Preliminary Steps

Before any specimen is collected, the phlebotomist must confirm the identity of the patient.⁴ Two or three items of identification should be used (e.g., [1] name, [2] medical record number, [3] date of birth, [4] address if the patient is an outpatient). In specialized situations, such as paternity testing or other tests of medico-legal importance, establishment of a chain of custody for the specimen may require additional patient identification, such as a photograph, provided as part of the identification process or taken to confirm the identity of the patient.

Identification must be an active process. Where possible, the patient should state his or her name, and the phlebotomist should verify information on the patient’s wrist band if the patient is hospitalized. If the patient is an outpatient, the phlebotomist should ask the patient to state his or her name and should confirm the information on the test requisition form with identifying information provided by the patient. In the case of pediatric patients, the parent or guardian should be present and should provide active identification of the child. In many institutions at this point in the process, the patient should be asked about latex allergies. If latex allergy is present and if latex gloves or a latex tourniquet may be used, the phlebotomist should secure an alternative tourniquet and put on gloves that are latex free. Finally, for some tests for genetic diseases, the performing laboratory may request a signed consent form from the patient; this should be completed at this time if it was not provided by the requesting physician.

Before collection of a specimen, a phlebotomist should dress in personal protective equipment (PPE), such as an impervious gown and gloves applied immediately before approaching the patient, to adhere to standard precautions against potentially infectious material and to limit the spread of infectious disease from one patient to another.¹⁴ If the phlebotomist is to collect a specimen from a patient in isolation in a hospital, the phlebotomist must put on a clean gown and gloves and a face mask and goggles before entering the patient’s room. The face mask limits the spread of potentially infectious droplets, and the goggles limit the possible entry of infectious material into the eye. The extent of the precautions required will vary with the nature of the patient’s illness and the institution’s policies and bloodborne pathogen plan, to which a phlebotomist must adhere. If airborne precautions are indicated, the phlebotomist must wear an N95 TB respirator.

If appropriate, the phlebotomist should verify that the patient is fasting, what medications are being taken or have been discontinued as required, and so forth. The patient should be comfortable, seated or supine (if sitting is not feasible), and should have been in this position for as long as possible before the specimen is drawn. For an outpatient, it is generally recommended that patients be seated before completion of the identification process to maximize their relaxation. At no time should venipuncture be performed on a standing patient. Either of the patient’s arms should be extended in a straight line from the shoulder to the wrist. An arm with an inserted intravenous line should be avoided, as should an arm with extensive scarring or a hematoma at the intended collection site. If a woman has had a mastectomy, arm veins on that side of the body should not be used, because the surgery may have caused lymphostasis (blockade of normal lymph node drainage), affecting the blood composition. If a woman has had double mastectomies, blood should be drawn from the arm of the side on which the first procedure was performed. If the surgery was done within 6 months on both sides, a vein on the back of the hand or at the ankle should be used.

Before performing a venipuncture, the phlebotomist should estimate the volume of blood to be drawn and should select the appropriate number and types of tubes for the blood, plasma, or serum tests requested. In many settings, this will be facilitated by computer-generated collection recommendations and should be designed to collect the minimum amount necessary for testing. The sections below on “Order of Draw for Multiple Collections” and “Collection With Evacuated Blood Tubes” discuss in greater detail the recommended order of draw for multiple specimens and types of tubes. In addition to tubes, an appropriate needle must be selected. The most commonly used sizes are 19 to 22 gauge. (The larger the gauge number, the smaller the bore.) The usual choice for an adult with normal veins is 20 gauge; if veins tend to collapse easily, a size 21 is preferred. For volumes of blood from 30 to 50 mL, an 18-gauge needle may be required to ensure adequate blood flow. A needle is typically 1.5 inches (3.7 cm) long, but 1-inch (2.5-cm) needles, usually attached to a winged or butterfly collection set, are also used. All needles must be sterile, sharp, and without barbs. If blood is drawn for trace element measurements, the needle should be stainless steel and should be known to be free from contamination.

Location

The median cubital vein in the antecubital fossa, or crook of the elbow, is the preferred site for collecting venous blood in adults because the vein is large and is close to the surface of the skin.12,20 Veins on the back of the hand or at the ankle may be used, although these are less desirable and should be avoided in people with diabetes and other individuals with poor circulation. In the inpatient setting, it is appropriate to collect blood through a cannula that is inserted for long-term fluid infusions at the time of first insertion to avoid the need for a second stick. For severely ill individuals and those requiring many intravenous injections, an alternative blood-drawing site should be chosen. Selection of a vein for puncture is facilitated by palpation. An arm containing a cannula or an arteriovenous fistula should not be used without consent of the patient’s physician. If fluid is being infused intravenously into a limb, the fluid should be shut off for 3 minutes before a specimen is obtained and a suitable note made in the patient’s chart and on the result report form. Specimens obtained from the opposite arm are preferred.¹² Specimens below the infusion site in the same arm may be satisfactory for most tests, except for those analytes that are contained in the infused solution (e.g., glucose, electrolytes).

