Verifies records and signs off on any deviations during thawing.

Lab Supervisor: Ensures environmental conditions and equipment used are compliant.

4. Accountability

The Head of Bioanalytical Operations is accountable for ensuring that sample integrity is maintained during thawing and equilibration and that procedures are compliant with regulatory expectations.

5. Procedure

5.1 Retrieval from Storage

1. Use the Sample Request Log to retrieve required biological samples from -20°C or -70°C freezers.
2. Record retrieval date, time, analyst name, and freezer temperature in Annexure-1: Sample Retrieval Log.
3. Ensure minimal disturbance and return unused samples to appropriate storage without delay.

5.2 Thawing Procedure

1. Place samples in labeled, secondary containment racks.
2. Allow thawing at controlled room temperature (20–25°C) on the benchtop.
3. Monitor thawing visually—do not expose samples to direct sunlight or warm baths.
4. Record start and end time of thawing; ensure thawing duration does not exceed 1 hour unless otherwise justified.
5. Do not thaw using high-temperature devices like heating blocks or ovens.

5.3 Equilibration

1. Once thawed, allow samples to equilibrate at room temperature for 10–15 minutes before vortexing.
2. Gently mix using a vortex mixer for 10 seconds to homogenize the matrix content.
3. Avoid repeated vortexing or agitation to prevent hemolysis or degradation.

5.4 Inspection and Handling

1. Inspect each sample for:
   • Presence of clots or precipitates
   • Color changes indicating hemolysis
   • Container integrity (leakage or cracks)
2. Document any abnormalities in Annexure-2: Thawing Observation Sheet.

5.5 Deviation Handling

1. If sample is found to be damaged, label it as “Do Not Use” and segregate for further review.
2. Initiate a deviation report and notify QA within 24 hours.
3. Replacement or backup samples (if available) may be used following protocol approval.

5.6 Post-Thaw Handling

1. Processed samples should be immediately used or aliquoted and returned to designated freezer if not used within 2 hours.
2. Repeat freeze-thaw cycles should be avoided unless validated during method development.
3. Document freeze-thaw events using Annexure-3: Freeze-Thaw Tracking Log.

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• LC-MS/MS: Liquid Chromatography-Mass Spectrometry/Mass Spectrometry
• QA: Quality Assurance

7. Documents

1. Sample Retrieval Log – Annexure-1
2. Thawing Observation Sheet – Annexure-2
3. Freeze-Thaw Tracking Log – Annexure-3

8. References

• ICH M10 – Bioanalytical Method Validation
• US FDA Bioanalytical Method Validation Guidance
• Internal Lab SOPs on Sample Management

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Sample Retrieval Log

Date	Sample ID	Freezer Temp	Retrieved By	Return Status
16/04/2025	PLM-1023	-70°C	Rajesh Kumar	Returned

Annexure-2: Thawing Observation Sheet

Sample ID	Condition	Color	Clots	Remarks
PLM-1023	Thawed	Pale Yellow	None	OK

Annexure-3: Freeze-Thaw Tracking Log

Sample ID	No. of Cycles	Dates	Authorized By
PLM-1023	1	17/04/2025	Sunita Reddy

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/01/2022	1.0	Initial release	New SOP	QA Head
17/04/2025	2.0	Expanded thawing procedures and added freeze-thaw log	Stability Compliance	QA Head