When collecting blood in an evacuated tube with an anticoagulant What is the importance of having the correct volume of blood?

Definition/Introduction

Laboratory tube collection is a process applied when withdrawing blood samples from patients before they get tested in the laboratory. It follows the principle, which is most commonly known as the "order of draw." Different tests and biochemical assays require varying types of sample collection tubes. The reason why tubes are color-coded is for practical and easy identification.

In-vitro analysis of blood samples can be performed in clinical laboratories. However, these blood samples come in different forms. Testing procedures can require any of the following blood sample types: serum or plasma. In performing multiple collections of venous and/or arterial blood samples in a single patient, a color sequence of withdrawing and its indications based on the Clinical & Laboratory Standards Institute (CLSI) is the recommended procedure.[1]

Yellow, Pink, and Blue

These colors indicate the color of the culture bottles necessary for blood culture. These tubes (bottles) contain sodium polyanethole sulfonate (SPS), which serves as an anticoagulant. The primary function of this anticoagulant is to inhibit the activation of complement function.[2] Following the collection of the blood, mixing the bottle 8 to 10 times is needed to mix the contents adequately. The color of blood culture bottles may vary depending on the manufacturer.

Light Blue

The "citrate tube" is for collecting blood for performing coagulation studies. It contains 3.2% sodium citrate as its anticoagulant.[3] Mixing the blood inside the tube a few times is also recommended to form the plasma.

Red

There is no anticoagulant or additive inside the tube. Because of the contact of blood with the surface of the tube, the coagulation cascade becomes activated. Blood is allowed to clot for 10 to 15 minutes before subjecting to centrifugation to separate the clot from the serum further. Clotting time differs between specimens and may range from 10 minutes to almost an hour (60 minutes).

Green

The green tubes contain heparin (can either be in the form of sodium heparin, lithium heparin, or ammonium heparin), which acts as an anticoagulant by inhibiting thrombin formation. Unlike red tubes, the type of blood produced in a green tube needed for testing is not serum but plasma. Green tubes are frequently used in requesting "stat" or immediate blood chemistry results for emergency cases.

Lavender

The lavender tube is also referred to as EDTA tubes because it uses ethylene-diamine-tetra-acetic acid. The primary role of EDTA in anticoagulation is by chelating calcium ions in the blood to prevent it from coagulation. Since the blood did not clot, the liquid portion (without the red cell) is a plasma. Lavender tubes are commonly used in collecting blood samples for hematologic assays.[4]

Grey

The grey tubes contain potassium oxalate that binds to calcium, thus, inhibiting coagulation. Another substance, sodium fluoride, is also contained in the tubes, which serve as an antiglycolytic agent.[5] That is why it is used for plasma glucose and lactic acid testing.

Apart from these more commonly used collection tubes, other colored tubes (gold, tiger, pink, gold, etc.) are also used in actual hospital and clinical practice. The use of these tubes and the order of draw vary among medical institutions.[6] Tubes with anticoagulants require adequate mixing with blood. Thus, the evacuated tubes can be inverted and follow a pattern of "figure-of-8". However, proper caution must be in mind when mixing tubes as overmixing can produce hemolysis. Inefficient mixing can result in forming small clots.[7] Improper laboratory tube collection can cause harm not only in the laboratory but more so in the clinical setting.

Issues of Concern

Issues inherent to the application of laboratory tube collection include, but are not limited, to the following:

• Problems with the evacuated tube system (ETS): Multiple blood collection will be difficult, especially when the vacutainer used is already damaged or malfunctioning. 

• Problems with the collection tube: The tubes may also be damaged. For instance, the vacuum inside the tube could be lost when it is uncapped. On the other hand, an anticoagulant may not be present in some tubes during packaging, which will mean incorrectly using serum instead of plasma for testing.

• Inadequately trained personnel: The laboratory staff should have training in laboratory tube collection. The order of draw is made easy with color-coding, but it can still lead to an incorrect collection if the phlebotomist does not follow the order of draw because of inadequate training.

• Contamination: If the tubes become contaminated from the beginning, during, or after the collection, these will yield incorrect and unreliable results. For example, the sterility of the cap of blood culture tubes must be ensured and should not be in contact with any potential source of contamination. Another way of contamination is when the anticoagulant from a tube can be transferred to another tube when it sticks with the needle of the vacutainer. 

• Lack of research studies on laboratory tube collection: While the CLSI made the recommendations for the proper sequence of tubes, additional studies are needed to reveal the actual amount of variability if improper order of collection occurs. Future studies can reveal underlying concepts to the laboratory tube collection process itself, which can help develop future standards on laboratory science practice in healthcare. Medical laboratory scientists working not only in the clinical laboratories but also in research and the academe should give adequate attention to laboratory tube collection as it is given less priority nowadays despite its clinical implications.

Clinical Significance

The correct application of the "order of draw" principle in multiple blood tube collection is an essential aspect of ensuring quality laboratory test results.[8] Falsely increased and false decreased values may result in specific analytes in biochemistry, hematology, serology, and clinical microbiology, to name a few.[9] These inaccurate and unreliable results can never be used in clinical correlation as they will alter the diagnosis and management of the patient's disease. 

