Specimen collection and processing

Preliminary steps.

Before venipuncture is started 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 specialized tests such as testing for genetic diseases, the performing laboratory may request the use of a special requisition. When these are required, in general they should be provided by the requesting physician and be brought by the patient to the collection.

Before collection of a specimen, the phlebotomist should dress in personal protective equipment (PPE), such as an impervious gown and gloves applied immediately before approaching the patient, and adhere to standard precautions against potentially infectious material; the goal is to limit the spread of infectious disease from one patient to another and to promote the safety of the patient and phlebotomist. Because small children are often frightened of anyone in a white coat or gown, pediatric phlebotomists often dress in bright, cheerful colors, including colored PPE rather than standard white. Pediatric drawing stations are also often brightly colored with lots of distracters for the patient. If the phlebotomist must 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 varies with the nature of the patient’s illness and the institution’s policies and bloodborne pathogen plan to which a phlebotomist must adhere. For example, if airborne precautions are indicated, the phlebotomist must wear an N95 tuberculosis respirator in the United States.

If required, the phlebotomist should verify that the patient has fasted, identify what medications are being taken or have been discontinued as required, and determine any other relevant information required. Chapter 5 describes in more detail the effects of diet and fluid intake and the recommended steps for patient preparation, including fasting, before phlebotomy. 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. The correct interpretation of certain tests (e.g., aldosterone, renin, plasma metanephrines) requires that the patient be in a supine position for at least 30 minutes before venipuncture. (For details on the effects of position, refer to Chapters 5 and 53 .) 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.

Infants and young children may need to be held in order to restrain them and prevent movement. Young children may be held sitting upright in a parent’s lap with the parent helping to support and hold the patient and arm still ( Fig. 4.1 ). Infants’ blood is often drawn with the infant in a supine position, and the infant may be swaddled in a blanket, or a papoose board may be used to restrain movement. Occasionally, the parents will be more anxious than the child or will wish not to be associated with a procedure which causes the child pain, and the phlebotomist will need to make the decision to request help from a colleague phlebotomist to properly and safely perform the collection. ^,

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 (IV) 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 a double mastectomy, 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 is facilitated by computer-generated collection recommendations and should be designed to collect the minimum amount necessary for testing. Estimating volume of blood to be drawn is especially critical in a pediatric setting. An average-weight newborn infant has a total blood volume of approximately 350 mL. Collecting too much blood from an infant in a hospital setting will eventually result in the need to give the infant blood back in the form of a transfusion, risking exposure to bloodborne pathogens. Blood collection in the pediatric population should not exceed recommended volumes for the pediatric patient’s weight. The later sections on “Order of Draw for Multiple Collections” and “Collection with Evacuated Blood Tubes” discuss in greater detail the recommended order in which to draw multiple specimens and the types of tubes to be used. Careful consideration should also be taken in the case of an adult patient, as one study showed that on average, every 100 mL of phlebotomy was associated with a decrease in hemoglobin of 7.0 g/L and hematocrit of 1.9%. Such iatrogenic blood loss can lead to the same possible unnecessary required blood transfusion and an increased risk of exposure to bloodborne pathogens in adults as in children.

In addition to tubes, an appropriate needle must be selected. The most commonly used sizes for adults 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. In pediatric patients, 23- to 25-gauge needles are most commonly used, with 23-gauge being the preferred size. Venipuncture on infants and children younger than 2 years old is often performed on dorsal hand veins rather than arm veins, and the veins in either place are very small in this age group. Even for larger volumes of blood, rarely will a needle larger than a 21 gauge be used because it will not fit into the vein easily. 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 and are common in pediatrics. All needles must be sterile and 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.

Finally, the phlebotomist should ensure that all postdraw safety devices are in place. These include (for the person drawing) quick, convenient, and safe access to proper disposal devices for all (now) contaminated needles and associated devices and (for the patient) the appropriate post–blood draw supplies (gauze and bandage) are in place to ensure no adverse events might affect the patient.

Timing.

