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No, but don't ask me what that MEANS: "Each ANTIGEN requires a DIFF TUBE".

No, but don't ask me what that means: "Each antigen requires a diff tube".

SELECTING to MEASURE INFO on only ONE group of cells.

Selecting to measure info on only one group of cells.

SIX. Forward scatter (size), 90 degree scatter (internal strx) and the four filters (filters have increasingly HIGHER frequency).

Six. Forward scatter (size), 90 degree scatter (internal strx) and the four filters (filters have increasingly higher frequency).

Expect MIX of Abs to be on PLASMA cells. Expect mix of light chains (kappa and lambda). When these EXPECTED patterns DEVIATE, can assess/identify clonality in these CELL types.

Expect mix of Abs to be on plasma cells. Expect mix of light chains (kappa and lambda). When these expected patterns deviate, can assess/identify clonality in these cell types.

Express specific ANTIGENS, we can LOOK closely at HEM to look for clonality.

Express specific antigens, we can look closely at hem to look for clonality.

LOOK at their LIGHT CHAIN EXPRESSION PATTERNS.

Look at their light chain expression patterns.

CD34 (CD45 – all).

CD34 (CD45 – all).

CD19, CD20 (CD45 – all).

CD19, CD20 (CD45 – all).

CD13, CD14, CD15 (CD45 - all).

CD13, CD14, CD15 (CD45 - all).

All LEUKOCYTES.

All leukocytes.

BLASTS / STEM CELLS.

Blasts / stem cells.

B CELLS.

B cells.

B CELLS.

B cells.

GRANULOCYTES, MONOCYTES.

Granulocytes, monocytes.

Monocytes.

Monocytes.

GRANULOCYTES, MONOCYTES.

Granulocytes, monocytes.

CYTOTOXIC T CELLS.

Cytotoxic T cells.

HELPER T CELLS.

Helper T cells.

T CELLS.

T cells.

• CD45 = LEUKOCYTE COMMON ANTIGEN, LCA.
• CD3 = T–cell marker.
• CD4 = helper T cells.
• CD8 = cytotoxic T cells.

• CD45 = LEUKOCYTE Common ANTIGEN, LCA.
• CD15 = Granulocyte marker.
• CD13.

CD38 (+): ANYTHING above horizontal line.

CD20 (+): anything to the RIGHT of the vertical line.

RED CELLS = CD38+ and CD20–.

BLUE cells = CD38– and CD20+.

CD38 (+): anything above horizontal line.

CD20 (+): anything to the right of the vertical line.

Red cells = CD38+ and CD20–.

Blue cells = CD38– and CD20+.

Normal: should see very specific AG EXPRESSION PATTERN. Abnormal: inappropriate expression of Ag (eg, BLAST INAPPROPRIATELY expressing a very mature marker).

Normal: should see very specific Ag expression pattern. Abnormal: inappropriate expression of Ag (eg, blast inappropriately expressing a very mature marker).

There is lots of cross-reactivity. Cells might have numerous amounts of antigens on them. What's EXPRESSED on the green cell might also be expressed on the blue cell (so Ab against green cell Ag will also BIND to the blue cell). Cells pass through and have VARIABLE amounts of DIFFERENT ABS attached to them.

There is lots of cross-reactivity. Cells might have numerous amounts of antigens on them. What's expressed on the green cell might also be expressed on the blue cell (so Ab against green cell Ag will also bind to the blue cell). Cells pass through and have variable amounts of different Abs attached to them.

Conjugate beads to just FITC, and beads to just PE and then you can SUBTRACT for COMPENSATION.

Conjugate beads to just FITC, and beads to just PE and then you can subtract for compensation.

DEPENDS on what fluorochrome you labeled on your reagent. Each FLUORESCENT marker gives of LIGHT at a DIFFERENT wavelength.

Depends on what fluorochrome you labeled on your reagent. Each fluorescent marker gives of light at a different wavelength.

X–axis: WAVELENGTH. Y–axis: INTENSITY.

X–axis: wavelength. Y–axis: intensity.

Green. Low to intermediate forward scatter (SMALL to medium SIZE). Very high SIDE scatter (LOTS of granules).

Green. Low to intermediate forward scatter (small to medium size). Very high side scatter (lots of granules).

