6.2 CLSI Order of Draw

6.2 CLSI Order of Draw

The Clinical and Laboratory Standards Institute (CLSI) establishes the standard protocol for blood collection sequence known as the Order of Draw. Collecting tubes in the precise CLSI order prevents cross-contamination of chemical additives (additive carryover) from one tube to another, which would alter laboratory results and lead to erroneous patient diagnoses.

Master CLSI Order of Draw & Additive Summary

+-------------------------------------------------------------------------------------------------------+
|                                 MASTER CLSI VENOUS ORDER OF DRAW                                      |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| #  | Stopper / Tube Color | Chemical Additive         | Mode of Action        | Lab Department        |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| 1  | Yellow               | SPS (Sodium Polyanethol   | Anticoagulant;        | Microbiology          |
|    | / Blood Cultures     | Sulfonate) / Media        | Binds calcium         | (Blood Cultures)      |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| 2  | Light Blue           | 3.2% Sodium Citrate       | Binds calcium         | Coagulation           |
|    |                      | (Liquid Anticoagulant)    | (Reversible)          | (PT/INR, aPTT)        |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| 3  | Red / Gold / SST     | Silica Clot Activator /   | Promotes clotting;    | Chemistry, Serology,  |
|    | (Tiger Top)          | Thixotropic Gel           | Gel separates serum   | Immunohematology      |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| 4  | Orange / Rapid Serum | Thrombin                  | Rapid clot activation | STAT Chemistry        |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| 5  | Green / PST          | Sodium / Lithium /        | Inhibits thrombin     | STAT & Routine        |
|    |                      | Ammonium Heparin          | formation             | Plasma Chemistry      |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| 6  | Lavender / Purple    | K2EDTA or K3EDTA          | Binds calcium         | Hematology            |
|    |                      |                           | (Irreversible)        | (CBC, ESR, Hgb)       |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| 7  | Pink                 | K2EDTA                    | Binds calcium         | Blood Bank            |
|    |                      | (Spray-dried)             | (Irreversible)        | (Type & Crossmatch)   |
+----+----------------------+---------------------------+-----------------------+-----------------------+
| 8  | Gray                 | Sodium Fluoride &         | Antiglycolytic;       | Chemistry             |
|    |                      | Potassium Oxalate         | Binds calcium         | (Glucose, Lactic Acid)|
+----+----------------------+---------------------------+-----------------------+-----------------------+

Detailed Breakdown by Tube Type

1. Yellow (SPS) / Blood Culture Bottles

• Additive: Sodium Polyanethol Sulfonate (SPS) in yellow stopper tubes; nutrient broth media in aerobic/anaerobic blood culture bottles.
• Mechanism: SPS prevents clotting by binding calcium and inhibits complement, phagocytosis, and certain antibiotics to allow bacterial growth.
• Primary Tests: Blood cultures to detect bacteremia or septicemia.
• Clinical Note: Always collected FIRST to maintain strict sterility and prevent bacterial contamination from needle contact with non-sterile tube stoppers.

2. Light Blue (Sodium Citrate)

• Additive: 3.2% Sodium Citrate.
• Mechanism: Binds calcium reversibly to prevent coagulation while preserving labile clotting factors (Factor V and Factor VIII).
• Blood-to-Anticoagulant Ratio: Requires an exact 9:1 blood-to-anticoagulant ratio. Underfilling the tube results in excess citrate in the plasma, which falsely prolongs coagulation times (PT/INR and aPTT).
• Butterfly Discard Tube Rule: When using a winged infusion set (butterfly), a discard tube (either a plain red or light blue tube) must be drawn prior to the diagnostic light blue tube to clear air from the tubing and ensure a full 9:1 blood draw.

3. Red, Gold & SST (Clot Activator & Serum Separator)

• Glass Red: No additive (siliconized interior surface).
• Plastic Red & SST / Gold: Contains silica particles as a clot activator. SST (Serum Separator Tubes) also contain a thixotropic polymer gel located at the tube bottom.
• Mechanism: Silica accelerates blood clotting (complete clot forms in 30 minutes). During centrifugation, the gel density shifts, forming a physical barrier between clotted RBCs and liquid serum.
• Department: Routine Chemistry (lipids, liver panels, metabolic panels), Infectious Disease Serology.

