Flow cytometry Interview Questions & Answers

Question 1. Flow Cytometry Is A Measure Of Two Things, What Are They?

Answer :

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

Question 2. In Flow Cytometry Are You Measuring In All Blood Cells?

Answer :

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

Question 3. What Is Flow Cytometry Used Commonly For?

Answer :

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

Question 4. What Is The Technique Of Flow Cytometry?

Answer :

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.

Question 5. What Are The Contests Of The Test Tube During Flow Cytometry?

Answer :

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.

Question 6. What Happens In The Flow Cytometer?

Answer :

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.

Question 7. What Kind Of Data Is Collected From Flow Cytometry?

Answer :

Forward scatter. Side scatter. Fluorescence emitted.

Question 8. What Does Forward Scatter Tell You?

Answer :

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.

Question 9. What Does Side Scatter Tell You?

Answer :

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.

Question 10. During Flow Cytometry, How Do You Assess The Size Of The Cell?

Answer :

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

Question 11. During Flow Cytometry, How Do You Assess The Complexity Of The Cell Cytoplasm?

Answer :

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

Question 12. What Are The Expected Findings For A Lymphocyte?

Answer :

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

Question 13. What Are The Expected Findings For Blasts Aka Stem Cells?

Answer :

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

Question 14. What Are The Expected Findings For Monocytes?

Answer :

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

Question 15. What Are The Expected Findings For Granulocytes (neutrophils)?

Answer :

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

Question 16. What Is On The Axises For A Graph Generated By The Fluorescence?

Answer :

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

Question 17. What Determines The Wavelength?

Answer :

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

Question 18. How Do You Deal/adjust For Compensation In Flow Cytometry Of More Than One Wavelength?

Answer :

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

Question 19. When You Use Many Different Ab Reagents Specific For The Cell Types You Want To Identify, What Can Make Understanding Results Difficult?

Answer :

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.

Question 20. What Would You Expect For Ag Expression In Normal Cells Vs Abnormal Cells?

Answer :

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

Question 21. How Do You Interpret This?

Answer :

CD38 (+): anything above horizontal line.

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

Red cells = CD38+ and CD20–.

Blue cells = CD38– and CD20+.

Question 22. What Are The Antibodies Expressed On Granulocytes?

Answer :

• CD45 = Leukocyte Common Antigen, LCA.
• CD15 = Granulocyte marker.
• CD13.

Question 23. What Are The Antibodies Expressed On T Cells?

Answer :

• CD45 = Leukocyte Common Antigen, LCA.
• CD3 = T–cell marker.
• CD4 = helper T cells.
• CD8 = cytotoxic T cells.

Question 24. What Is Cd3 Expressed On?

Answer :

T cells.

Question 25. What Is Cd4 Expressed On?

Answer :

Helper T cells.

Question 26. What Is Cd8 Expressed On?

Answer :

Cytotoxic T cells.

Question 27. What Is Cd13 Expressed On?

Answer :

Granulocytes, monocytes.

Question 28. What Is Cd14 Expressed On?

Answer :

Monocytes.

Question 29. What Is Cd15 Expressed On?

Answer :

Granulocytes, monocytes.

Question 30. What Is Cd19 Expressed On?

Answer :

B cells.

Question 31. What Is Cd20 Expressed On?

Answer :

B cells.

Question 32. What Is Cd34 Expressed On?

Answer :

Blasts / stem cells.

Question 33. What Is Cd45 Expressed On?

Answer :

All leukocytes.

Question 34. What Are The Antibodies Expressed On Monocytes?

Answer :

CD13, CD14, CD15 (CD45 - all).

Question 35. What Are The Antibodies Expressed On B Cells?

Answer :

CD19, CD20 (CD45 – all).

Question 36. What Are The Antibodies Expressed On Blasts / Stem Cells?

Answer :

CD34 (CD45 – all).

Question 37. How Do You Assess B Cell Clonality?

Answer :

Look at their light chain expression patterns.

Question 38. How Do You Assess T Cell Clonality?

Answer :

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

Question 39. How Do You Assess Plasma Cell Clonality?

Answer :

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.

Question 40. If You Are Measuring With 4 "filters", How Many Measurements Will You Have In The End?

Answer :

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

Question 41. What Is "gating"?

Answer :

Selecting to measure info on only one group of cells.

Question 42. Can Multiple Antigens Be In The Same Tube?

Answer :

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