SCYM Sample Preparation & Staining Techniques 3 — Questions and Answers
Question 1: What is the recommended maximum time between blood draw and staining for optimal flow cytometric immunophenotyping?
- 30 minutes
- 2 hours
- 24-48 hours (Correct answer)
- 72 hours
Correct answer: 24-48 hours
EDTA-anticoagulated whole blood can be stained within 24-48 hours of collection when stored at room temperature, though same-day processing is ideal.
Question 2: Which anticoagulant is preferred for flow cytometric analysis of platelet activation markers?
- EDTA
- Heparin
- Sodium citrate (Correct answer)
- ACD (acid-citrate-dextrose)
Correct answer: Sodium citrate
Sodium citrate is preferred for platelet studies because EDTA chelates calcium and can activate or alter platelet surface antigens, while citrate better preserves platelet function.
Question 3: When staining bone marrow aspirates, which step is critical to perform BEFORE antibody addition?
- Heat treatment at 56°C
- Counting viable cells and adjusting to the target cell concentration (Correct answer)
- Addition of secondary antibodies
- Red cell lysis only after antibody staining
Correct answer: Counting viable cells and adjusting to the target cell concentration
Adjusting to a consistent cell concentration (typically 1×10⁶ cells per test) before antibody addition ensures accurate antibody-to-cell ratios and prevents under- or over-saturation.
Question 4: Which viability dye is excluded by live cells and enters only cells with compromised membranes?
- Annexin V
- Calcein AM
- 7-Aminoactinomycin D (7-AAD) (Correct answer)
- DiOC6
Correct answer: 7-Aminoactinomycin D (7-AAD)
7-AAD is a membrane-impermeant DNA intercalating dye that only enters cells with damaged membranes, making it an effective dead cell exclusion dye.
Question 5: What is the purpose of adding DNase to dissociated tissue samples prior to flow cytometry?
- To digest surface glycoproteins
- To reduce cell clumping caused by free DNA from dead cells (Correct answer)
- To permeabilize live cells
- To fix intracellular proteins
Correct answer: To reduce cell clumping caused by free DNA from dead cells
Free DNA released from dead cells is sticky and causes cell clumping; DNase digests this free DNA to produce a single-cell suspension.
Question 6: Which technique is used to assess cell cycle distribution by flow cytometry?
- Annexin V/PI staining
- Propidium iodide (PI) staining of fixed, permeabilized cells (Correct answer)
- CFSE dilution assay
- BrdU incorporation only
Correct answer: Propidium iodide (PI) staining of fixed, permeabilized cells
PI stains total DNA stoichiometrically after fixation and permeabilization, allowing G0/G1, S, and G2/M phases to be distinguished by DNA content.
Question 7: A laboratory receives a lymph node biopsy for flow cytometry. What is the first step in sample preparation?
- Freeze the tissue immediately at -80°C
- Mechanical disaggregation to create a single-cell suspension (Correct answer)
- Fix the tissue in formalin
- Add antibodies directly to the intact tissue
Correct answer: Mechanical disaggregation to create a single-cell suspension
Solid tissue must first be mechanically disaggregated (through a cell strainer or by mincing) to produce a single-cell suspension suitable for flow cytometric analysis.
SCYM Sample Preparation & Staining Techniques 3
What is the recommended maximum time between blood draw and staining for optimal flow cytometric immunophenotyping?