The time at which a specimen is obtained is important for blood constituents that undergo marked diurnal variation (e.g., corticosteroids, iron), for those for which a fasting sample has been requested, and for those used to monitor drug therapy. In each case, the timing should match the conditions under which reference intervals or clinical decision points were determined (see Chapter 9 ). Furthermore, timing is important in relation to specimens for alcohol or drug measurements in association with medicolegal considerations.

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 (CLSI GP41, see Table 4.1 ). 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. However, in infants and children younger than 2 years old, collection from superficial veins is recommended, and these sites may be preferred over the median cubital vein. 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. This method of collection may increase the chances of a hemolyzed sample and contamination of the collected sample with fluids being infused. Careful adherence to withdrawal of a discard volume and discussions with the clinical team on alternative site for phlebotomy can greatly reduce these preanalytical variables. For severely ill individuals and those requiring many IV 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 at least 3 minutes (with clinician consent) before a specimen is obtained and a suitable note made in the patient’s chart and on the result report form and the recommencement of the infusion must be ensured. Specimens obtained from the opposite arm are preferred. Specimens below the infusion site in the same arm may be satisfactory for most tests, except for analytes that are contained in the infused solution (e.g., glucose, electrolytes).

Preparation of the 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 (e.g., Zephiran chloride solution, 1:750). Cleaning of the puncture site should be done with a circular motion 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. After the skin has been cleaned, it should not be touched until after the venipuncture has been completed.

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. If a dorsal hand vein is being accessed in infants and young children, no tourniquet is used. The phlebotomist applies enough pressure with the hand holding the patient’s wrist and hand to occlude and distend the vein.

It is rarely necessary to leave a tourniquet in place for longer than 1 minute after venous access is secured and the tourniquet is removed, but even within this short time, the composition of blood changes, and adherence to institutional policies must be followed. Although the changes that occur in 1 minute are slight, marked changes have been observed after 3 minutes for some chemistry analytes. The composition of blood drawn first—that is, the blood closest to the tourniquet—is most representative of the composition of circulating blood and the least affected by fluid shifts where protein bound components and other large molecules will be concentrated; water-soluble smaller molecules such as electrolytes may be less affected. The first-drawn specimen should therefore be used for analytes such as calcium and other analytes that are both protein bound and pertinent to critical medical decisions and that may be affected by the collection process. ^, A uniform procedure for the order of draw for tests should therefore be established (see later discussion). If it is only possible to collect a small volume of blood, the priority of which tests to perform should be established.

Two special notes on the collection process: Pumping of the fist before venipuncture should be avoided because it causes an increase in plasma potassium, phosphate, and lactate concentrations. The lowering of blood pH by accumulation of lactate also causes the plasma ionized calcium concentration to increase. The ionized calcium concentration reverts to normal 10 minutes after the tourniquet is released. Importantly, the stress associated with blood collection and/or hospitalization can have effects on patients at any age. As a consequence, plasma concentrations of analytes affected by stress, such as cortisol, thyroid-stimulating hormone, 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 are prevented by using only sterile tubes 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. When collecting multiple specimens with an evacuated tube system, one of the primary concerns is to prevent cross-contamination between tubes. For example, potassium ethylenediaminetetraacetic acid (EDTA) contamination can cause an erroneously reported hyperkalemia or hypocalcemia when an inappropriate tube type is used. To minimize problems if backflow occurs and to optimize the quality of specimens by preventing cross-contamination with anticoagulants, blood should be collected into tubes in the order outlined in Table 4.2 , which generally follows a process of no anticoagulant to mild anticoagulant to strong anticoagulant. 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. In addition, completing a blood collection within 2 minutes of starting, and getting the tubes mixed correctly as soon as possible, helps to prevent clotting in anticoagulated tubes. The order of collection when multiple tubes are drawn from a skin puncture is different than when an evacuated tube system is used (see the later section on skin puncture).

Collection with evacuated blood tubes.