Preparation of Site

The area around the intended puncture site should be cleaned with whatever cleanser is approved for use by the institution. Three commonly used materials are a prepackaged alcohol swab, a gauze pad saturated with 70% isopropanol, and a benzalkonium chloride solution (Zephiran chloride solution, 1 : 750). Cleaning of the puncture site should be done with a circular motion and from the site outward. The skin should be allowed to dry in the air. No alcohol or cleanser should remain on the skin because traces may cause hemolysis and invalidate test results. Once the skin has been cleaned, it should not be touched until after the venipuncture has been completed.

Timing

The time at which a specimen is obtained is important for those blood constituents that undergo marked diurnal variation (e.g., corticosteroids, iron) and for those used to monitor drug therapy (see Chapter 34). For most current molecular diagnostic tests, the time of day is unlikely to contribute to altered or invalid test results. Furthermore, timing is important in relation to specimens for alcohol or drug measurements in association with medico-legal considerations.

Venous Occlusion

After the skin is cleaned, a blood pressure cuff or a tourniquet is applied 4 to 6 inches (10 to 15 cm) above the intended puncture site (distance for adults). This obstructs the return of venous blood to the heart and distends the veins (venous occlusion). When a blood pressure cuff is used as a tourniquet, it is usually inflated to approximately 60 mm Hg (8.0 kPa). Tourniquets typically are made from precut soft rubber strips or from Velcro. It is rarely necessary to leave a tourniquet in place for longer than 1 minute, but even within this short time the composition of blood changes. Although the changes that occur in 1 minute are slight, marked changes have been observed after 3 minutes for many chemistry analytes (Table 7-1). No known changes affect molecular diagnostics.

The composition of blood drawn first—that is, the blood closest to the tourniquet—is most representative of the composition of circulating blood. The first-drawn specimen should therefore be used for those analytes such as calcium that are pertinent to critical medical decisions.²⁵ Blood drawn later shows a greater effect from venous stasis. Thus the first tube may show a 5% increase in protein, whereas the third tube may show a 10% change.²² The concentration of protein-bound constituents is also influenced by stasis. Prolonged stasis may increase the concentration of protein or protein-bound constituents by as much as 15%. A uniform procedure for the order of draw for tests should therefore be established (see later). If it is possible to collect only a small volume of blood, the priority of which tests to perform should be established.

The increase in activity of creatine kinase and aspartate aminotransferase in serum seen after venipuncture may be caused by hemoconcentration, by slight trauma to tissue as the needle pierces the skin, and by stasis of blood in the tissue.

Pumping of the fist before venipuncture should be avoided because it causes an increase in plasma potassium, phosphate, and lactate concentrations. Lowering of blood pH by accumulation of lactate causes the plasma ionized calcium concentration to increase.²⁴ The ionized calcium concentration reverts to normal 10 minutes after the tourniquet is released.

Stress associated with blood collection can have effects on patients at any age. As a consequence, plasma concentrations of cortisol and growth hormone may increase. Stress occurs particularly in young children who are frightened, struggling, and held in physical restraint. Collection under these conditions may cause adrenal stimulation leading to an increased plasma glucose concentration or may create increases in the serum activities of enzymes that originate in skeletal muscle.

Order of Draw for Multiple Blood Specimens

In a few patients, backflow from blood tubes into veins occurs owing to a decrease in venous pressure. The dangerous consequences of this occurrence may be prevented if only sterile tubes are used for collection of blood. Backflow is minimized if the arm is held downward and blood is kept from contact with the stopper during the collection procedure. To minimize problems if backflow should occur, and to optimize the quality of specimens—especially to prevent cross-contamination with anticoagulants—blood should be collected into tubes in the order outlined in Table 7-2. This table also provides the recommended number of inversions for each tube type because it is critical that complete mixing of any additive with the blood collected be accomplished as quickly as possible.

Collection With Evacuated Blood Tubes

Evacuated blood tubes are usually considered to be less expensive and are more convenient and easier to use than syringes, and thus are the collection device of choice in many institutions. Evacuated blood tubes may be made of soda-lime or borosilicate glass or plastic (polyethylene terephthalate). Because of the decreased likelihood of breakage and subsequent exposure to infectious materials, many laboratories have converted from glass tubes to plastic tubes. Several types of evacuated tubes may be used for venipuncture collection.¹² They vary by the type of additive added and the volume of the tube. The different types of additives are identified by the color of the stopper used (Table 7-3). Serum or plasma separator tubes are available that contain an inert, thixotropic, polymer gel material with a specific gravity of approximately 1.04. Aspiration of blood into the tube and subsequent centrifugation displace the gel, which settles like a disk between cells and supernatant when the tube is centrifuged. A minimum relative centrifugal force (RCF) of 1100 ×g is required for gel release and barrier formation in most tubes. Release of intracellular components into the supernatant is prevented by the barrier for several hours or, in some cases, for a few days. These separator tubes may be used as primary containers from which serum or plasma can be directly aspirated by a number of analytical instruments. Additional tubes, not listed, are sold for special applications, such as RNA isolation. These less common tubes must be validated by each laboratory before use if not approved by the manufacturer for the specific analysis to be conducted.

