Phlebotomy Order of draw
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Phlebotomy: Order of Draw
The order of draw is the standardized sequence for filling blood collection tubes during venipuncture. Its purpose is to prevent cross-contamination of additives between tubes, which can cause spurious lab results (e.g., EDTA carry-over causing false hyperkalemia, or heparin interfering with coagulation tests).
Standard Order of Draw for Venipuncture (Evacuated Tube System)
Based on CLSI guidelines (GP41), as documented in the Tietz Textbook of Laboratory Medicine (Table 4-2):
| # | Stopper Color | Additive / Contents | Inversions | Common Tests |
|---|---|---|---|---|
| 1 | Yellow | Sterile media (blood culture) | 8 | Blood cultures (anaerobic/aerobic) |
| 2 | Royal blue (no additive) | None | 0 | Trace elements, toxicology |
| 3 | Clear (discard) | None - fills dead space in collection set | 0 | Discard tube (used with butterfly sets before coagulation tube) |
| 4 | Light blue | Sodium citrate 3.2% | 3-4 | PT, aPTT, coagulation studies |
| 5 | Gold / Red-Gray | Serum separator tube (SST) with clot activator + gel | 5 | Chemistry, serology |
| 6 | Red | Serum tube, with or without clot activator | 5 | Serum chemistry, immunology |
| 7 | Green | Lithium heparin (with or without gel) | 8 | Plasma chemistry, stat tests |
| 8 | Tan (glass) | Sodium heparin | 8 | Lead levels |
| 9 | Royal blue (additive) | Sodium heparin or EDTA (trace metal free) | 8 | Trace metals |
| 10 | Lavender / Pink / White | EDTA (K2 or K3), with or without gel | 8 | CBC, hematology, blood bank |
| 11 | Gray | Sodium fluoride / potassium oxalate (glycolytic inhibitor) | 8 | Glucose, lactate |
| 12 | Yellow (glass) | ACD (acid-citrate-dextrose) | 8 | Molecular studies, cell culture |
Modified from CLSI GP39-A6 and GP41, as cited in Tietz Textbook of Laboratory Medicine, 7th Ed., p. 128
Memory Aid
A common mnemonic: "Stop Looking Right There, Grab Large Elephants Gracefully"
Or simply remember the principle: No anticoagulant → mild anticoagulant → strong anticoagulant
Key Rules & Rationale
1. Blood Cultures First
- Collected first to minimize contamination risk from skin flora and additives.
- Requires careful antiseptic skin prep.
2. Light Blue (Citrate) - Critical Rules
- Must maintain a 9:1 blood-to-anticoagulant ratio (fill to the line).
- Underfilling yields excess citrate → falsely elevated clotting times.
- With a straight needle: no discard tube is needed even if it is the first/only tube.
- With a butterfly/winged set: a discard tube (clear) MUST be drawn first to fill the dead space of the tubing and prevent underfilling of the citrate tube.
- EDTA and heparin are NOT acceptable for coagulation tests - they cause erroneous results.
(Tietz Textbook of Laboratory Medicine, 7th Ed., p. 186)
3. Why Serum (Red/Gold) Before Heparin (Green) and EDTA (Lavender)?
- Heparin contamination of a serum tube would interfere with many assays.
- EDTA chelates calcium; EDTA carry-over can cause false hyperkalemia/hypocalcemia and interfere with metal-dependent enzyme assays.
4. Gray (Fluoride/Oxalate) is Always Last
- Oxalate is a strong anticoagulant that also inhibits enzymes; it must not contaminate any other tube.
Order of Draw for Catheter Lines (Central Venous Access)
When drawing from an indwelling catheter (per Henry's Clinical Diagnosis and Management by Laboratory Methods):
- Draw 3-5 mL and discard (clears the line of heparin/IV fluids)
- Blood cultures
- Anticoagulated tubes (lavender, green, light blue)
- Clot tubes (red, SST)
Note: Coagulation tests drawn from catheters require even larger discard volumes due to heparin sensitivity. If heparin contamination is suspected, heparin neutralization of the sample should be considered.
