Blood Withdrawal, Serum Preparation, Clotting (Standing) Time, and Serum Storage
1.0 Purpose
1.1 To
describe the standardized procedure for blood withdrawal, serum preparation,
clotting (standing) time, centrifugation, serum separation, and storage to
ensure high-quality serum samples for biochemical, immunological, molecular,
and toxicological analyses.
2.0 Scope
2.1 This
SOP applies to all laboratory personnel involved in blood collection and serum
preparation from experimental animals or human samples.
3.0 Responsibilities
3.1 Laboratory
Scientist
3.2 Laboratory
Technician
3.3 Principal
Investigator
3.4 Quality
Assurance Personnel
4.0 Materials Required
4.1 Plain
Blood Collection Tube (Red Top)
4.2 Serum
Separator Tube (SST, Yellow/Gold Top)
4.3 Rapid
Serum Tube (RST, Orange Top; optional)
4.4 Sterile
Syringe/Needle
4.5 Micropipettes
and Sterile Tips
4.6 Refrigerated
Centrifuge
4.7 Cryovials
4.8 Labels
and Marker
4.9 Personal
Protective Equipment (PPE)
5.0 Procedure
5.1 Blood Withdrawal
5.1.1. Collect
blood using aseptic technique.
5.1.2. Transfer
blood gently into the appropriate collection tube.
5.1.3. Avoid
vigorous shaking.
5.1.4. Mix
tubes containing additives by gentle inversion (5–8 times).
5.1.5. Keep
plain tubes upright.
Note: Do not centrifuge immediately
after blood collection.
5.2 Standing (Clotting)
Time Before Centrifugation
5.2.1. Allow
blood to stand upright at 20–25°C (Room Temperature).
5.2.2. Important
5.2.2.1. Do
not disturb the clot.
5.2.2.2. Do
not refrigerate before clot formation.
5.2.2.3. Avoid
standing for more than 2 hours before centrifugation.
5.3 Centrifugation
5.3.1. Centrifuge
after complete clot formation.
5.3.1.1 Recommended
Conditions
- 1500–2000 × g
- 10–15 minutes
- Room temperature or
refrigerated centrifuge (as per assay requirement)
5.4 Serum Separation
5.4.1. Carefully
remove the serum without disturbing the clot.
5.4.2. Transfer
serum into labeled sterile cryovials.
5.4.3. Avoid
transferring red blood cells or fibrin.
5.4.4. Prepare
aliquots whenever possible.
6.0 Serum Storage Condition
6.1 Immediate Analysis
- Store at 2–8°C
- Analyze preferably within 24–48 hours
6.2 Short-Term Storage
6.5 Recommended
for:
- Cytokines
- Hormones
- Biomarkers
- Proteins
- DNA/RNA studies
- Metabolomics
7.0 Storage Precautions
7.1 Label
every aliquot clearly.
7.2 Store
in small aliquots.
7.3 Avoid
repeated freeze–thaw cycles.
7.4 Thaw
samples only once whenever possible.
7.5 Mix
gently after thawing.
7.6 Do not use hemolyzed serum unless validated.
8.0 Sample Rejection
Criteria
8.1 Reject
samples showing:
8.1.1. Severe
hemolysis
8.1.2. Lipemia
(if unsuitable for assay)
8.1.3. Incorrect
labeling
8.1.4. Insufficient
volume
8.1.5. Incomplete
clotting
8.1.6. Leakage
or contamination
9.0 Quality Control
9.1 Verify
centrifuge speed and calibration.
9.2 Monitor
refrigerator (2–8°C) daily.
9.3 Monitor
freezer temperatures (−40°C and −80°C) continuously.
9.4 Maintain
sample storage logs.
10.0 Blood Withdrawal to Serum Flow Chart
11.0 References
- CLSI GP44 (formerly H18-A4):
Procedures for the Handling and Processing of Blood Specimens for
Common Laboratory Tests.
- CLSI GP41: Collection of
Diagnostic Venous Blood Specimens.
- WHO. Guidelines on
Drawing Blood: Best Practices in Phlebotomy.
- Tietz Textbook of Clinical
Chemistry and Molecular Diagnostics, Latest Edition.
- ISO 15189: Medical
Laboratories—Requirements for Quality and Competence.
Note: Storage duration depends on the
analyte being measured. For highly sensitive biomarkers (e.g., cytokines,
hormones, RNA), follow the specific assay manufacturer's recommendations if they
differ from this general SOP.
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