What is the correct method for collecting a venous blood sample for plasma ammonia measurement, including patient positioning, minimal tourniquet time, needle gauge, use of a pre‑chilled lithium‑heparin (green‑top) tube, ice handling, and timing of transport, centrifugation, and analysis?

Blood Collection for Ammonia Level Measurement

Blood for ammonia measurement must be collected from free-flowing venous or arterial blood into a lithium heparin or EDTA tube, transported immediately on ice, and processed within 15 minutes to prevent falsely elevated results. 1

Critical Collection Technique

Tourniquet Use and Blood Flow

• Minimize or avoid tourniquet use entirely – tourniquets cause blood cell activation and hemolysis, which artificially elevate ammonia levels 1
• If a tourniquet must be used, apply it only briefly to locate the vein with gentle pressure, then release or maintain minimal pressure during collection 1
• Ensure free-flowing blood without intermediate stopping to prevent cellular activation and ammonia release 1

Needle Selection and Sample Handling

• Use a large-bore 21-gauge needle to facilitate smooth blood flow and minimize hemolysis 1
• Discard the first 2-3 mL of blood to avoid tissue thromboplastin contamination and cellular activation 1
• Collect blood directly into the tube rather than using a syringe, as syringes lack anticoagulant and promote coagulation activation 1

Tube Selection and Mixing

• Use plastic collection tubes (lithium heparin or EDTA) rather than glass to avoid contact activation 1
• Fill tubes completely to achieve the proper blood-to-anticoagulant ratio 1
• Immediately invert tubes gently 8-10 times after collection to mix blood with anticoagulant 1
• Keep tubes in a vertical upright position during transport 1

Immediate Post-Collection Handling

Temperature Control

• Place tubes on ice immediately after collection – ammonia increases by approximately 0.09 μmol/L per minute at room temperature in healthy subjects 2
• Transport samples on ice to the laboratory without delay 1, 3
• Avoid direct contact of tubes with ice to prevent cell lysis, but maintain cold temperature 1

Processing Timeline

• Process samples within 15 minutes of collection – this is the critical window to prevent in vitro ammonia generation 1, 3
• If immediate processing is impossible, samples stored at room temperature show significant ammonia increases after just 1 hour 4
• Centrifuge at 1500-2000 × g for 10 minutes at 4°C to separate plasma 1
• Analyze plasma immediately after separation – ammonia is not stable even when refrigerated or frozen 4

Common Pitfalls and Critical Warnings

Pre-Analytical Errors to Avoid

• Never delay processing beyond 15 minutes – delayed recognition and treatment of hyperammonemia can lead to irreversible neurological damage, and falsely elevated results from poor sample handling can confuse clinical decision-making 1, 3
• Reject hemolyzed samples – hemolysis releases intracellular ammonia and produces falsely elevated results 1
• Samples with elevated ALT or GGT show accelerated ammonia generation (3.6 μmol/L per hour vs 0.5 μmol/L per hour in normal samples) when stored at 4°C 5

Storage Limitations

• Ammonia cannot be reliably stored – even at -20°C, average bias exceeds acceptable limits after just 3 hours of storage 4
• If analysis must be delayed, specialized stabilization methods (sodium borate/L-serine additives) can extend stability to 12 hours at 4°C, but this requires laboratory-specific protocols 5
• Standard freezing at -80°C provides some stability (0.2 μmol/L per hour increase), but immediate analysis remains the gold standard 5

Clinical Context

• Normal ammonia levels are ≤35 μmol/L, with hyperammonemia defined as >100 μmol/L in neonates or ≥50 μmol/L in older patients 6
• Duration of hyperammonemic coma >3 days or ammonia levels >1,000 μmol/L predict irreversible neurological damage, making accurate and timely measurement critical for patient outcomes 1, 6
• Some laboratories will accept non-ideal samples with appropriate interpretive comments rather than reject them and delay diagnosis, but this should be discussed with the laboratory director 2