Order of Draw for Skin Puncture (Capillary Blood)
The order is different from venipuncture:
| Order | Usage / Additive | Tube Color |
|---|---|---|
| 1st | Blood gases (heparin) | Microhematocrit / capillary tubes |
| 2nd | EDTA | Lavender |
| 3rd | Other heparin | Green |
| 4th | Other additives | Light blue, gray |
| 5th | Non-additives | Red, tiger, yellow |
Tietz Textbook of Laboratory Medicine, 7th Ed., Table 4.5, p. 128
The rationale differs: EDTA tubes are drawn early in capillary collection because platelet clumping begins rapidly after skin puncture, and hematology samples (CBC) require the most accurate cell counts.
Sources
- Tietz Textbook of Laboratory Medicine, 7th Edition, p. 128, 186 (Table 4-2, 4-5)
- Henry's Clinical Diagnosis and Management by Laboratory Methods - Chapter 3, Box 3.2 and Box 3.7
- Based on CLSI GP41 guidelines
Teach me vacutaners first
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Excellent - I now have rich textbook data from both Tietz and Henry's. Let me put together a thorough, visual teaching guide.
Vacutainers - Complete Guide
A Vacutainer is an evacuated (pre-vacuumed) blood collection tube. The vacuum draws a precise volume of blood, and the stopper color tells you everything: what additive is inside, what specimen type it produces, and what tests it's used for.
The Core Concept: What Happens Inside the Tube?
There are only 3 outcomes when blood enters a tube:
| Outcome | How | Tubes |
|---|---|---|
| Clots → Serum | No anticoagulant, or clot activator added | Red, Gold/SST, Orange |
| Stays liquid → Plasma | Anticoagulant binds or inhibits clotting factors | Light blue, Green, Lavender, Gray, Pink |
| Stays whole blood | No separation needed | Lavender (for CBC), Pink |
Serum = plasma minus clotting factors (fibrinogen etc.) Plasma = fluid portion of unclotted blood (retains clotting factors)
Tube-by-Tube Breakdown
🟡 Yellow - Blood Cultures
| Additive | Sodium polyanethol sulfonate (SPS) in sterile culture media |
| Specimen | Whole blood for microbiology |
| Inversions | 8 |
| Tests | Aerobic & anaerobic blood cultures |
| Why first | Needs sterile collection; drawn before any additive contamination |
🔵 Light Blue - Coagulation
| Additive | Sodium citrate 3.2% (0.105 M) |
| Mechanism | Chelates (binds) calcium - calcium is required for clotting cascade |
| Specimen | Plasma (citrated plasma) |
| Inversions | 3-4 gentle |
| Tests | PT, aPTT, INR, fibrinogen, D-dimer, thrombin time, factor assays |
| Blood:Anticoagulant ratio | 9:1 - CRITICAL - must fill to the line |
Key facts:
- Underfilling = too much citrate relative to blood = falsely prolonged PT/aPTT
- With a butterfly needle, draw a clear discard tube first to fill the dead space of the tubing
- EDTA and heparin are NOT acceptable substitutes for coagulation testing
"Coagulation tests require the correct ratio of blood and anticoagulant; thus containers should be adequately filled. An insufficient blood collection volume yields excess citrate in plasma and falsely elevated clotting times." - Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 48
⚫ Black - ESR (Erythrocyte Sedimentation Rate)
| Additive | Buffered sodium citrate |
| Blood:Anticoagulant ratio | 4:1 (different from light blue!) |
| Specimen | Whole blood |
| Tests | Westergren sedimentation rate |
🔴 Red (Glass) - Serum, No Additive
| Additive | None (silicone-coated interior only) |
| Mechanism | Blood clots naturally |
| Specimen | Serum (after 30-60 min clot time + centrifugation) |
| Inversions | 0 |
| Tests | Chemistry, serology, immunology, blood bank, drug levels |
🔴 Red (Plastic / Hemogard) - Serum with Clot Activator
| Additive | Silica clot activator |
| Mechanism | Silica particles accelerate clot formation |
| Specimen | Serum |
| Inversions | 5 |
| Tests | Same as glass red; faster clot time (~15-30 min) |
🟡🔴 Gold / Tiger-top / SST (Serum Separator Tube)
| Additive | Clot activator + thixotropic polymer gel |
| Mechanism | Gel separates serum from clot during centrifugation (rises and forms a barrier) |
| Specimen | Serum |
| Inversions | 5 |
| Tests | Chemistry panels (BMP, CMP), LFTs, lipids, thyroid, hormones, serology |
The gel's trick: During centrifugation, the gel (originally at the bottom) liquefies, rises between the clot and serum, then re-solidifies - creating a physical barrier. This makes it ideal for transport and storage.
