TL;DR Vacutainers
| Tube Top Color | Additive | Mode of Action | Clotting Time | Commonly Associated Tests |
| Red (Plain) | None | N/A (allows natural clotting) | Allow complete clotting (15-30 minutes) | Chemistry panels (after serum separation) |
| Red (Speckled) | Clot activator (silica particles) | Promotes clotting | Allow complete clotting (15-30 minutes) | Chemistry panels (after serum separation) |
| Gold | Clot activator (silica particles) & Gel separator | Promotes clotting & Separates serum from RBCs after centrifugation | Allow complete clotting (15-30 minutes) | Chemistry panels (after serum separation) |
| Light Blue | Sodium citrate 3.2% | Binds calcium ions, preventing clotting | No clotting required | Coagulation studies (PT, PTT, INR) |
| Green (Darker) | Sodium heparin | Inhibits thrombin formation, preventing clotting | No clotting required (gentle inversion) | Chemistry panels (especially “stat” tests), Blood gas analysis |
| Green (Lighter) | Lithium heparin (with or without gel) | Similar to sodium heparin | No clotting required (gentle inversion) | Chemistry panels (especially “stat” tests), Blood gas analysis |
| Lavender | Potassium EDTA (K2EDTA) | Binds calcium ions, preventing clotting | No clotting required (gentle inversion) | Hematology (CBC), Blood bank testing |
| Pink | Potassium EDTA (K2EDTA) | Similar to lavender top | No clotting required (gentle inversion) | Hematology (CBC), Blood bank testing |
| Gray | Sodium Fluoride & Potassium Oxalate / Sodium Oxalate | Sodium Fluoride: inhibits glycolysis; Potassium Oxalate/Sodium Oxalate: binds calcium to prevent clotting | No clotting required (gentle inversion) | Blood glucose, Blood alcohol (sometimes) |
| Royal Blue (No Additive) | None | N/A (relies on natural clotting) | Allow complete clotting (15-30 minutes) | Trace element and heavy metal testing (less common) |
| Royal Blue (EDTA) | Potassium EDTA (K2EDTA) | Similar to lavender/pink top | No clotting required (gentle inversion) | Trace element and heavy metal testing (more common) |
Introduction
Blood tests are a cornerstone of modern medicine, providing vital information for diagnosis, monitoring treatment, and overall health assessment. However, the accuracy of these tests hinges heavily on proper blood collection technique and the use of the right blood collection tubes (vacutainer) with their specific additives.
Why does proper collection matter?
Minimizing pre-analytical errors: Blood collection is the first step in the testing process. Errors at this stage can significantly impact the results, leading to misdiagnosis, unnecessary interventions, or delays in treatment.
Ensuring sample integrity: Blood is a complex mixture of cells and components. Proper collection methods and tubes help maintain the sample’s integrity, preventing contamination, hemolysis (red blood cell destruction), or activation of unwanted cellular processes that can alter test results.
The role of blood collection tubes (vacutainers) and additives
Blood collection tubes (vacutainers) are not simply containers; they play a critical role in maintaining sample quality.
- Types of tubes: Different tubes (vacutainers) are designed for specific purposes. Some contain no additives (serum separator tubes) and allow blood to clot for serum analysis. Others contain anticoagulants (like heparin or EDTA) that prevent clotting for tests requiring whole blood or plasma analysis.
- Additives and their purpose:
- Clot activators: Promote clotting for serum separation using serum separator tubes (vacutainers).
- Anticoagulants: Prevent clotting for whole blood or plasma analysis (e.g., sodium citrate, heparin, EDTA). These work by different mechanisms, like binding calcium (essential for clotting) or inhibiting clotting factors.
- Other additives: Some tubes (vacutainers) may contain additional components like gel separators (to isolate serum after centrifugation) or specific inhibitors (e.g., sodium fluoride in gray-top tubes to preserve glucose levels during transport).
By using the correct tube (vacutainer) and handling it appropriately, we ensure optimal blood sample quality, leading to:
- Accurate test results: Reliable test results are crucial for informed clinical decisions and patient care.