Stoppers may contain zinc, invalidating the use of evacuated blood tubes for zinc measurement, and TBEP [tris(2-butoxyethyl) phosphate], a constituent of rubber, which may interfere with the measurement of certain drugs. With time, the vacuum in evacuated tubes is lost and their effective draw diminishes. The silicone coating also decays with age. Therefore the stock of these tubes should be rotated and careful attention paid to the expiration date. Blood collected into a tube containing one additive should never be transferred into other tubes, because the first additive may interfere with tests for which a different additive is specified. Additionally, transfer of the additive from one tube to another should be minimized (or adverse effects reduced) through strict adherence to recommendations for order of tube use (see Table 7-2).

A typical system for collecting blood in evacuated tubes is shown in Figure 7-1.¹⁷ This is an example of a commonly used single-use device that incorporates a cover that is designed to be placed over the needle when collection of the blood is complete, thereby reducing the risk of puncture of the phlebotomist by the now contaminated needle. A needle or winged (butterfly) set is screwed into the collection tube holder, and the tube is then gently inserted into this holder. The tube should be gently tapped to dislodge any additive from the stopper before the needle is inserted into a vein; this prevents aspiration of the additive into the patient’s vein.

After the skin has been cleaned, the needle should be guided gently into the patient’s vein (Figure 7-2); once the needle is in place, the tube should be pressed forward into the holder to puncture the stopper and release the vacuum. As soon as blood begins to flow into the tube, the tourniquet should be released without moving the needle (see earlier discussion on venous occlusion). The tube is filled until the vacuum is exhausted. It is critically important that the evacuated tube be filled completely. Many additives are provided in the tube based on a “full” collection; deviation or short draws can be a source of preanalytical error because they can significantly affect test results.⁷ Once the tube is filled completely, it should be withdrawn from the holder, mixed gently by inversion, and replaced by another tube, if this is necessary. Other tubes may be filled using the same technique with the holder in place. When several tubes are required from a single blood collection, a shut-off valve—consisting of rubber tubing that slides over the needle opening—is used to prevent spillage of blood during exchange of tubes.

Blood Collection With Syringe

Syringes are customarily used for patients with difficult veins. If a syringe is used, the needle is placed firmly over the nozzle of the syringe, and the cover of the needle is removed. If the syringe has an eccentric nozzle, the needle should be arranged with the nozzle downward but the bevel of the needle upward. The syringe and the needle should be aligned with the vein to be entered and the needle pushed into the vein at an angle to the skin of approximately 15 degrees. When the initial resistance of the vein wall is overcome as it is pierced, forward pressure on the syringe is eased, and the blood is withdrawn by gently pulling back the plunger of the syringe. Should a second syringe be necessary, a gauze pad may be placed under the hub of the needle to absorb the spill; the first syringe is then quickly disconnected, and the second put in place to continue the blood draw. Using the same needle or a new needle, the cap of the evacuated tube should be punctured and the evacuated tube allowed to fill passively. Uncapping the evacuated tube is not recommended. Vigorous withdrawal of blood into a syringe during collection or forceful transfer from the syringe to the receiving vessel may cause hemolysis of blood. Hemolysis is usually less when blood is drawn through a small-bore needle than when a larger-bore needle is used.

Completion of Collection

When blood collection is complete and the needle withdrawn, the patient should be instructed to hold a dry gauze pad over the puncture site, with the arm raised to lessen the likelihood of leakage of blood. The pad may then be held in place by a bandage or by a nonadhesive strap (which avoids pulling hairs on the arm when it is removed); these are removed after 15 minutes. With a collection device, such as that shown in Figure 7-1, the needle is covered, and the needle and the tube holder are immediately discarded into a sharps container. In the event that a winged (butterfly) set is used, the wings are pushed forward to cover the needle, or with newer available equipment, a button is pressed, releasing a spring that retracts the needle. If a syringe was used, the needle and syringe (still attached) should be discarded in a hazardous waste receptacle.

All tubes should then be labeled per institutional policy. Most institutions have a written procedure prohibiting the advance labeling of tubes because this is seen as providing the potential for mislabeling, one of the most common sources of preanalytical error. Some institutions recommend showing the labeled tube to the patient to further confirm correct identification. Gloves should be discarded in a hazardous waste receptacle if visibly contaminated, or in noncontaminated trash if not visibly contaminated. Before applying new gloves and proceeding to the next patient, and depending on institutional policy, clinicians should use an alcohol-based cleanser or soap and water to wash their hands.

Venipuncture in Children

The techniques for venipuncture in children and adults are similar. However, children are likely to make unexpected movements, and assistance in holding them still is often desirable. A syringe or an evacuated blood tube system may be used to collect specimens. A syringe should be the tuberculin type or should have a 3-mL capacity, except when a large volume of blood is required for analysis. A 21- to 23-gauge needle or a 20- to 23-gauge butterfly needle with attached tubing is appropriate to collect specimens. In general, in the pediatric population, alternative collection through skin puncture is often used.