Evacuated blood tubes are usually considered to be safer, less expensive, 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, if not most, laboratories have converted from glass to plastic tubes. Several types of evacuated tubes may be used for venipuncture collection. 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. Color coding of specimen collection tubes is not yet harmonized and may vary according to manufacturers. Table 4.3 presents the most common forms of color codes of various tube types. Serum or plasma separator tubes are available that contain an inert, polymer gel material with a specific gravity of approximately 1.04. Aspiration of blood into the tube and subsequent centrifugation displaces 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 many cases, for 7 days or more, allowing for additional testing (“add-ons”) from samples collected at a specific time in the patient’s care. However, all laboratories need to review the specific manufacturers’ recommendations of what may be allowed based on provided data or perform their own validation studies. Most importantly, these separator tubes may be used as primary containers from which serum or plasma can be directly aspirated by a number of analytical instruments, avoiding aspiration of red blood cells (RBCs) or possible errors of patient or sample identification during aliquoting. Additional tubes, not listed, are sold for special applications, such as RNA isolation. As with all specimen collection containers, 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 tris(2-butoxyethyl) phosphate (TBEP), also a constituent of the stopper, 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, cross-contamination of additives from one tube to another during multiple tube draws should be minimized (or adverse effects reduced) through strict adherence to recommendations for order of tube use (see Table 4.2 ).

Typical systems for collecting blood are shown in Fig. 4.2 . Single-use devices incorporate 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; when 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, particularly for coagulation testing, are provided at concentrations in the tube based on a specified volume requirement; both short and too-full draws can be a source of preanalytical error because they can significantly affect the established testing parameters that are based on a properly collected sample. Therefore a vacuum tube should always be filled using the vacuum that is designed to fill it correctly. These tubes should never be opened and filled from a syringe or other source. After the tube is filled completely, it should be withdrawn from the holder, mixed gently by inversion, and replaced by another tube if necessary. Other tubes may be filled using the same technique with the holder in place.

Blood collection with a syringe.

Syringes are customarily used for patients with difficult veins, including very small veins, and for blood gas analysis. 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 very gently pulling back the plunger of the syringe. If a second syringe is 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 is put in place to continue the blood draw.

After filling the syringe and completing the collection, if the sample needs to be transferred to an evacuated tube, a transfer device should be used to puncture the cap of the tube. Use of transfer devices prevents having to puncture an evacuated tube with a needle and risking a needle-stick injury. The tube should be allowed to fill passively using its vacuum; uncapping the evacuated tube is not recommended for the reasons stated earlier. Vigorous withdrawal of blood into a syringe during collection or forceful transfer from the syringe to the receiving vessel may cause hemolysis of blood and will likely make the sample not valid for testing. Communication of this common preanalytical error to those not trained in routine sample collection is the responsibility of all laboratory directors and the experts, the phlebotomy team. Although safe use and disposal of sharps is important with any collection device, this is particularly important with the use of a needle and syringe. The phlebotomist must ensure an appropriate sharps disposal bin is available at the point of collection, that the location is free of interference or distractions that may increase the risk of a needle-stick injury, and that he or she has been trained in all procedures.

Completion of collection.

When blood collection is complete and the needle withdrawn, the patient should be instructed to hold a dry gauze pad tightly over the puncture site with the arm raised to stop residual bleeding and promote the clotting process. The pad should then be held in place firmly by a bandage or by a nonadhesive strap (which avoids pulling hairs on the arm when it is removed); these may be removed after 15 minutes. With a collection device, such as that shown in Fig. 4.2 , the needle is covered, and the needle and the tube holder are immediately discarded into a sharps container that should be conveniently and safely positioned. 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 should not be removed because of the danger of a needlestick on the part of the phlebotomist. All used supplies 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. Collectors should ensure that the correct labels are applied. Incorrect labels which have been mistakenly filed in patient notes, files, or are in the room of a hospitalized patient, are a major source of mislabeling and preanalytical error. Many US institutions recommend showing the labeled tube to the patient to further confirm correct identification. At the conclusion of this process, gloves should be discarded in a hazardous waste receptacle if visibly contaminated or in uncontaminated trash if not visibly contaminated. Before applying new gloves and proceeding to the next patient, and depending on institutional policy, all caregivers including phlebotomists should use an alcohol-based cleanser or soap and water to wash their hands.