- Improved patient outcomes: Accurate diagnoses and timely treatment interventions can significantly impact patient well-being.
- Cost-effectiveness: Avoiding unnecessary repeat tests due to pre-analytical errors saves healthcare resources.
Common Blood Collection Tubes (Vacutainers)
Serum Separator Tubes (SSTs)
Serum separator tubes (SSTs) are a type of blood collection tube (vacutainer) commonly used in clinical laboratories for tests requiring serum.
Color Tops of Serum Separator Tubes (Vacutainers)
- Red (plain): No additives, allows for natural clotting.
- Red with clot activator (speckled): Contains silica particles that promote clotting.
- Gold (with gel): Similar to the red speckled top with clot activator, but also includes a gel separator that aids in separating serum from red blood cells after centrifugation.
Additives of Serum Separator Tubes (Vacutainers)
- Plain red top: None.
- Red speckled top and Gold top: Clot activator (silica particles).
Mode of Action of Serum Separator Tubes (Vacutainers)
- Plain red top: Blood clots naturally without any additives.
- Red speckled top: Silica particles activate the clotting cascade, accelerating clot formation.
- Gold top: It has silica particles to activate the clotting cascade. The gel separator in the gold top tube acts as a physical barrier after centrifugation, isolating the clear serum layer above the clot and red blood cells.
Clotting Time for Serum Separator Tubes (Vacutainers)
- Allow blood to clot completely before centrifugation. This typically takes 15-30 minutes at room temperature. Insufficient clotting can lead to hemolysis (red blood cell rupture) and contaminate the serum.
Commonly Associated Tests for Serum Separator Tubes (Vacutainers)
- Chemistry panels: These measure electrolytes, enzymes, proteins, and other analytes in the blood serum.
- Serology: Tests for antibodies against various infectious agents.
- Immunology: Tests related to the immune system, such as autoimmune diseases or allergies.
Additional Notes
- While plain red top serum separator tubes (vacutainers) can be used, red-speckled top serum separator tubes (vacutainers) are more common as they ensure complete clotting even with slight variations in collection technique.
- Gel serum separator tubes (gold top) are generally preferred for routine chemistry testing as they simplify the separation process and minimize handling time.
Coagulation Tubes (Light Blue Top)
Coagulation tubes (vacutainers), also known as citrate tubes, are specifically designed for tests that assess blood clotting function.
Color Top: Light Blue
Additive: Sodium citrate 3.2%
Mode of Action: Sodium citrate works as an anticoagulant by chelating calcium ions. Calcium plays a vital role in the blood coagulation cascade. By binding calcium, sodium citrate effectively prevents the formation of blood clots in the tube.
Clotting Time: No clotting required. In fact, clotting in a coagulation tube (vacutainer) would invalidate the test results. After blood collection, the tube needs to be gently inverted several times to ensure proper mixing of the blood with the sodium citrate.
Commonly Associated Tests
- Prothrombin Time (PT): Measures the time it takes for blood plasma to clot after the addition of clotting factors (thrombin and thromboplastin).
- Activated Partial Thromboplastin Time (aPTT): Similar to PT, but assesses clotting time by adding only thromboplastin, evaluating a different part of the clotting cascade.
- International Normalized Ratio (INR): A standardized ratio derived from PT results, used to monitor patients on warfarin (blood thinner) therapy.
Additional Notes
- The specific amount of sodium citrate and the blood-to-citrate ratio are crucial for accurate coagulation testing.
- Improper mixing of blood with sodium citrate can lead to inaccurate results due to incomplete anticoagulation.
- Coagulation tubes are essential for monitoring patients with bleeding disorders, those on anticoagulant medications, or undergoing procedures that require blood clotting control.
Heparin Tubes (Green Top)
Heparin tubes are another type of blood collection tube (vacutainer) used for tests requiring whole blood or plasma analysis, but unlike citrate tubes (vacutainers), they prevent clotting through a different mechanism.
