Specimen Requirements

Introduction to Specimen Requirements

• Explanation of the specimens and tubes needed for blood collection and their color meanings.

Lesson Objectives

1. Introduction to the types of blood collection additives and the chemical composition of each.

2. Describe the ETS tube stopper color coding used to identify additives.

3. Connect additives and stopper colors with laboratory departments and tests (focus on Hematology and Chemistry).

4. Indicate the main samples needed for Hematology and Chemistry.

5. Distinguish between serum and plasma.

6. Discuss specimen quality issues: Hemolysis, Icterus, and Lipemia.

Evacuated Tube System (ETS)

• Definition: A closed system where blood flows through a needle inserted into a vein and directly into a collection tube.

• Benefits:

  • Prevents exposure to air and outside contaminants.

  • Allows collection of multiple tubes with a single venipuncture.

Evacuated Tubes

• Characteristics:

  • Available in different sizes and volumes.

  • Tube size depends on:

  • Age of patient.

  • Volume of blood required for the test.

  • Size and condition of the patient’s vein.

  • Available materials:

  • Glass and plastic (plastic is preferred for safety).

Evacuated Tubes: Continued

• Vacuum:

  • Defined as negative pressure from which air is pulled to draw blood into the tube.

• Types of tubes:

  • Additive tubes: contain substances such as anticoagulants or clot activators.

  • Non-additive tubes: contain no substances (rare).

• Stoppers:

  • Made of rubber or rubber with a plastic covering.

  • Color coding: indicates the type of additive in the tube.

• Expiration dates: printed on tube labels for additives and vacuum checks.

Anticoagulants

• Purpose: To prevent blood from clotting and maintain it in a liquid state.

• Mechanisms of action:

  • Precipitating or binding calcium to prevent clot formation.

  • Inhibiting thrombin formation, which is crucial for clotting.

Preservation of Blood in Liquid State

• Mechanism:

  • Removal of calcium allows blood to remain liquid post-collection, crucial for tests requiring plasma.

  • Enables separation of plasma from blood cells without clotting.

Coagulation Cascade

• Importance:

  • Understanding the coagulation cascade is crucial for grasping clot formation.

• Objective:

  • To create fibrin and stabilize the clot.

• Key components:

  • Calcium: Critical for the coagulation cascade and clot formation.

  • Thrombin: Central to the cascade; essential for converting fibrinogen to fibrin.

• Mechanisms to prevent clotting by stopping:

  • Thrombin generation or calcium removal from the cascade.

Types of Anticoagulants

1. Ethylenediaminetetraacetic acid (EDTA)

   • Color Code: Lavender top.

   • Mechanism: Chelating agent binding calcium, preventing activation of clotting factors (like conversion of prothrombin to thrombin).

   • Uses: Hematology (CBC, Reticulocyte count, Differential), HbA1C, Molecular Testing.

   • Mixing requirement: Must be mixed immediately to avoid micro-clots (8-10 times).

   • Filling rule: Underfilled tubes may lead to inaccurate results due to incorrect blood-to-additive ratios.

2. Sodium Citrate

   • Color Code: Powder blue top.

   • Mechanism: Chelates calcium ions, prevents clotting factor activation.

   • Uses: Coagulation testing (PT/INR, PTT, etc.).

   • Mixing requirement: Gently mix 3-4 times.

   • Blood-to-additive ratio: Critical ratio of 9:1; tubes underfilled are rejected.

3. Heparin

   • Color Code: Green top.

   • Nature: A naturally occurring anticoagulant that enhances antithrombin III activity.

   • Mechanism: Inhibits thrombin activity, preventing conversion of fibrinogen to fibrin.

   • Mixing requirement: Mix 5-10 times.

   • Consideration: Hemolyzed specimens unsuitable for many tests.

4. Oxalates

   • Color Code: Grey top.

   • Mechanism: Binds calcium ions to prevent clotting, often contains sodium fluoride to control glycolysis to preserve glucose levels for 3 days.

5. Clot Activators

   • Purpose: Enhance coagulation in serum specimen tubes.

   • Can include silica particles, gel, or clotting factors.

   • Common Colors: Red or gold (mottled/tiger top).