When the laboratory medicine department does not address these problems, incorrect results may reach the clinicians, nurses, and other clinical personnel without being noticed.[10] This situation is even worse in the wards and emergency departments. However, additional studies concerning the role of laboratory-related errors on laboratory tube collection and its impact in clinical settings may still be necessary to strengthen these claims, and thus, their clinical relevance.

Nursing, Allied Health, and Interprofessional Team Interventions

Even though laboratory tube collection is mainly the responsibility of the clinical laboratory and its staff, team interventions in the clinic and hospital settings can still help in the total quality implementation of such laboratory practices. For instance, non-laboratory personnel such as nurses and clinicians should at least become oriented or aware of the order of draw so that when they get to see laboratory staff or phlebotomists at the bedside engaging in improper procedures, they can immediately call attention to it before there is an adverse clinical impact to the patient. Assisting a colleague at work can have a significant effect on patient care. 

Moreover, during conferences or department meetings, the laboratory manager (or pathologist) can discuss the importance of proper laboratory tube collection with the administration, clinical staff, purchasing department, and human resources. Aside from non-laboratory health personnel, non-clinical personnel must be included in the team to increase the scope of knowledge. While interprofessional teams' goal is to improve overall patient care, it should be emphasized that helping other colleagues should not be intended to blame a health professional but rather to lift everyone in the team and provide corrective action.

Nursing, Allied Health, and Interprofessional Team Monitoring

Team monitoring is not standard practice because of the departmentalized system in healthcare. On the other hand, interprofessional teams require collaboration among different healthcare professionals and should solely rely upon the clinicians, specialists, and other providers. Nursing, allied health (laboratory, pharmacy, etc.), and non-health (administration staff) can create teams with clinicians and specialists and conduct team monitoring via regular "peer review" (as a recommendation) so that each member of the team will get to realize the interconnectedness of the work they do in healthcare and its collective impact to patients. The specific steps in conducting a peer review of the interprofessional team, in the case of laboratory tube collection, for example, must be further studied.

Review Questions

References

Bowen RA, Remaley AT. Interferences from blood collection tube components on clinical chemistry assays. Biochem Med (Zagreb). 2014;24(1):31-44. [PMC free article: PMC3936985] [PubMed: 24627713]

Palarasah Y, Skjoedt MO, Vitved L, Andersen TE, Skjoedt K, Koch C. Sodium polyanethole sulfonate as an inhibitor of activation of complement function in blood culture systems. J Clin Microbiol. 2010 Mar;48(3):908-14. [PMC free article: PMC2832435] [PubMed: 20042630]

Lima-Oliveira G, Lippi G, Salvagno GL, Montagnana M, Picheth G, Guidi GC. Sodium citrate vacuum tubes validation: preventing preanalytical variability in routine coagulation testing. Blood Coagul Fibrinolysis. 2013 Apr;24(3):252-5. [PubMed: 23160241]

Banfi G, Salvagno GL, Lippi G. The role of ethylenediamine tetraacetic acid (EDTA) as in vitro anticoagulant for diagnostic purposes. Clin Chem Lab Med. 2007;45(5):565-76. [PubMed: 17484616]

Li G, Cabanero M, Wang Z, Wang H, Huang T, Alexis H, Eid I, Muth G, Pincus MR. Comparison of glucose determinations on blood samples collected in three types of tubes. Ann Clin Lab Sci. 2013 Summer;43(3):278-84. [PubMed: 23884222]

Jacobsen KK, Brandt I, Christensen AV, Rimsø BA, Krøier CJ, Sørensen M, Smith J, Jensen KOF, Larsen JM. Order of draw practices in venous blood sampling at clinical biochemistry departments in the Danish health care system. Clin Biochem. 2018 Jun;56:113-116. [PubMed: 29684368]

Heireman L, Van Geel P, Musger L, Heylen E, Uyttenbroeck W, Mahieu B. Causes, consequences and management of sample hemolysis in the clinical laboratory. Clin Biochem. 2017 Dec;50(18):1317-1322. [PubMed: 28947321]

Cornes M, van Dongen-Lases E, Grankvist K, Ibarz M, Kristensen G, Lippi G, Nybo M, Simundic AM., Working Group for Preanalytical Phase (WG-PRE), European Federation of Clinical Chemistry and Laboratory Medicine (EFLM). Order of blood draw: Opinion Paper by the European Federation for Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for the Preanalytical Phase (WG-PRE). Clin Chem Lab Med. 2017 Jan 01;55(1):27-31. [PubMed: 27444170]

Bowen RA, Sattayapiwat A, Gounden V, Remaley AT. Blood collection tube-related alterations in analyte concentrations in quality control material and serum specimens. Clin Biochem. 2014 Feb;47(3):150-7. [PMC free article: PMC3944914] [PubMed: 24240064]

Giavarina D, Lippi G. Blood venous sample collection: Recommendations overview and a checklist to improve quality. Clin Biochem. 2017 Jul;50(10-11):568-573. [PubMed: 28242283]

Why is it important to draw blood in the correct tube order?

Why is it important that tubes with an anticoagulant additive are full?

How should the anticoagulant be mixed with a blood specimen in an evacuated tube?

How does anticoagulant affect the blood draw?