Color Top: Green (darker shade for sodium heparin, lighter shade for lithium heparin)
Additive: Sodium heparin or lithium heparin (may be present with or without a gel separator)
Mode of Action: Heparin, both sodium and lithium forms, functions as an anticoagulant by inhibiting the formation of thrombin and thromboplastin. These are key enzymes involved in the clotting cascade. By preventing their formation, heparin effectively stops the blood from clotting within the tube (vacutainer).
Clotting Time: No clotting required. Similar to citrate tubes, clotting in a heparin tube (vacutainer) would compromise the test. However, after blood collection, immediate gentle inversion of the tube 8-10 times is crucial to ensure proper mixing of the blood with heparin to prevent clotting.
Commonly Associated Tests
- Chemistry Panels (especially “stat” tests): Heparinized plasma is preferred for some chemistry tests, particularly those needed urgently (“stat” tests). Heparin does not interfere with most chemistry analytes.
- Blood Gas Analysis: Measures blood pH, oxygen and carbon dioxide levels, and electrolytes. Heparinized blood is preferred for blood gas analysis as it maintains cellular integrity and minimizes clotting artifacts that can affect the results.
- Other Tests: Heparinized blood or plasma may also be used for specific tests like hormone testing or therapeutic drug monitoring.
Additional Notes
- The type of heparin used (sodium or lithium) generally doesn’t significantly impact most tests. However, lithium heparin may interfere with specific tests, so consulting laboratory guidelines is recommended.
- The presence or absence of a gel separator (often found in light green top tubes) is for convenience during processing, not related to the anticoagulant function for example to separate the plasma for biochemical testing.
- Delayed mixing of blood with heparin can lead to partial clotting, compromising the sample.
EDTA Tubes (Lavender or Pink Top)
EDTA tubes are another widely used type of blood collection tube (vacutainer) known for their effectiveness in preserving whole blood for various analyses. Here’s a breakdown of their key features:
Color Top: Lavender or Pink
Additive: Potassium EDTA (K2EDTA)
Mode of Action: EDTA functions as an anticoagulant by chelating calcium ions. Similar to sodium citrate in coagulation tubes (vacutainers), calcium plays a critical role in blood clotting. By binding free calcium, K2EDTA effectively prevents the clotting cascade from initiating, thus preserving the cellular components of whole blood.
Clotting Time: No clotting required. Gentle inversion of the tube 8-10 times after collection is essential to ensure thorough mixing of blood with the EDTA to prevent any microclotting.
Commonly Associated Tests
- Hematology (CBC): A complete blood count (CBC) analyzes various blood cell types and their characteristics. EDTA tubes (vacutainers) are the preferred choice for CBCs as they maintain the integrity of red blood cells (RBCs), white blood cells (WBCs), and platelets for accurate analysis.
- Blood Bank Testing: Pre-transfusion compatibility testing and blood typing require whole blood preserved with EDTA to ensure accurate assessment of cell surface antigens.
- Other Tests: EDTA tubes (vacutainers) may also be used for specific tests like flow cytometry (analyzing cell populations), lead level testing, or certain parasite detection methods.
Additional Notes
- Overfilling the EDTA tube (vacutainer) can dilute the blood sample and affect test results. Proper blood-to-EDTA ratio is crucial.
- Prolonged tourniquet application during blood collection can lead to artificial cell activation and potentially alter some test results.
- EDTA tubes (vacutainers) are not suitable for coagulation studies or tests requiring serum analysis as they prevent clotting.
Sodium Fluoride/Potassium Oxalate Tubes (Gray Top)
Sodium Fluoride/Potassium Oxalate (NaF/K Oxalate) tubes (vacutainers), also sometimes referred to as gray-top tubes due to their color, are specifically designed for tests that require preserved levels of certain analytes, particularly blood glucose (sugar).
Color Top: Gray
Additives: This tube can contain two possible combinations:
- Sodium Fluoride & Potassium Oxalate
- Sodium Oxalate (alone)
Mode of Action: The combination of additives works in two ways:
- Sodium Fluoride: Inhibits the process of glycolysis, the breakdown of glucose by red blood cells. This helps preserve blood glucose levels in the sample even during transport or slight delays in analysis, which is crucial for accurate measurement.
