1.3 Order of Draw, Additives & Tube Inversions

1.3 Order of Draw, Additives & Tube Inversions

To ensure accurate diagnostic results, blood collection tubes contain specialized chemical additives designed to prevent coagulation, preserve specific biochemical compounds, accelerate clot formation, or separate blood components. However, drawing tubes in the wrong sequence leads to additive carryover—a condition where chemical additives from one tube cross-contaminate the needle tip and enter a subsequent tube. To prevent cross-contamination, the Clinical and Laboratory Standards Institute (CLSI) established a standardized Order of Draw that must be rigorously followed for all blood collection procedures.

The CLSI Order of Draw Standard

The CLSI order of draw is designed to prevent carryover of anticoagulants, clot activators, and preservatives that could alter laboratory test results.

1. Sterile Blood Cultures (SPS / Yellow)
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2. Light Blue (Sodium Citrate - Coagulation)
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3. Red / Gold / SST (Clot Activator / Gel Separator)
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4. Green (Heparin - Lithium / Sodium)
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5. Lavender / Purple (EDTA - K2 / K3)
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6. Gray (Sodium Fluoride / Potassium Oxalate)
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7. Yellow (ACD / SPS Special Genetics)

Figure 1.4: Standard CLSI Order of Draw for Evacuated Tube System Collection.

Detailed Breakdown of the Order of Draw

1. Blood Culture Bottles / Yellow SPS Tubes

• Additive: Sodium Polyanethol Sulfonate (SPS) or nutrient broth media.
• Primary Function: Preserves viable bacteria or fungi while inhibiting human complement factors, phagocytosis, and certain antibiotics.
• Why First: Drawn first to prevent bacterial contamination from non-sterile tube stoppers or skin flora on subsequent equipment.

2. Light Blue Top Tubes (Coagulation Panel)

• Additive: 3.2% Sodium Citrate.
• Primary Function: Binds (chelates) calcium reversibly to prevent blood clotting while preserving coagulation factors. Used for Prothrombin Time (PT/INR) and Activated Partial Thromboplastin Time (aPTT).
• The Critical 9:1 Ratio: Must be filled to the etched fill line to achieve an exact 9 parts blood to 1 part sodium citrate ratio. Underfilled tubes contain excess free citrate, which binds reagent calcium during testing, causing falsely prolonged coagulation times.

3. Red, Gold, and Speckled SST Tubes (Serum Tubes)

• Additive: Plastic Red tops contain silica clot activators; Gold and Tiger-top Serum Separator Tubes (SST) contain silica clot activators plus a inert polymer gel.
• Primary Function: Silica particles accelerate natural blood clotting (typically within 30 minutes). The polymer gel shifts during centrifugation to form a physical barrier between the red blood cell clot and the liquid serum. Used for Chemistry panels, Serology, and Immunology.

4. Green Top Tubes (Heparin Plasma Tubes)

• Additive: Lithium Heparin or Sodium Heparin.
• Primary Function: Anticoagulant that enhances the activity of antithrombin, which directly inhibits thrombin and Factor Xa. Preferred for STAT Chemistry determinations because blood can be centrifuged immediately without waiting for a clot to form.

5. Lavender / Purple Top Tubes (Hematology Tubes)

• Additive: Dipotassium (K2) EDTA or Tripotassium (K3) EDTA (Ethylenediaminetetraacetic Acid).
• Primary Function: Irreversibly chelates calcium, preserving blood cell morphology and preventing platelet clumping. Essential for Complete Blood Count (CBC), Differential, Hemoglobin A1c, and Blood Bank testing.

6. Gray Top Tubes (Glycolytic Inhibitor Tubes)

• Additive: Sodium Fluoride (antiglycolytic agent) co-added with Potassium Oxalate (anticoagulant).
• Primary Function: Sodium fluoride inhibits the enzyme enolase, stopping red blood cells from consuming glucose (glycolysis) for up to 24 hours. Potassium oxalate binds calcium to stop clotting. Used for Glucose Tolerance Tests (GTT), Blood Alcohol Levels, and Lactate measurements.

7. Yellow Top Tubes (ACD / Specialty)

• Additive: Acid Citrate Dextrose (ACD).
• Primary Function: Preserves red cell viability and DNA for Human Leukocyte Antigen (HLA) phenotyping, paternity testing, and DNA studies.

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Additive Mechanisms of Action

Understanding how additives act chemically is vital for troubleshooting specimen rejections:

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Mixing Requirements & Inversion Counts

Immediately after drawing an additive tube, the phlebotomist must gently invert the tube to mix the blood with the additive. Inadequate mixing leads to micro-clot formation in EDTA/Heparin tubes or incomplete clotting in SST tubes, while vigorous shaking causes severe mechanical hemolysis.

Specific Inversion Protocols

• Light Blue (Sodium Citrate): 3 to 4 gentle inversions. Minimal mixing prevents premature platelet activation and clot cascade triggering.
• Red Glass (No Additive): 0 inversions.
• Plastic Red / SST / Gold (Clot Activator): 5 gentle inversions to distribute silica particles thoroughly throughout the blood.
• Green (Heparin): 8 to 10 gentle inversions to prevent micro-clot formation.
• Lavender / Purple (EDTA): 8 to 10 gentle inversions to ensure complete calcium binding.
• Gray (Fluoride / Oxalate): 8 to 10 gentle inversions to dissolve oxalate crystals and distribute fluoride.

⚠️ CRITICAL PRACTICE RULE: Inversion Technique One complete inversion consists of turning the tube upside down 180 degrees until the air bubble travels to the bottom, and then returning it right-side up. Never shake blood collection tubes vigorously.

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Cross-Contamination & Additive Carryover Hazards

Additive carryover occurs when blood containing an additive touches the stopper end of the posterior needle or when tiny droplets cling to the needle tip as tubes are swapped.

Classic Carryover Error Scenarios

1. EDTA Carryover to Heparin or Serum Tubes

If a lavender EDTA tube is drawn before a green heparin or gold serum tube:

• EDTA contains high concentrations of potassium (K2/K3 EDTA) and chelates calcium.
• Carryover into a serum tube causes a falsely sky-high Potassium result and a falsely depleted Calcium result. This dangerous lab error can lead clinicians to misdiagnose hyperkalemia or cardiac emergency.

2. Heparin Carryover to Coagulation Tubes

If a green heparin tube is drawn before a light blue sodium citrate tube:

• Heparin enters the sodium citrate tube and inhibits thrombin.
• Test results will show falsely prolonged PT and aPTT times, leading clinicians to believe a patient is over-anticoagulated.