PENGANTAR PRAKTIKUM FCM LM tata carayang

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1. What is FCM? F CM: examining physical and chemical properties of cells, beads, or particles as they pass in a fluid stream through a measuring apparatus (flow cytometer). Measurements are made on a per-cell basis at routine rates of 500 to 4000 cells per second Fluidic : To introduce and focus the cells for interrogation Optic : To generate and collect the light signals Electronic : To convert the optical signals to proportionate electronic signals and digitize them for further analysis on the computer Princi ples

2. Fluidics–Hydrodynamic Focusing Low Sample Pressure High Sample Pressure sheath sheath sample sheath sheath sample

3. Flowcytometry

4. Forward Scatter—diffracted light Related to cell surface area Detected along axis of incident light in the forward direction Side Scatter—reflected and refracted light Related to cell granularity and complexity Detected at 90° to the laser beam Right Angle Light Detector a Cell Complexity Forward Light Detector a Cell Surface Area Incident Light Source Properties of FSC and SSC

5. Fluorescence Emitted Fluorescence Intensity   Binding Sites FITC FITC FITC FITC FITC FITC FITC FITC FITC FITC Fluorescent Intensity Number of Events

6. T T T C H CD3 CD3 CD3 CD8 CD4 T-Cell Subsets

7. Immunophenotyping CD # = cluster designation number Since FSC ≈ size and SSC ≈ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogeneous cell population FSC SSC Lymphocytes Monocytes Granulocytes RBCs, Debris, Dead Cells Why Look at FSC v. SSC

8. GOAL: to identify the leukemic population The abnormal sample contains a prominent population of blast cells (red) with low SSC and less expression of CD45 than normal lymphocytes (green) CD45 PerCP-Cy5.5 SSC Height SSC Height CD45 PerCP-Cy5.5 Normal Abnormal

9. Classification of Acute Leukemia

10. AML: FAB CLASSIFICATION Subclass Description M0 Acute non-differentiated leukemia – immature blast with minimal differentiated M1 Acute myeoloblastic leukemia without maturation M2 Acute myeloblastic leukemia with granulocytic maturation M3 Promyelocytic or acute promyelocytic leukemia M4 Acute myelomonocytic leukemia M4Eo Variant  increase in abnormal marrow eosinophils M5 M5a  acute monocytic leukemia without maturation M5b  acute monocytic leukemia with partial maturation M6 Acute erythromyelosis M7 Acute megakaryoblastic

11. ALL: FAB CLASSIFICATION

12. Diagnosis of Hematological Malignancy Clinical Symptoms and signs Laboratory findings Cytogenetics Immunophenotyping Molecular biology / FISH Morphology + cytochemistry

13. Bone Marrow Morphology SMEAR SMEAR TOUCH PREPS

14. Morphologies of AML & ALL ALL AML

15. Immunophenotyping

16. Indication for Immunophenotyping

17. Sampel Whole blood EDTA, 2 -3 mL room temp, stability 48 hours. Bone Marrow Sampel Peripheral blood sampel LCS sampel Other body Fluid Limph node

18. Phenotyping in Acute Leukemia Hoffbrand AV, Moss PAH. Essential Haematology , 6 th ed. London: Wiley BlackWell , 2011. Stem Cells cMPO cCD79a

19. Precursor B-cell ALL

20. Gating at blast area was positive for CD34 cyCD79a CD19 CD10 CD38 B lineage cMPO cCD79a HLA-DR

21. Precursor T-cell ALL

22. Gating at blast area was positive for CD34 cyMPO HLA-DR CD13+33 CD14 CD64 CD117 cyCD3 CD3 CD7 Mixed phenotype (myeloid/T) cMPO cCD79a HLA-DR

23. Multiple Myeloma Definition: A bone marrow-based , multifocal neoplastic proliferation of plasma cells , usually associated with M protein in serum and/or urine and evidence of end organ damage related to plasma cell neoplasm Epidemiology: 1% of malignancy tumors 10 – 15% of hematopoietic neoplasm 20% death from hematological malignancies Incidence increases progressively with age, 90% cases aged > 50 years (median diagnosis ~ 70 years) WHO/IARC. WHO classification of tumours of haematopoietic and lymphoid tissues. Revised 4t. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al., editors. Lyon: IARC; 2017.

24. Detection of Malignant Plasma Cells

25. Immunophenotyping by FCM Monotypic cytoplasmic Ig (monoclonal) AND lack surface Ig Expressing CD38  dimmer CD138  brighter Negative CD45 or low leve l s CD19 in 95% cases CD27 and CD81 or underexpressed Aberrant expression (90% of cases) CD56 (75-80%) CD200 (60-75%) CD28 (~40%) CD117 (20-35%) CD20 (10-20%) CD52 (8-14%) WHO/IARC. WHO classification of tumours of haematopoietic and lymphoid tissues. Revised 4t. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al., editors. Lyon: IARC; 2017.

26. Immuno phenotyping