- Potassium Oxalate (or Sodium Oxalate): Acts as an anticoagulant by chelating calcium ions, similar to EDTA and sodium citrate in other tubes (vacutainers). This prevents clotting and preserves the cellular components of the blood.
Clotting Time: No clotting required. Gentle inversion of the tube 8-10 times after collection is important to ensure proper mixing of the blood with the additives.
Commonly Associated Tests
- Blood Glucose: The primary use of NaF/K Oxalate tubes (vacutainers) is for measuring blood glucose levels. Preserving glucose levels with sodium fluoride is particularly important if there’s a potential delay between blood collection and analysis.
- Blood Alcohol: These tubes (vacutainers) can also be used for blood alcohol testing as sodium fluoride helps maintain the integrity of the sample.
- Lactate Levels: In some cases, NaF/K Oxalate tubes (vacutainers) may be used for measuring lactate levels, although specific tests might have different tube preferences.
Additional Notes
- Underfilling the tube can significantly alter the blood-to-additive ratio, compromising the effectiveness of the preservatives and potentially leading to inaccurate results.
- Improper mixing after collection can lead to localized clotting and affect test results, especially for glucose levels.
- NaF/K Oxalate tubes are not suitable for most routine blood tests as the additives can interfere with certain analytes.
Royal Blue Top Tubes
Royal blue top tubes are used for trace elements and heavy metal testing in blood. There are actually two variations of the royal blue top tube, with slightly different characteristics.
1. Royal Blue Top with No Additive
Additives: None
Mode of Action: Relies on the natural clotting process. The clot formation separates the cellular components from the serum, which is then used for analysis. However, this type of tube is less common due to potential contamination issues.
2. Royal Blue Top with EDTA
Additive: Potassium EDTA (K2EDTA)
Mode of Action: Similar to the lavender or pink top EDTA tubes, K2EDTA chelates calcium ions, preventing blood clotting and preserving whole blood for analysis. This is the more common variant of the royal blue top tube (vacutainer).
Clotting Time: The clotting time depends on the specific type of royal blue top tube.
- No additive: Allow blood to clot completely (typically 15-30 minutes).
- EDTA: No clotting required, but gentle inversion 8-10 times after collection is essential for proper mixing.
Commonly Associated Tests: Trace element and heavy metal testing in blood. These tests measure the levels of specific metals, such as lead, arsenic, mercury, or iron, in the blood.
Additional Notes
- Royal blue top tubes are manufactured under clean room conditions to minimize contamination from dust or other environmental factors, which is crucial for accurate trace element and heavy metal analysis.
- The specific type of royal blue top tube used (with or without EDTA) may depend on the specific test being ordered.
- These tubes are not suitable for routine blood tests like chemistry panels or CBCs.
Specimen Quality Concerns
Specimen quality is paramount when it comes to blood tests. Improper blood collection or handling can significantly impact the results, leading to misdiagnosis or unnecessary procedures.
Serum Separator Tubes (SSTs)
- Incomplete clotting: If blood doesn’t clot completely before centrifugation, red blood cells (RBCs) can lyse (rupture), releasing hemoglobin into the serum. This hemolysis can alter the results of various tests, especially those measuring enzymes or bilirubin.
- Excessive mixing: Vigorous or prolonged mixing can also cause hemolysis, especially with clot activator tubes (red-speckled top). Gentle inversion 8-10 times is sufficient for proper mixing.
Coagulation Tubes (Light Blue Top)
- Underfilling or overfilling: The blood-to-sodium citrate ratio is crucial for accurate coagulation testing. Underfilling concentrates the anticoagulant, while overfilling dilutes it, both leading to unreliable results.
- Improper mixing: Failure to mix the blood with sodium citrate upon collection can lead to localized clotting and inaccurate results in coagulation tests.
Heparin Tubes (Green Top)
- Underfilling: Similar to coagulation tubes (vacutainers), underfilling heparinized tubes can alter the blood-to-anticoagulant ratio, affecting test results.
- Presence of air bubbles: Air bubbles in the tube (vacutainer) can interfere with some tests, like blood gas analysis, as they alter the gas composition of the sample.
