Hematology: LabCe Introduction to Flow Cytometry: Blood Cell Identification

One of the most widely used marker combinations for chronic (mature) lymphocyte analysis is

CD5(t cell marker) and CD19(b cell marker) Abnormal B cells can express CD5

Absolute lymphs

WBC count x 1000 x percent lymphs

Flow cytometry analyzes which blood cells?

White blood cells RBCs removed by lysis

Granulocytes/neutrophils

purple

Lympocytes

red

Fluorescent signal

reflects what MoAb was bound to on the cell surface

B cells, monocytes, and neutrophils should not express either antigen

thus they should not emit a fluorescent signal when they intersect the laser.

Monocytes

yellow

Which of the following represents the percent population of T-helper cells in this sample data? Absolute lymphocyte count= 2.4 x 109/L CD3 = 60% CD4 = 40% CD8 = 20%

40% T-helper cells are CD4 positive. The flow results reflected 40% CD4.CD3 is a pan T cell marker (and should reflect CD4 plus CD8).CD8 is the cytotoxic T cell marker.

All of the following competencies are needed to interpret a case using the principles of flow cytometry:

- Knowledge of CD markers and which CD markers are associated with which cell populations. - Ability to establish a gated cell population. - Skill in reading the cytogram and histogram data.

Dead cells cause complication by

- Non-specifically binding monoclonal antibodies and emitting false fluorescent signals. - Falling in inappropriate portions of the light scatter graph and falsely elevating event/cell counts

What is a flow cytometer used for?

1. To isolate and fluorescently label blood cells based on molecules of interest 2. Analyzes and stores information about the cells based on amount of light scatter & fluorescent light that is emitted

Sample prepation

1. prepare sample according to cell type 2. Obtain a WBC count 3. Adjust the WBC count by concentrating the WBCs for staining 4. Incubate 5. Lyse red blood cells with ammonium chloride 6. Centrifuge 7. Pour off supernatant 8. Wash in phosphate buffered saline 9. Resuspend cells in PBS 10. Analyze the cells using flow cytometer

WBC count

2.5 x 10^3/mcL (2500 cells/mcL)

% Lymphs

30%

Gate

A boundary that is set up around a subset of data points to segregate those events for analysis, or to exclude other events from the analysis.

Cluster designation or cluster differentiation (CD)

A group of antibodies that recognize the same antigen

What is flow cytometry?

A laboratory method that allows for the simultaneous measurement of multiple physical characterics of cells & particles

acquired immunodeficiency syndrome (AIDS)

After the T-helper cell count falls below 200 cells/mm^3

Monoclonal antibody (MoAb)

An antibody that is produced from a single clone of cells and therefore has high purity and reproducibility. A monoclonal antibody that is used in flow cytometry is directed against a single antigen. For example, the CD2 MoAb will bind to the CD2 antigen on the cell surface of a T cell.

human immunodeficiency virus (HIV)

Attacks and destroys T-helper cells (CD4)

How is the intereaction recorded?

By focused laser beam

Select each marker listed below that is expressed by a normal mature B cell.

CD19 CD20 Kappa or Lambda Normal mature B cells express CD19, CD20, and Kappa or Lambda. CD2 is expressed by mature T cells.

Which of the following CD markers is NOT present on a normal mature T cell?

CD20 CD20 is not present on mature T cells. CD20 is expressed by B cells.Normal mature T cells should express CD2, CD3, CD5 and CD7.

A population of small lymphocytes is gated for further evaluation in the flow cytometry laboratory. The scatterplot for one of the evaluations is shown on the right. How would you interpret this scatterplot?

Cells in the gated population are coexpressing CD5 and CD20. The scatterplot represents coexpression of CD5 and CD20. Q2 of the scatterplot contains CD5/CD20 + cells.

A monoclonal B-cell population (Kappa or Lambda predominant) with expression of CD19, CD20, CD23, and co-expression of CD5 is consistent with which of the following?

Chronic lymphocytic leukemia If Kappa or Lambda is predominant and CD5 is co-expressed with CD19 (CD19/CD5 dual positive lymphocyte population), and CD23 is expressed, chronic lymphocytic leukemia is a probable diagnosis. CD19 is normally found on normal B cell populations and CD5 is normally found on mature T cell populations. However, CD5 is present on B cells in B-chronic lymphocytic leukemia or Mantle Cell Lymphoma, both abnormal B cell malignant processes. CD23 expression is not consistent with Mantle cells as shown on the decision tree on the right.

Extrinsic cell characteristics are reflected by the forward and side scatter on a flow cytometry cytogram.

False Intrinsic cell characteristics are represented by forward and side scatter. Extrinsic cell characteristics are represented by the fluorescent signal produced as a result of tagged monoclonal antibodies binding to antigens on the cell surface.

Monoclonal antibodies are designed to bind to all antigens on cell surfaces

False Monoclonal antibodies are designed to bind only to specific matching antigens. This principle is what allows for sorting of individual cell lines and populations within a sample containing a mix of different types of cells.

Immunofluorescence

Fluorescent expression of immune reactions between antigens and antibodies

Fluorochrome (fluorophore)

Fluorescent marker that is excited by light of one wavelength (generated by the laser of the flow cytometer) and emits light of a different wavelength (fluorescent light).

When analyzing the raw data of a sample on the flow cytometer, CD marker information on the histograms represents data inside the _____ population.

Gated Gating sets up boundaries around a subset of data points to segregate those events for analysis.

Side scatter

Granularity

Which of these white blood cell populations would have the MOST side scatter when analyzed using flow cytometry?

Granulocytes

3 major types of mature T cells

Helper Cytotoxic Regulatory

Flow cytometry applications include

Immunophenotyping HIV disease assessment Transplant cellular distribution determination and CD3 suppression Leukemia/lymphoma diagnosis, staging, and minimal residual disease testing CD34 quantitation/Stem cell quantitation Fetal hemoglobin detection on red blood cells (RBCs) DNA ploidy determination Bacterial identification Research Cell sorting, population filtering Other

During the flow cytometry analysis process, cells exiting the flow cell intersect a laser, and produce what kind of data?

Intrinsic cell characteristicsExtrinsic cell characteristicsForward scatterFluorescence

How does single file happen?

Isotonic sheath fluid

During the 'staining' process prior to flow cytometry analysis of white blood cells, erythrocytes are _______ .

Lysed. Erythrocytes are lysed prior to flow cytometry analysis of white blood cells.

CD5 antigen is normally found on which of the following lymphocyte populations?

Mature T cells

Appropriate same types are

Peripheral blood Bone marrow Lymph nodes & other tissues Fluids Manually disaggregated tissue biopsy speciments

The scatterplot that is pictured below represents results from a normal peripheral blood sample that was analyzed using flow cytometery. The cell that is indicated by the arrow on the right would fall into which of the circled areas?

Population C

Lymphocytes

T cells or B cells

All helper cells are T cells. Consequently, in a normal lymphocyte population, it is reasonable to expect all CD4-positive cells to be CD3-positive as well. True or False?

True

A true cytotoxic T cell would emit

both orange and blue colors

Forward scatter

cell size

Helper T cells (CD3- and CD4-bound) will

fluoresce both orange and green

Cytotoxic T cells (CD3 bound) will

fluoresce orange, but not green

Gating is a method that is used in flow cytometry to

isolate cells of interest by placing an electronic gate around cells that display specific fluorescent or light scattering characteristics