DARK BLUE. HIGHER forward scatter (BIGGER cells in blood and BM). Intermediate side scatter (some granularity in CYTOPLASM).

Dark blue. Higher forward scatter (bigger cells in blood and BM). Intermediate side scatter (some granularity in cytoplasm).

Red. Higher forward scatter (very LARGE cells). Low SIDE scatter (do not have PROMINENT GRANULES).

Red. Higher forward scatter (very large cells). Low side scatter (do not have prominent granules).

Teal and pink. Low FORWARD SCATTER (SMALLEST cells in blood and BM). Low side scatter (lowest complexity CYTOPLASM – no granules).

Teal and pink. Low forward scatter (smallest cells in blood and BM). Low side scatter (lowest complexity cytoplasm – no granules).

Based on SIDE SCATTER: *Lowest COMPLEXITY = lowest side scatter. *Highest complexity = most side scatter.

Based on Side Scatter: *Lowest complexity = lowest side scatter. *Highest complexity = most side scatter.

BASED on Forward Scatter: *SMALLEST cells = LOWEST AMOUNT of forward scatter. *Larger cells = most forward scatter.

Based on Forward Scatter: *Smallest cells = lowest amount of forward scatter. *Larger cells = most forward scatter.

Reflects the complexity of the cell cytoplasm. Neutrophil: MANY GRANULES → very complex cytoplasm → high amount of side scatter. Reactive LYMPHOCYTE: few granules → less complex cytoplasm → LOW amount of side scatter.

Reflects the complexity of the cell cytoplasm. Neutrophil: many granules → very complex cytoplasm → high amount of side scatter. Reactive lymphocyte: few granules → less complex cytoplasm → low amount of side scatter.

REFLECTS the SIZE of the cell. As these different sized cells pass through Flow Cytometer and go past the laser, laser collects forward SCATTER BASED on how much light gets scattered when these cells go past the beam. *Smallest cells = lowest AMOUNT of forward scatter. *Larger cells = most forward scatter.

Reflects the size of the cell. As these different sized cells pass through Flow Cytometer and go past the laser, laser collects forward scatter based on how much light gets scattered when these cells go past the beam. *Smallest cells = lowest amount of forward scatter. *Larger cells = most forward scatter.

FORWARD SCATTER. SIDE scatter. FLUORESCENCE EMITTED.

Forward scatter. Side scatter. Fluorescence emitted.

Cells go through CHAMBER and ideally PASS through 1–by–1. Laser light source shines on cells. If ABS (from reagent you added) are attached to cells they will give off their fluorochrome and you will SEE fluorescence. Detects forward scatter properties and SIDE scatter properties on different cells.

Cells go through chamber and ideally pass through 1–by–1. Laser light source shines on cells. If Abs (from reagent you added) are attached to cells they will give off their fluorochrome and you will see fluorescence. Detects forward scatter properties and side scatter properties on different cells.

Cells from whatever SPECIMEN you're testing. Often BLOOD, BM, or LNs in hematopathology. Tissue SOLUTIONS in which cells are disaggregated. Abs with fluorochromes ATTACHED to them.

Cells from whatever specimen you're testing. Often blood, BM, or LNs in hematopathology. Tissue solutions in which cells are disaggregated. Abs with fluorochromes attached to them.

Fluorescence-labeled ABS INCUBATED with cells. Laser EXCITES fluorochromes (on Abs), which emits fluorescence. Fluorescence of Ag-Ab complex is MEASURED, along with scatter properties of cells.

Fluorescence-labeled Abs incubated with cells. Laser excites fluorochromes (on Abs), which emits fluorescence. Fluorescence of Ag-Ab complex is measured, along with scatter properties of cells.

IDENTIFY Ag expression patterns on CELLS. DETERMINE if cells are normal or ABNORMAL based on recognized patterns.

Identify Ag expression patterns on cells. Determine if cells are normal or abnormal based on recognized patterns.

No, you LYSE the RBC's PRIOR to the PROCEDURE.

No, you lyse the RBC's prior to the procedure.

LIGHT SCATTERED by CELLS and FLUORESCENCE from fluorochromes ATTACHED to cells.

Light scattered by cells and fluorescence from fluorochromes attached to cells.