- Prolonged mixing delay: Heparin can slightly alter certain analytes over time. Prompt processing or analysis of the blood sample is recommended to minimize this effect.
EDTA Tubes (Lavender or Pink Top)
- Prolonged tourniquet application: Keeping the tourniquet on for an extended period can lead to artificial cell activation and potentially alter some blood cell parameters in a CBC.
- Overfilling: Similar to other tubes, overfilling an EDTA tube (vacutainer) can dilute the blood sample and affect test results.
- Presence of clots: Even small clots in an EDTA tube (vacutainer) can interfere with complete blood cell analysis, especially for white blood cell counts and differentials.
Sodium Fluoride/Potassium Oxalate Tubes (Gray Top)
- Underfilling: Underfilling significantly alters the blood-to-additive ratio, compromising the effectiveness of the preservatives and potentially leading to inaccurate glucose or lactate measurements.
- Improper mixing: Localized clotting due to inadequate mixing can affect test results, especially for glucose levels.
General Concerns
- Improper labeling: Incorrect labeling of blood collection tubes (vacutainers) can lead to sample mix-up and compromise patient safety and test accuracy.
- Improper handling and storage: Blood samples need to be stored and transported under specific temperature conditions depending on the test. Failing to do so can alter the stability of certain analytes.
Order of Proper Draw
The importance of collecting blood tubes (vacutainers) in a specific order, often referred to as the “order of draw,” focuses on preventing contamination of the blood sample by additives present in different collection tubes. Here’s a breakdown of the rationale behind this practice:
Contamination Risks
Each blood collection tube (vacutainer) contains a specific additive (anticoagulant, clot activator, etc.) designed to preserve the sample for a particular type of test. If the order of draw is not followed, residual amounts of additives from a previously filled tube can be introduced into the next tube during the blood collection process.
This contamination can significantly alter test results:
- For example, if some heparin from a green-top tube (vacutainer) mixes with blood in a subsequent red-top serum separator tube (SST) (vacutainer), it can interfere with clot formation, leading to hemolysis (red blood cell rupture) and affecting various test results.
- Similarly, residual sodium citrate from a light blue coagulation tube (vacutainer) could contaminate a subsequent EDTA tube (lavender or pink top) (vacutainer), potentially affecting cell counts in a complete blood count (CBC).
Maintaining Sample Integrity
The order of draw ensures that the blood sample maintains its original composition as much as possible, minimizing the influence of additives from previous tubes. This allows for accurate analysis of various blood components and reliable test results across different tests ordered.
Universal Order of Draw
While specific protocols may vary slightly between institutions, a generally accepted order of draw exists:
- Coagulation Tubes (Light Blue Top) (Vacutainers): Contains sodium citrate, an anticoagulant for clotting studies.
- Serum Separator Tubes (SSTs) – Red Top (plain), Red Speckled Top (clot activator), Gold Top (with gel) (Vacutainers): Allow blood to clot for serum analysis. Order within serum separator tubes (SSTs) might vary depending on the presence of clot activator or gel separator.
- Heparin Tubes (Green Top) (Vacutainers): Contain heparin, an anticoagulant for whole blood or plasma analysis (e.g., blood gas).
- EDTA Tubes (Lavender or Pink Top) (Vacutainers): Contain EDTA, an anticoagulant for whole blood analysis (e.g., CBC).
- Sodium Fluoride/Potassium Oxalate Tubes (Gray Top) (Vacutainers): Contain additives to preserve specific analytes like glucose.
Benefits of Following Order of Draw
- Ensures accurate and reliable test results for various blood tests.
- Minimizes the risk of misdiagnosis or the need for repeat blood draws.
- Promotes patient safety by providing a clear and standardized collection procedure.
Disclaimer: This article is intended for informational purposes only and is specifically targeted towards medical students. It is not intended to be a substitute for informed professional medical advice, diagnosis, or treatment. While the information presented here is derived from credible medical sources and is believed to be accurate and up-to-date, it is not guaranteed to be complete or error-free. See additional information.
