Order of Draw – Blood Collection Tubes

Order of Draw – Overview

• The order in which you fill blood tubes is important. It stops different chemicals from mixing together and causing wrong test results.

• Always remember this order: Blood Cultures → Coagulation → Serum → Heparin → EDTA → Glycolytic Inhibitor → Specialized Tubes.

• After filling, gently turn each tube upside down the right number of times right away. This makes sure the added chemicals mix well without harming the blood cells.

• Using clean methods, filling tubes correctly, and sending them to the lab quickly are very important for getting accurate results and keeping patients safe.

Yellow Stopper – Blood Culture Tube (SPS)

• What’s inside: Sodium Polyanethol Sulfonate (SPS).

• What it does: It keeps blood from clotting. It also helps bacteria grow better for easy detection by stopping some of the body’s defenses and preventing antibiotics from working temporarily.

• How to collect: MUST be collected using a very sterile (clean, like surgery) method.

  • Clean the skin with povidone-iodine or chlorhexidine.

• How many times to flip: 8 times.

• Common tests: Blood cultures – to check if there are bacteria in the blood (bacteremia, septicemia).

• Important tips: This tube is collected first to prevent sk in germs from getting into other tubes. Make sure to collect both air-loving (aerobic) and no-air (anaerobic) bottles if needed.

Light Blue Stopper – Coagulation Tube

• What’s inside: Sodium Citrate (Na Citrate) 3.2\%.

• What it does: It stops blood from clotting by binding with calcium, which is essential for blood clotting.

• Important handling: This tube MUST be filled exactly to the mark (9 parts blood to 1 part chemical) to get correct results.

  • Tests need to be done within 2 hours (or the liquid part of blood, plasma, must be separated and frozen).

• How many times to flip: 3 times.

• Main tests: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT/ PTT), INR, fibrinogen, D-dimer, and tests for clotting factors.

• Connections: If you don’t fill it enough, PT/APTT results will appear longer than they are. If you overfill it, they’ll look shorter.

Red Stopper – Plain Serum Tube

• What’s inside: Nothing added (for glass tubes); some plastic tubes have a chemical (silica) that helps blood clot faster.

• What it does: It allows the blood to clot naturally. After spinning, you get serum (the liquid part of blood without clotting factors).

• How many times to flip: 0 for glass tubes (do not flip!); plastic tubes might need 5 gentle flips if they contain a clot activator.

• Key tests: Routine blood chemistry, tests for diseases (serology), blood typing (ABO/Rh), pregnancy tests (BHCG), HIV tests, syphilis tests (RPR).

• Notes: Needs to sit for 30–60 minutes to clot before being spun.

Red/Gray (Tiger), Gold, Marble, Speckled – SST (Serum Separator Tube)

• What’s inside/How it’s made: Contains a chemical (silica) to help blood clot and a special gel in the middle.

• What it does: The silica makes blood clot quickly (about 15 minutes). The gel forms a barrier between the blood cells and the serum when spun, preventing them from mixing.

• How many times to flip: 5 times.

• Typical tests: Comprehensive metabolic panel (CMP), basic metabolic panel (BMP), cholesterol tests, thyroid tests, tests for certain viruses (HIV, Hep B).

• Special Note: Not to be used for blood typing because the gel can interfere with some antibodies.

Green Stopper Family – Heparin Tubes

• Colors & What’s inside: Dark Green – Sodium Heparin; Light Green – Lithium Heparin + PST gel; Green/Gray mottled – Ammonium Heparin.

• What it does: Heparin stops various steps in the clotting process, keeping the blood from clotting.

• How many times to flip: 8 times.

• Key Uses: Urgent (STAT) blood chemistry tests (like electrolytes, kidney function, heart enzymes, urgent CMP), ammonia, lactic acid (if the lab allows), and blood gas tests.

• Things to watch out for: These tubes are NOT interchangeable – the type of heparin can affect some tests (e.g., lithium heparin can interfere with lithium drug levels). Fill quickly and place on ice for ammonia tests.

Lavender (Purple) Stopper – EDTA Tube

• What’s inside: Ethylene-Diamine-Tetra-Acetic Acid (K2 or K3).

• What it does: It permanently binds to calcium, which stops blood from clotting and helps keep blood cell shapes normal.