   • Mixing requirement: Tubes mixed 5X; total clotting time ranges 15-30 minutes.

   • Importance: If not clotted for a specified time, separation issues may occur.

6. Thixotropic Gel Separator

   • Definition: A non-reactive substance with an intermediate density.

   • Function: Moves between cells and serum/plasma upon centrifugation, preventing cellular metabolism.

   • Common applications: SSTs (gold top, mottled grey/red) and PSTs (light green top).

7. Trace Element-Free Tubes

   • Characteristics: Made of materials that do not contaminate trace elements.

   • Uses: For trace element tests, toxicology, and nutrient determinations (Royal-blue tops).

Order of Draw for Blood Collection

1. Blue top coagulation tube.

2. Serum tube with or without clot activator or gel.

3. Heparin tube with or without gel plasma separator.

4. EDTA tube.

5. Glycolytic inhibitor tube.

Carryover and Cross-Contamination Issues

• Causes of cross-contamination:

  • Blood in additive tubes touching needles during ETS collection.

  • Transferring blood from a syringe into ETS tubes.

• Order of draw helps minimize carryover risks.

• Important to fill specimen tubes from bottom to top to avoid carryover.

• Note: Never transfer blood from one additive tube to another as this is detectable in Chemistry tests.

Sources of Error and Interference in Blood Samples

1. IV Contamination:

   • Description: Occurs when samples collected from an arm with active IV can lead to dilution or contamination.

   • Implication: Such samples are unacceptable and must be canceled. Proper protocols must be followed when dealing with IV patients.

2. Hemoconcentration:

   • Definition: Increased relative number of red blood cells due to decreased plasma volume (e.g., dehydration), or increased erythrocyte production (polycythemia).

   • Causes: Prolonged tourniquet use (max 1 minute), massaging, squeezing the collection site.

3. Fibrin Interference:

   • Description: Insoluble protein produced during bleeding; micro-clots can appear in samples due to improper mixing during collection.

   • Effects: If micro-clots or fibrin are present, they can significantly hinder sample analysis, leading to inaccurate results and potential need for recollection.

Specimen Quality Issues

1. Hemolysis:

   • Definition: Damage or destruction of RBCs, releasing hemoglobin into the plasma.

   • Severity classifications: slight (pink), moderate (dark pink), gross (red).

   • Effects: Causes falsely elevated potassium, CK, LDH, AST, iron, Mg, and phosphorus levels, while also reducing RBC counts.

   • Causes: Poor collection techniques, such as shaking samples, fist pumping, or improper venipuncture.

2. Lipemia:

   • Description: Physiological variability leading to increased lipids in patient samples, causing turbidity.

   • Causes: Most common after non-fasting (lipid-rich meals); appears milky-white.

   • Effects: Can interfere with some testing methodologies.

3. Icterus (Bilirubin):

   • Definition: Caused by excess bilirubin in blood due to various forms of hemolysis or hepatic dysfunction.

   • Appearance: Jaundice leading to yellowish appearances in serum, plasma, and urine.

   • Effects: Color can interfere with testing results.

Serum vs Plasma

• Plasma:

  • Definition: Resulting when blood is collected with an anticoagulant; remains in liquid form.

  • Compositions involved: Lavender, Pink, Light Blue, Light Green (SST), Dark Green tubes all contain plasma.

  • Process: Centrifugation is used to separate cells from plasma.

• Serum:

  • Definition: Resulting after blood sits for 15-30 minutes to clot.

  • Compositions involved: Gold, Red, and mottled red/grey (tiger top) tubes contain serum; tubes may contain SST gel.

  • Process: Serum is separated from cells through centrifugation.

Summary of Tube Uses and Colors

• Hematology Testing: Lavender tubes.

• Coagulation Testing: Light Blue tubes.

• Chemistry Testing: Gold, Red (serum), or Light Green (plasma) tubes.

• Molecular Testing: Lavender tubes.

• Blood Transfusion Testing: Lavender or Pink tubes (both contain EDTA).

• Specialized Testing: Can utilize any tube color depending on test requirements.

References

• Phlebotomy Essentials, 7th edition

• Rodak's Hematology: Clinical Principles and Applications