Blood Aliquoting Procedure for a 9-Month-Old Child
Transfer blood from the collection tube to an intermediary conical tube after centrifugation, mix thoroughly by inverting 8-10 times, then aliquot 250 μL to 1 mL volumes into labeled cryovials with O-ring-sealed screw caps. 1
Critical Pre-Aliquoting Steps
Centrifugation and Initial Processing
- Centrifuge blood collection tubes for 10 minutes at 1500 to 2000 × g at 4°C 1
- For plasma collection (EDTA or heparin tubes): Immediately after blood draw, gently invert tubes 8-10 times, then centrifuge within 2 hours 1
- For serum collection: Allow blood to clot upright at room temperature for 30-60 minutes before centrifugation 1
Transfer to Intermediary Tube
- Use a disposable transfer pipette to carefully transfer the plasma or serum (top layer) to a 15 mL conical tube, avoiding disturbance of the buffy coat layer or red blood cells 1
- This intermediary step is essential because direct aliquoting from blood collection tubes after centrifugation leads to heterogeneity among aliquots due to centrifugation forces 1
- Leave the bottom ~10% of plasma/serum behind to prevent cross-layer contamination 1
Aliquoting Procedure
Mixing for Homogeneity
- Before aliquoting, gently invert the conical intermediary tube 8-10 times to ensure homogeneous distribution 1
- Alternatively, pipette up and down multiple times to achieve uniform mixing 1
- This mixing step is critical to prevent heterogeneity among aliquots 1
Volume and Container Specifications
- Aliquot 250 μL to 1 mL volumes into labeled microtubes or cryovials with O-ring-sealed screw caps 1
- Use printed labels or barcodes rather than handwritten labels to avoid sample mix-up 1
- Label tubes in advance of the blood collection visit 1
Storage
- Store all aliquots upright in a specimen box in an -80°C or colder freezer 1
- Aliquoting minimizes the impact of freeze/thaw cycles on sample quality 1
Pediatric-Specific Considerations
Blood Volume Context
- Estimated blood volume in a 9-month-old infant is approximately 80-100 mL/kg 2
- For a typical 9-month-old weighing 8-9 kg, total blood volume is approximately 640-900 mL
- Blood culture volumes for this age/weight should be 1-1.5 mL per bottle 3
- Each 10 mL of blood loss represents a clinically significant proportion of total blood volume in infants 4
Safety and Quality Control
- Always use proper personal protective equipment (PPE) as all blood samples should be considered potentially infectious 1
- Inspect samples for hemolysis using a reference chart (such as CDC Hemolysis Reference Palette), as hemolysis significantly deteriorates sample quality 1
- Good pipetting skills are essential: gently draw liquid from the top and gradually move the pipette down with the liquid 1
Common Pitfalls to Avoid
- Never aliquot directly from blood collection tubes after centrifugation without first transferring to an intermediary tube and mixing 1
- Avoid disturbing the buffy coat (whitish middle layer) or red blood cell layer when pipetting 1
- Do not include personal information on specimen labels to protect confidentiality 1
- Avoid prolonged delays: blood should be centrifuged within 2 hours of collection and stored at 4°C if immediate processing is not possible 1