation patients receive immunosuppresion such as anti thymocyte globulin for a short time (<1 year). The screening T cell (CD3 and CD4) is performed at this time. After this time the immunosuppresva is switched and the full panel of T cell is performed (more time available) but in lower frequency.
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