Flow Cytometryand its role in diagnosing acute leukemia.pptx

A presentation about the role of flow cytometry in Acute and chronic leukemia

1. Flow cytometry

2. Flow Cytometry

3. DMs allow the light of corresponding wavelengths to pass while reflecting the light of other wavelengths. OFs further narrow the wavelengths reaching a detector. The detectors are usually photomultiplier tubes (PMTs) or photodiode arrays (PDAs) that convert the signals to electrical impulses, which are measured and converted to digital information by the electronics system. The digital information is collected and interpreted by the analysis software . The connected computer system directly interfaces with the flow cytometer and controls its functions. Data analysis can be performed either on the computer connected to the flow cytometer or on other computers that have access to the data.

4. Specimens and reagents/ antibody panels Specimens suitable for FCM analysis include PB, BM, cerebrospinal fluid, serous effusions, fine needle aspirations (FNAs), and fresh unfixed tissue. PB and BM aspirates must be anticoagulated. Sample preparation for FCM analysis varies according to the specimen types and the antigens to be analyzed. The cell suspension generated by mincing tissue should be filtered to remove large particles that may clog the cytometer tubing and/or bind antibodies non-specifically. In any specimen containing a large amount of PB, the red blood cells should be removed through a lysis process using either a commercially available reagent or a homemade ammonium- chloride solution before running the sample in a flow cytometer.

5. In general, the antibody against a weakly expressed antigen should be conjugated with a bright fluorochrome to increase the chance of detecting this antigen. Color compensation to minimize the spillover between different fluorochromes is very important in multicolor tubes. For diagnostic purposes, antibodies are typically combined as panels to answer specific questions about a cell population The screening panels should evaluate sufficient antigens to distinguish a neoplastic cell population from normal/reactive cells with a high degree of sensitivity.

6. Light Scatter and Fluorescence Signals In flow cytometry, light scatter and fluorescence are the two main signals used to analyze cells as they pass through a laser beam.Forward scatter (FSC)measures light scattered at narrow angles and is proportional to cell size, while side scatter (SSC) measures light scattered at 90° angles and reflects the cell’s granularity and internal complexity. FSC and SSC together allow the differentiation of cell types in heterogeneous populations, such as leukocytes. Fluorescent dyes attached to cells emit light after being excited by the laser, and the fluorescence is detected to quantify antigen expression levels on the cells.

7. Data analysis

8. Gating

9. Acute Leukemia

10. CLL

11. 4. Guiding Treatment Decisions:   - Targeted Therapy Decisions: Based on flow cytometry results, specific therapies can be chosen. For example, in CLL, therapies targeting CD20 (e.g., rituximab) or CD19 (e.g., CAR-T cell therapies) can be applied based on the expression of these markers.   - Immunophenotypic Response to Treatment:Flow cytometry can measure the response to immunotherapy or chemotherapy by analyzing changes in the leukemic cell population.

12. CML In Chronic Myeloid Leukemia (CML), flow cytometry has a more limited but still valuable role compared to other hematologic malignancies such as chronic lymphocytic leukemia (CLL). The primary diagnostic tool for CML remains cytogenetic and molecular tests (e.g., detecting the Philadelphia chromosome or BCR-ABL1 fusion gene). However, flow cytometry can still contribute in specific contexts:

13. 1. Diagnosis of Blast Phase (Blast Crisis) in CML:- Identification of Blast Cells: Flow cytometry is useful for characterizing the immunophenotype of blast cells when CML progresses from the chronic phase to the blast phase (blast crisis). This progression resembles acute leukemia, and flow cytometry can help identify whether the blast crisis is myeloid (AML-like) or lymphoid (ALL-like) in nature.   - Immunophenotyping of Blasts:In blast crisis, flow cytometry identifies surface markers characteristic of immature cells (blasts), such as CD34, CD117 (for myeloid lineage), or CD19 and CD10 (for lymphoid lineage). This is crucial for distinguishing between myeloid and lymphoid blast crisis, as the treatment approach differs.

14. 6. Assessing Response to Targeted Therapy:   - While not the primary method for monitoring BCR-ABL1 transcript levels (which is done by quantitative PCR), flow cytometry can complement molecular tests by assessing the immunophenotypic changes in leukemic cells, particularly during targeted therapy with tyrosine kinase inhibitors (TKIs).

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