🟠 Orange / Gray-Yellow - Stat Chemistry
| Additive | Thrombin (dry) |
| Mechanism | Thrombin rapidly activates clotting - serum ready in ~5 minutes |
| Specimen | Serum |
| Inversions | 5-6 |
| Tests | Stat/emergency chemistry tests |
🟢 Green - Heparin (Plasma)
| Additive | Lithium heparin (LiHep) or sodium heparin (NaHep) |
| Mechanism | Heparin activates antithrombin III → neutralizes thrombin → prevents fibrin formation |
| Specimen | Plasma (heparinized) |
| Inversions | 8 |
| Tests | Stat plasma chemistry, ammonia, chromosomes, HLA typing |
Lithium vs Sodium Heparin:
- LiHep (green) - used for most chemistry tests. Cannot be used for lithium levels (will falsely elevate them)
- NaHep - used for trace elements, lead, toxicology. Cannot be used for sodium levels
- Heparin should NOT be used for coagulation or hematology testing
- Heparin has an advantage over EDTA: it does not affect calcium ion levels
"Heparinized plasma is preferred for potassium measurements to avoid an elevation due to release of potassium from platelets as the blood clots." - Henry's, p. 48
🟢⚫ Light Green / PST (Plasma Separator Tube)
| Additive | Lithium heparin + polymer gel separator |
| Specimen | Plasma (with gel barrier after centrifugation) |
| Tests | Stat chemistry, plasma chemistry panels |
💜 Lavender / Purple - EDTA (Hematology)
| Additive | K₂EDTA (spray-dried) or K₃EDTA (liquid) |
| Mechanism | EDTA chelates (strongly binds) calcium → blocks the coagulation cascade completely |
| Specimen | Whole blood / EDTA plasma |
| Inversions | 8 |
| Tests | CBC, differential, reticulocyte count, hemoglobin A1c, blood film |
K₂ vs K₃ EDTA:
- K₂EDTA (spray-dried) - preferred; does NOT dilute the sample
- K₃EDTA (liquid) - dilutes sample by ~1-2%; less preferred
Important: EDTA cannot be used for coagulation or chemistry tests involving calcium, iron, or many enzymes that depend on metal ions.