• How many times to flip: 8 times.

• Routine tests: Complete Blood Count (CBC), examining blood cells under a microscope, ESR, Hemoglobin A1c (for diabetes), Reticulocyte count (new red blood cells), blood cross-match (another choice besides pink tubes).

• Important things to know: If you overfill it, blood might clot. If you don’t fill it enough, blood cells might shrink and appear smaller than they are.

Gray Stopper – Glycolytic Inhibitor Tube

• What’s inside: Potassium Oxalate (stops clotting) + Sodium Fluoride (stops sugar from being used up).

• What it does: Oxalate binds to calcium to stop clotting; fluoride prevents glucose (sugar) in the blood from changing for up to 24 hours.

• How many times to flip: 8 times.

• Key tests: Glucose Tolerance Test (GTT), Fasting glucose, Blood alcohol (EtOH), Lactic acid (needs ice).

• Handling: MUST be filled exactly to the line to prevent issues like blood cell damage or incorrect dilution.

Yellow Stopper (ACD) – Specialty Genetics Tube

• What’s inside: Acid Citrate Dextrose (Solution A or B).

• What it does: Citrate stops clotting, and dextrose provides nutrients to keep red and white blood cells alive for a long time.

• How many times to flip: 8 times.

• Uses: Human Leukocyte Antigen (HLA) typing (important for organ transplants), bone marrow transplant pre-checks, paternity tests, DNA studies.

• Note: This is NOT the same as the yellow blood culture tube, even though they are both yellow.

Royal Blue Stopper – Trace Element Tube

• What's inside choices: Nothing (red label band) – for serum tests; EDTA (lavender band) – for whole blood tests; Sodium Heparin (green band) – for plasma tests.

• What it does: These tubes are extremely clean and have very low levels of metals. The EDTA/heparin works the same way as in other tubes.

• How many times to flip: None for the plain serum tube (0); 8 for EDTA/Heparin tubes.

• Primary tests: Tests for toxic metals (lead, mercury, arsenic), trace minerals (zinc, copper), and nutrition studies.

• Processing: Let the plain royal blue tube clot for 30–60 minutes before spinning.

Tan Stopper – Lead Determination Tube

• What’s inside: Lithium Heparin (which contains very little lead itself).

• What it does: It stops blood from clotting, and the tube is certified to be lead-free inside.

• How many times to flip: 8 times.

• Only test: Lead (Pb) level; very important for testing children.

• Tip: Use a special needle and collection set to avoid environmental lead contaminating the sample.

Orange Stopper – Rapid Serum Tube (RST)

• What’s inside: Thrombin (a chemical that helps blood clot).

• What it does: It makes blood clot in less than 5 minutes, allowing quick separation of serum for urgent tests.

• How many times to flip: 8 times.

• Typical Use: Urgent (STAT) serum tests when results are needed very fast (e.g., emergent CMP, urgent pregnancy test).

Black Stopper – ESR Tube (Westergren Method)

• What’s inside: Sodium Citrate 0.109\,M (a different amount and ratio than the light blue tube).

• How many times to flip: 3–4 times.

• Only test: Westergren Sedimentation Rate (ESR).

• Important: This tube is designed to be filled with exactly 4 parts blood to 1 part citrate; do not use blood from other tubes that have anticoagulants.

Cross-Tube & Procedural Reminders

• Filling Accuracy: Not filling light blue or black citrate tubes exactly to the line changes the blood-to-chemical ratio, which can mess up results.

• Mixing Method: Gently flip 180 degrees; a full flip counts as one. Shaking hard can damage blood cells.

• Why Order of Draw: It prevents chemicals from one tube from getting into another and causing false results (e.g., chemical from an EDTA tube can make calcium appear low and potassium appear high).

• Temperature Needs: Ammonia and lactic acid tests need to be placed on ice; tests for cold agglutinins need to be kept warm (37^\circ\text{C}); potassium levels can increase if tubes are not spun quickly.

• Blood Culture Bottle Amounts: For adults: 8–10\,mL per bottle; for children: 1–3\,mL. Always write down where the blood was taken from and the time.

• Patient ID & Labeling: Always use two ways to identify the patient, write the date/time, who collected it, and add special labels for blood bank and legal samples.