BIMM18 Dec 20 th - Flow cytometry in clinical diagnostics

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1 BIMM18 Dec 20 th - Flow cytometry in clinical diagnostics I. B cell leukemia and lymphomas Immunophenotyping as part of the diagnostic work- up of hematologic malignancies offers a rapid and effective means of providing a diagnosis. The ability to analyze multiple cellular characteristics, along with new antibodies and gating strategies, has substantially enhanced the utility of flow cytometry in the diagnosis of leukemias and lymphomas. 1. What are the advantages of flow cytometry in the diagnosis of leukemias compared to only assessing morphological changes by histology? 2. Immunophenotyping by flow cytometry allows for the identification of abnormal tumor cells, for example in the blood or in the bone marrow. Abnormalities can occur in different ways. Look at the figures below and specify the type of abnormality and in what way flow cytometry could be helpful in identifying it. Assume this is the normal phenotype in the organ. Two types of cells, the green ones are much less frequent than the purple ones. What is wrong in the other samples? A B C D

2 3. Flow cytometry plays a big role for the diagnosis and classification of the different types of B cell leukemias and lymphomas. The first step in the diagnosis of a B cell malignancy is to find the abnormal B cells. Apart from checking for abnormal expression of antigens on the B cells it is also important to check for the presence of monoclonal B cells. What is special about monoclonal cells and why is the presence of a monoclonal population an indication for leukemia? 4. A B- cell carries either kappa- or lambda- light chains on its surface. Normal polyclonal B- cells are a mixture of kappa- B- cells and lambda B- cells, while a monoclonal population consist of either kappa or lambda- B cells. With this in mind, how would you design an experiment to test a patient sample for monoclonality? Draw histograms of the expected outcome for a healthy control and a patient with a B cell leukemia. healthy control patient

3 5. Two problems might occur when staining for monoclonal B cells: other cells in the tissue sample need to be excluded from the analysis and the monoclonal population might be very small. How can you make sure to only look at B cells and what needs to be considered in the case that the monoclonal population is very small? Instead of a histogram, design two- parameter dot plots (like the ones you worked with in the FlowJo workshops) to identify monoclonal B cells in a patient compared to a healthy control. Which additional marker would you use? healthy control patient

4 6. As mentioned above, malignant cells can downregulate their normally expressed antigen. This feature can be helpful in identifying small monoclonal B cells. In the first plot below, identify the malignant monoclonal B cells, normal polyclonal populations, and non- B cells. Assuming the second plot shows the same cells, draw the corresponding gates for the different populations in the second plot. Are the malignant cells kappa- or lambda- positive? 7. Once it has been established that there is a monoclonal, most likely malignant population the next step to define its immunophenotype, which means to check which antigens are expressed on these cells and which are not. Different B cell malignancies are characterized by specific immunophenotypes, so flow cytometry is an integral part of determining a specific type of B cell leukemia or lymphoma. Assuming the cell population in the red box are the malignant cells - define their immunophenotype and use the table below to identify what type of B cell malignancy they come from.

5 CLL/SLL IC PLL MCL HCL FL CD5 + - to weakly + - to weakly CD CD11c - to + - /+ - /+ - strongly + - CD CD20 (weakly) CD22 - to weakly + + +/ CD / /- CD24 + +/ CD25 - to mod. + - to weakly CD38 - to mod. + +/- - rarely + CD CD79b CD FMC CLL: Chronic Lymphocytic Leukemia SLL: Small Lymphocytic Lymphoma IC: Immunocytoma PLL: Prolymphocytic Leukemia MCL: Mantle Cell Leukemia HCL: Hairy Cell Leukemia FL: Follicular Lymphoma 8. What problems can occur when using these kind of tables for matching the immunophenotype with a specific malignancy?

6 II. Autoimmunity Systemic lupus erythromatosus (SLE) is an autoimmune disease triggered by large anti- dsdna and anti- nuclear proteins autoantibody complexes causing kidney pathogenesis and other systemic disorders. In the blood of SLE patients, high numbers of a certain immune cell population (see FACS plots) can be observed and has been implicated in the disease progression. Naïve B cells Memory B cells Plasma blasts/plasma cells CD19 + CD38 - /low CD19 + CD38 +/low CD19 low CD38 high 1. Based on the FACS plots and table above determine the population appearing in the blood of SLE patients. What is the function of those cells? 2. What could be the - potentially pathological - consequence of increased numbers of this cell population? 3. Is the presence of this population in high numbers a direct proof for being involved in the disease progression? Why?

7 4. How would you test for auto- reactivity of those cells using flow cytometry? What controls would you need to include? 5. How could you check whether abundance of those cells is correlated to disease severity? 6. Think about an idea how to help patients with SLE: what immune- based therapy would you think might be helpful if those cells are indeed involved in disease progression?