🩷 Pink - Blood Bank EDTA
| Additive | Spray-dried K₂EDTA |
| Specimen | Whole blood |
| Tests | ABO grouping, Rh typing, antibody screening, crossmatch |
| Special | Has a special crossmatch label; FDA-approved for blood bank collections (AABB requirements) |
⬜ White - Molecular Diagnostics EDTA
| Additive | EDTA + gel |
| Specimen | Plasma |
| Tests | Molecular diagnostics (PCR, viral load, genotyping) |
🩶 Gray - Glucose / Fluoride-Oxalate
| Additive | Sodium fluoride (antiglycolytic agent) + potassium oxalate (anticoagulant) |
| Mechanism | Fluoride inhibits enolase - an enzyme in the glycolytic pathway → prevents cells from metabolizing glucose |
| Specimen | Plasma or whole blood |
| Inversions | 8 |
| Tests | Glucose, lactate, blood alcohol |
Key facts:
- Fluoride preserves glucose for up to 3 days at room temperature
- In bacterial septicemia, fluoride may fail to preserve glucose (bacteria overwhelm inhibition)
- Gray tubes contain no heparin - oxalate is the anticoagulant here
🔵🟤 Royal Blue - Trace Elements / Toxicology
| Additive | Either: no additive (serum), sodium heparin, or EDTA - check label |
| Special feature | Tube is trace metal-free (special stopper, no contaminating metals) |
| Tests | Heavy metals (lead, mercury, arsenic, zinc), trace elements, toxicology |
🟤 Tan - Lead
| Additive | Sodium heparin (glass) or K₂EDTA (plastic) |
| Specimen | Plasma or whole blood |
| Tests | Lead levels specifically |
Visual Summary Table
| Color | Additive | Mechanism | Specimen | Common Tests |
|---|---|---|---|---|
| Yellow | SPS (sterile media) | - | Whole blood | Blood cultures |
| Light Blue | Sodium citrate 3.2% | Ca²⁺ chelation | Plasma | PT, aPTT, INR, coagulation |
| Black | Sodium citrate | Ca²⁺ chelation | Whole blood | ESR (Westergren) |
| Red (glass) | None | Clots naturally | Serum | Chemistry, serology |
| Red (plastic) | Silica clot activator | Faster clotting | Serum | Chemistry, serology |
| Gold / SST | Clot activator + gel | Gel barrier | Serum | BMP, CMP, LFTs, hormones |
| Orange | Thrombin | Rapid clotting | Serum (stat) | Stat chemistry |
| Green | Lithium/sodium heparin | Inhibits thrombin | Plasma | Stat chemistry, ammonia |
| Light Green / PST | LiHep + gel | Inhibits thrombin + barrier | Plasma | Stat chemistry |
| Lavender | K₂ or K₃EDTA | Ca²⁺ chelation | Whole blood | CBC, differential, HbA1c |
| Pink | K₂EDTA | Ca²⁺ chelation | Whole blood | Blood bank (type & screen) |
| White | EDTA + gel | Ca²⁺ chelation | Plasma | Molecular diagnostics |
| Gray | Sodium fluoride + oxalate | Enolase inhibition | Plasma | Glucose, lactate |
| Royal Blue | None / heparin / EDTA | Varies | Serum/plasma | Trace metals, toxicology |
| Tan | Heparin or EDTA | Varies | Plasma | Lead levels |
Key Mechanisms to Remember
There are really only 3 anticoagulant mechanisms in use:
-
Calcium chelation (citrate, EDTA, oxalate) - most common
- Clotting cascade needs Ca²⁺ at multiple steps. Remove calcium = no clot.
- Citrate is reversible (used in blood banking - add back Ca²⁺ and blood can clot again)
- EDTA is irreversible for lab purposes
-
Thrombin inhibition (heparin)
- Heparin activates antithrombin III → inactivates thrombin and factor Xa
- Does NOT remove calcium, so calcium levels are unaffected
-
Glycolysis inhibition (sodium fluoride)
- Not really an anticoagulant - it's a metabolic inhibitor
- Oxalate (the actual anticoagulant in gray tubes) chelates Ca²⁺
- Fluoride blocks enolase → stops cells from consuming glucose
Practical Tips
- Always invert tubes the correct number of times immediately after filling - this mixes the additive with blood. Too few inversions = microclots. Too many = hemolysis.
- Never shake tubes - rolling/inverting only.
- Fill to the line - especially critical for light blue (citrate) tubes. Underfilling is the #1 preanalytical error for coagulation tests.
- Hemolysis (ruptured RBCs) from traumatic draws or improper handling releases potassium from cells and falsely elevates K⁺.
- A tube should be gently tapped before insertion to dislodge additive from the stopper, preventing aspiration of additive into the patient's vein during backflow.
Sources: Tietz Textbook of Laboratory Medicine, 7th Ed., Table 4.3; Henry's Clinical Diagnosis and Management by Laboratory Methods, Table 3.4
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