4. Orange / Rapid Serum Tube (RST)

• Additive: Thrombin-based clot activator.
• Mechanism: Directly converts fibrinogen to fibrin, inducing full clot formation within 5 minutes.
• Department: Emergency / STAT Serum Chemistry testing.

5. Green / PST (Heparin & Plasma Separator)

• Additive: Lithium Heparin, Sodium Heparin, or Ammonium Heparin (with or without thixotropic gel for PST).
• Mechanism: Heparin inhibits clotting by enhancing antithrombin III, which neutralizes thrombin and Factor Xa.
• Department: STAT Plasma Chemistry, Electrolytes, Troponin, Arterial Blood Gases.
• Clinical Note: Lithium Heparin is preferred for general chemistry; Sodium Heparin must not be used for electrolyte testing (falsely elevates sodium levels).

6. Lavender / Purple (EDTA)

• Additive: Dipotassium EDTA (K2EDTA) or Tripotassium EDTA (K3EDTA).
• Mechanism: Prevents coagulation by irreversibly chelating (binding) calcium ions.
• Department: Hematology—Complete Blood Count (CBC), Hemoglobin & Hematocrit (H&H), Erythrocyte Sedimentation Rate (ESR), Platelet Count.
• Clinical Note: Preserves cell morphology and prevents platelet aggregation better than any other anticoagulant.

7. Pink (EDTA - Blood Bank)

• Additive: Spray-dried K2EDTA.
• Department: Blood Bank (Immunohematology) for ABO/Rh blood typing, antibody screening, and crossmatching.
• Labeling Standard: Requires specialized blood bank identification labels with signature of collector, date, time, and patient ID.

8. Gray (Sodium Fluoride / Potassium Oxalate)

• Additive: Sodium Fluoride (antiglycolytic agent) combined with Potassium Oxalate (anticoagulant).
• Mechanism: Sodium fluoride inhibits the enzyme enolase, stopping glycolysis (cellular glucose breakdown by RBCs). Potassium oxalate binds calcium to block clotting.
• Department: Blood Glucose tolerance tests (GTT), Lactic Acid, Blood Alcohol Concentration (BAC).
• Clinical Note: Sodium fluoride preserves glucose stability for up to 72 hours at room temperature.

Tube Inversion Protocols & Carryover Hazards

+-----------------------------------------------------------------------------------+ 
|                         REQUIRED TUBE INVERSION FREQUENCY                         |
+-----------------------+-----------------------------------+-----------------------+
| Tube Stopper Color    | Additive                          | Required Inversions   |
+-----------------------+-----------------------------------+-----------------------+
| Light Blue            | 3.2% Sodium Citrate               | 3 - 4 Inversions      |
+-----------------------+-----------------------------------+-----------------------+
| Red (Glass)           | None                              | 0 Inversions          |
+-----------------------+-----------------------------------+-----------------------+
| Red (Plastic) / SST   | Silica Clot Activator / Gel       | 5 - 8 Inversions      |
+-----------------------+-----------------------------------+-----------------------+
| Green (Heparin)       | Lithium / Sodium Heparin          | 8 - 10 Inversions     |
+-----------------------+-----------------------------------+-----------------------+
| Lavender / Pink       | K2EDTA / K3EDTA                   | 8 - 10 Inversions     |
+-----------------------+-----------------------------------+-----------------------+
| Gray                  | Sodium Fluoride / Oxalate         | 8 - 10 Inversions     |
+-----------------------+-----------------------------------+-----------------------+

Proper Inversion Technique

One complete inversion consists of turning the filled tube upside down 180 degrees so the air bubble travels to the bottom, then returning it right-side up. Inversions must be performed gently immediately after drawing each tube.

• Vigorous Shaking Hazard: Shaking tubes causes mechanical hemolysis (rupture of red blood cells), releasing intracellular potassium and enzymes into plasma, rendering specimens unusable.
• Incomplete Mixing Hazard: Failing to invert additive tubes results in micro-clot formation, invalidating CBC and coagulation results.

Additive Carryover Risks

If tubes are drawn out of order, additive transfer from needle tip to subsequent tubes creates severe diagnostic errors:

• EDTA Carryover to SST/Serum: EDTA binds calcium and contains potassium. Drawing EDTA before Serum/SST causes falsely decreased calcium levels and falsely elevated potassium levels.
• Heparin Carryover to Light Blue: Heparin transferred into Sodium Citrate tubes falsely prolongs PT and aPTT coagulation times.