Section 6: Order of Draw

Anticoagulants vs Clot Activators

As a phlebotomist it is imperative to learn the correct order of draw when collecting blood samples in multiple tubes. This is important because each tube contains certain additives. The additives in the tubes mix with the blood to prepare it for specific laboratory testing. The additives are either anticoagulants or clot activators. For blood to coagulate means that the blood changes from a liquid state to a semisolid or solid state. This is natural for blood to do. In the body this process is called Hemostasis. Hemostasis is the body's healing process. You have seen this happen anytime you have gotten a cut, watched a scab form, and then new skin cells are produced. This happens in not only our skin, but our blood vessels and all tissue that experiences injury or damage. Hemostasis happens in four phases. The third stage is the coagulation phase which is the blood clotting phase. When blood is removed from the body it clots. If a tube contains anticoagulant, the blood will not clot. To be sure the blood mixes properly with the additive you must invert the tube a certain number of times. Do not shake the tube. To invert the tube once, gently turn your wrist 180 degrees and back up again. 

More details on each tube...

Blood Cultures (Bottles or Tubes)

• Sometimes a blood culture can be run off of a yellow top tube containing SPS (Sodium Polyanethol Sulfonate) to test for bacterial and fungal blood cultures.

• The nutrient broth in the blood culture bottle allows for rapid growth of microorganisms. 

• A common test for blood cultures is the BCx2. This is a 2 bottle test. The first bottle is aerobic (with oxygen) and the second is anaerobic (without oxygen).

• Blood cultures are usually always STAT

• Blood culture requires strict aseptic technique when preparing the draw site. This means more than an alcohol swab. Usually Chloraprep is used. You'll want to also clean the top of the bottle as well to avoid contamination. 

• A blood culture bottle must be filled to the required amount. Keep the bottle upright so that none of the additive spills into the line and enters the body's blood stream. 

Light-Blue Top Tube

• Sodium Citrate bonds to the calcium in the blood preventing the coagulation and preserves the clotting factors. This allows for testing the blood's ability to coagulate.

• MUST be filled completely to the line to complete the needed ratio of 9 parts blood to 1 part liquid additive. 

• If drawing with a butterfly, draw an additive-free (or additional light blue top) discard tube before drawing the tubes for testing.

  • Common tests ordered are Prothrombin Time (PT) and Partial Thromboplastin Time (PTT). These tests evaluate the intrinsic and extrinsic system of coagulation cascade in the body. 

Royal Blue Top (Serum) Tube

• Always check the tube label as there are two kinds of Royal Blue tubes that are drawn at different places in the order of draw. A red stripe indicates a Silica additive that acts as a clot activator. These are drawn after light blue and before other serum tubes. 

  • Common tests ordered are trace element, trace metal and toxicology studies.

Red Top Tube

• This is the "plain vacuum tube." In GLASS tubes, there are no additives, no anticoagulants / no clot activators. However, in PLASTIC tubes, there is a Silica additive that acts as a clot activator.

• Let the blood sit 30-60 minutes before spinning in a centrifuge. Make sure the blood is completely clotted before spinning.

• There is no serum separator in the red tube. This means after you centrifuge the blood, you must keep the tube up right and handle it gently. If you invert it, it will remix leading to issues with testing the serum. Usually after you spin down a red top you must very carefully pipette the serum off of the top and put it in a pour off tube to be sent to the lab for testing. The rest of the sample is usually discarded unless the test indicates differently. This is the same for any serum tube with no serum separator gel additive. 

Gold / Tiger / Speckled Top Tube

• SST stands for Serum Separator Tube and is the most common tube to draw for testing.

• The Silica clot activator in these tubes causes the blood to clot faster than normal like in the (glass) red top tubes. They can clot anywhere from 15-30 minutes.

• When you spin the blood in the centrifuge the gel serum separator will sit between the serum and the formed elements. This will help prevent contamination and remixing of the components. It also makes it very easy to pour off the serum and use it for testing. 

  • Common tests are the Basic Metabolic Panel (BMP), Comprehensive Metabolic Panel (CMP), Serum Pregnancy Tests, Thyroid tests and many, many more. 

Green Top Tubes

• Heparin is an anticoagulant that inhibits thrombin in the coagulation cascade which therefore prevents clotting.

Lithium Heparin

• • Common tests are routine chemistry, electrolyte and metabolic testing. This is the standard tube used in acute settings, such as hospitals. Can be spun down and run immediately, not having to clot first. Often used for STAT testing.

Sodium Heparin

• Used for more sensitive testing: toxicology, cytogenetic & karyotyping, flow cytometry and trace element testing. 

Royal Blue Top (plasma) Tube

• Contains EDTA to prevent clotting, therefore must be drawn like a lavender unlike the red striped royal blue tube which is drawn before serum tubes. 

  • Also used to test for trace element, trace metal and toxicology studies.

Lavender Top Tube

• K2 EDTA additive inhibits coagulation by binding to the calcium in the blood. This ensures the blood will remain 'Whole' or liquid for most common testing.

• Must be filled at least 1/2 full in order to ensure accurate test results.

  • Common tests run on whole blood are CBC (complete blood count), ESR (tests for inflammation), HgbA1c

• Can be spun down to produce  plasma

  • Common tests run on EDTA plasma (whole blood that is spun down) are molecular or biochemical testing, ie: PCR or DNA

Pink Top Tube

• Similar to Lavender Top Tubes, however the EDTA contained here is K3, instead of K2. As K3 tends to dilute the blood more than K2, this is used in fewer tests.

• Must be filled 2/3 full. 

  • Common uses: by blood banks for blood typing and antibodies, cross-matches for blood transfusions. Also used for molecular diagnostics and testing viral loads.

Pearl Top (PPT) Tube

• PPT = Plasma Preparation Tube

• Contains K2 EDTA and a gel barrier

  • Not as common of a tube to use. Used for collection of plasma for molecular (PCR) tests. 

Tan Top Tube

• • Contains Sodium EDTA as an anticoagulant that binds to metal ions in the blood.

• • DO NOT SPIN. Must be run on whole blood.

  • Commonly used for blood lead analysis.

Gray Top Tube

• The Sodium Fluoride in the tube preserves the glucose by preventing the breakdown of sugar. It can preserve the glucose for 3 days. 

• Potassium oxalate prevents clotting by binding to the calcium in the blood.

• Glucose tests are often run on gray plasma tubes. However, glucose can also be run on serum from a SST or red top tube.

  • Common glucose tests are the Oral Glucose Tolerance Test (OGTT) where blood and/or urine is tested for glucose after ingesting a glucose drink. This blood is drawn several times over a period of time, like 3 hour, or 5 hour tests. It tests the blood sugar levels in the body. Pregnant women have this test to rule out gestational diabetes. A baseline blood draw is performed before the glucose drink is ingested. 

  • Lactic acid tests can be run off gray tubes but must be drawn without stasis (without a tourniquet). Abnormal lactic acid can mean the body is not receiving proper oxygenation. 

  • Alcohol tests can also be run on gray tubes. Do not use an alcohol wipe to clean the site when doing an alcohol test.

Yellow Top (ACD) Tubes

**Not to be confused with the yellow colored blood culture tubes. Remember the yellow blood culture tubes will have SPS as an additive.** 

• Acid Citrate Dextrose (ACD) anticoagulant binds with calcium to prevent clotting

  • Commonly used for flow cytometry and tissue typing assays.