Sample Inegrity

Overview of Sample Integrity

Definition: Sample integrity refers to the degree to which a sample accurately represents the biological state of the cells or molecules of interest at the time of analysis
Importance:
- Accurate Results: Poor sample integrity can lead to inaccurate and unreliable results
- Reproducibility: Maintaining sample integrity is essential for reproducible experiments
- Valid Conclusions: Only high-quality samples can provide valid conclusions about the biological system being studied
Key Factors Affecting Sample Integrity:
- Collection
- Handling
- Storage
- Processing

Best Practices:
- Proper Identification: Clearly label all sample tubes with patient or sample identifiers, date, and time of collection
- Appropriate Collection Tubes: Use appropriate collection tubes that are compatible with the sample type and downstream analysis
- Anticoagulants: Use appropriate anticoagulants (e.g., EDTA, heparin) to prevent clotting of blood samples
- Standardized Procedures: Follow standardized collection procedures to minimize variability and ensure consistency
- Quick Action: Record date and time and send to lab for timely processing.
Specific Considerations:
- Blood: Collect blood samples using a standardized phlebotomy technique
- Bone Marrow: Collect bone marrow aspirates using sterile techniques
- Solid Tissue: Collect tissue samples using appropriate surgical techniques
- Body Fluids: Collect body fluids using sterile techniques
Actions to Avoid:
- Collection delays
- Over or Under filling tubes

Best Practices:
- Gentle Handling: Handle samples gently to avoid cell damage and aggregation
- Temperature Control: Maintain samples at appropriate temperatures to preserve cell viability and phenotype
- Proper Mixing: Mix samples gently to ensure that cells are evenly distributed
- Minimize Delays: Process samples as quickly as possible after collection
Specific Considerations:
- Light Exposure: Protect light-sensitive samples from prolonged exposure to light
- Vortexing: Avoid vigorous vortexing of samples, which can damage cells
Actions to Avoid:
- Forceful pipetting
- Leaving specimen out of collection container for long period of time

Best Practices:
- Appropriate Temperature: Store samples at appropriate temperatures to preserve cell viability and phenotype
  - Room Temperature (18 - 25°C): Good for some whole blood samples for short-term storage (check specific assay requirements)
  - Refrigerated (2 - 8°C): Common for short-term storage of many sample types
  - -20°C or -80°C: Often used for long-term storage of serum, plasma, or purified cell fractions
  - Liquid Nitrogen (-196°C): For cryopreservation of cells, requires specific protocols and cryoprotective agents
- Cryopreservation: Cryopreserve samples in liquid nitrogen or at -80°C for long-term storage
- Stabilization Reagents: Use appropriate stabilization reagents (e.g., cell stabilizers) to prevent degradation during storage
- Controlled Thawing: Thaw cryopreserved samples rapidly and gently to minimize cell damage
Actions to Avoid:
- Thawing then refreezing specimen

Best Practices:
- Standardized Protocols: Follow standardized processing protocols to minimize variability
- Reagent Quality: Use high-quality reagents that are free from contaminants
- Appropriate Techniques: Use appropriate techniques to isolate and purify cells
Specific Considerations:
- Red Blood Cell Lysis: Use appropriate lysing buffers and incubation times to remove red blood cells without damaging target cells
- Cell Washing: Use appropriate washing buffers and centrifugation speeds to remove unwanted debris and reagents
Actions to Avoid:
- Extended processing times
- Using harsh chemicals during processing

Cell Viability Assays:
- Trypan Blue Exclusion: Differentiates between live and dead cells based on membrane integrity
- Propidium Iodide (PI) Staining: Identifies cells with compromised membranes by binding to DNA
- 7-AAD Staining: Another DNA-binding dye used to exclude non-viable cells
- Annexin V Apoptosis Detection: Detects cells undergoing apoptosis by binding to phosphatidylserine on the cell surface
Visual Inspection:
- Microscopic Evaluation: Examine samples under a microscope to assess cell morphology, aggregation, and presence of debris
Automated Cell Counters:
- Determine cell concentration and viability using automated cell counters with viability assessment capabilities
Flow Cytometry Scatter Properties:
- Forward Scatter (FSC) and Side Scatter (SSC): Assess cell size, granularity, and homogeneity. Shifts in these parameters can indicate changes in cell integrity
RNA Integrity Number (RIN):
- For RNA analysis, use RIN to assess the degradation of RNA
Assessment of Surface Marker Expression:
- Evaluate the expression of specific surface markers known to be sensitive to changes in cell state.

Low Event Count:
- Possible Causes:
  - Cell death during collection, handling, or storage
  - Loss of cells during processing
- Troubleshooting Steps:
  - Optimize collection, handling, and storage protocols
  - Minimize cell loss during processing
High Background Noise:
- Possible Causes:
  - Dead cells and debris
  - Non-specific antibody binding
- Troubleshooting Steps:
  - Remove dead cells and debris
  - Use blocking reagents
Poor Resolution:
- Possible Causes:
  - Cell aggregates
  - Degraded antigens
- Troubleshooting Steps:
  - Minimize cell aggregates
  - Optimize staining protocols
Inaccurate Results:
- Possible Causes:
  - Incorrect cell processing, incorrect storage temperature
- Troubleshooting Steps:
  - Verify cell processing methods and storage temperature

Sample Integrity: The degree to which a sample accurately represents the biological state of the cells
Anticoagulant: A substance that prevents blood from clotting
Cryopreservation: Preserving cells by freezing them at very low temperatures
Cell Viability: The percentage of live cells in a sample
Apoptosis: Programmed cell death