Which of the following tubes contains an anticoagulant that works by binding calcium?

Blood Tubing

The tubing used to connect the various components and conduct blood into and out of the patient’s vascular system is medical-grade polyvinyl chloride. For decades, the blood-tubing interface was untreated polyvinyl chloride. However, the newer generation of polyvinyl chloride tubing has surface coatings and other modifications that significantly alter the bioreactivity of the surface. Collectively, these coatings have been shown to reduce plasma levels of markers of subclinical coagulation, attenuate the increase of cytokines and other inflammatory markers, and shorten intubation times.268-270

Glass versus Plastic Tubes for Collection of Blood

Plastic blood collection tubes are lighter than glass, unbreakable, and provide easy disposal. Correlation studies between glass and plastic blood collection tubes for common analytes and some hormones and tumor markers have been published [51, 52]. Many laboratories have performed their own evaluations of plastic tubes to document analyte compatibility before converting, but haven’t always published their findings. Dasgupta et al. examined the stability of 13 therapeutic drugs stored in plastic tubes compared to glass tubes, using quality control material [53]. The authors found no significant reduction in concentrations of caffeine, primidone, procainamide, NAPA, acetaminophen, salicylate, amikacin, valproic acid, methotrexate or cyclosporine. When comparing volume of serum in the tube, they observed significant reductions in concentrations of phenytoin, phenobarbital, carbamazepine, quinidine and lidocaine after storage of 500 μl of serum versus 1 mL in both glass and plastic gel tubes. The poor recovery of these drugs was attributed to the gel in the tubes, rather than the tube material.

Faynor and Robinson examined the suitability of plastic blood collection tubes for cyclosporine measurement [54]. Specimens from renal transplant patients were collected into glass and plastic BD Vacutainer EDTA tubes. Tubes were stored at room temperature and 4°C after collection, and between testing intervals of 0, 1, 4 and 7 days. The drug levels in the plastic tubes were slightly higher than those from the glass tubes at both storage temperatures, and did not appear to be time dependent. All of the differences for individual pairs of samples were within 10%. The authors concluded that cyclosporine levels are stable in plastic tubes over 7 days at room temperature or refrigerated. Boeynaems et al. compared Terumo’s Venoject® glass tubes with Venosafe® PET tubes with clot activator and heparin for several therapeutic drugs at 2 and 24 hours post-collection [55]. Blood was spiked with the parent drug to low, mid and high therapeutic levels. The authors found no consistent significant differences among the tube types for the panel of drugs tested. These data agree with other comparisons between Venoject II and BD Vacutainer plastic tubes [56].

Tubing

Tubing should be adapted to the patient's size. The extracorporeal volume, consisting of the arterial and venous segments and the dialyzer, should not exceed 10% of the patient's blood volume. For patients weighing less than 6 kg this goal cannot be achieved even with the smallest available tubing, and priming of the extracorporeal system is needed, either with whole blood or with packed red blood cells diluted with normal saline to an estimated hematocrit of 33% to 36%. Although blood units can be divided so a single unit can be used for four consecutive HD sessions, there still is a high exposure to blood products. Iron overload may occur despite the fact that at the end of each session the blood in the extracorporeal system is not routinely washed back to the patient, unless the patient requires a transfusion.

Exposing a patient to many units of blood increases the risk of viral infection and of human leukocyte antigen (HLA) sensitization. However, the incidence of sensitization in infants treated with chronic HD necessitating multiple transfusions has not been systematically studied.

In patients weighing more than 6 kg the tubing is primed with either 5% albumin solution (in smaller patients) or physiological saline solution in those weighing more than 10 kg. In those patients, the blood in the tubing is washed back to the patient at the end of each session. This procedure deserves special consideration as the blood volume filling the tubing is equivalent to 0.5 to 1 weight-adjusted blood unit. Even with the slowest possible pump speed the whole volume will be washed back within 2 to 3 minutes, which is exceptionally fast. This may cause an abrupt increase in the right atrial pressure and if the patient has a patent foramen ovale (PFO) it would increase the risk for possible microthrombi paradoxical embolism. Despite this concern, as well as the known relative cognitive deficiency in patients on dialysis and the frequent (18%–33%) finding of ischemic lesions on brain MRI studies in pediatric ESRD patients, this point has not been explored.80,83 In adult patients there was no evidence of faster cognitive deterioration with PFO on HD compared with those without PFO, although atrial pressures during blood return at the end of dialysis may not be as high in adults.134

Surfactants

Commercially available BCT may contain different types of surfactants that are often not listed in the manufacturer’s package insert but are commonly silicone-based polymers. Although these are considered to be inert, there have been reports of interferences in clinical assays. Generally, surfactants can bind nonspecifically, displace from solid matrix, and complex with or mask detection of signal antibodies in immunoassay reagents, contributing to increases in absorbance and turbidity to cause interferences [14]. Bowen et al. [15,16] showed that the surfactant Silwet L-720 used in Becton Dickinson SST collection tubes gave falsely elevated total triiodothyronine (TT3) by the Immulite 2000/2500 immunoassay. One mechanism shown was that increasing surfactant concentrations dose dependently desorbed the capture antibody from the solid phase among other nonspecific effects. However, this was manufacturer method dependent because TT3 levels were unaffected by the AxSYM immunoassay, in which antibodies are adsorbed onto the solid phase with more robust binding [15,16]. This manufacturer confirmed similar interferences for a variety of other immunoassays (folate, vitamin B12, follicle-stimulating hormone, hepatitis B surface antigen, cancer antigen 27, and cortisol) on a variety of different instrument platforms and has since decreased the surfactant content to reduce this interference [17,18].

Aneurysms and Arterial Wall Stress

When the structural components of the arterial wall are weakened, aneurysms may form. Rupture occurs when the tangential stress within the arterial wall becomes greater than the tensile strength. The tangential stress (τ) within the wall of a fluid-filled cylindrical tube can be expressed as:

(8.6)τ=Prδ,

whereP is the pressure exerted by the fluid,r is the internal radius, andδ is the thickness of the tube wall. Thus tangential stress is directly proportional to pressure and radius but inversely proportional to wall thickness. Stress (τ) has the dimensions of force per unit area of tube wall (dynes per square centimeter).Eq. 8.6 is similar to Laplace's law, which defines tangential tension (T) as the product of pressure and radius:

(8.7)T=Pr.

Tension is given in units of force per tube length (dynes per centimeter). The termsstress andtension have different dimensions and describe the forces acting on the tube wall in different ways. Laplace's law can be used to characterize thin-walled structures such as soap bubbles; however, it is not suitable for describing the stresses in arterial walls.

Fig. 8.13 shows a tube with an outside radius of 1.0 cm and a wall thickness of 0.2 cm, dimensions similar to those of atherosclerotic aortas. If the internal pressure is 150 mm Hg, the tangential wall stress is 8.0 × 105 dynes/cm2. Expansion of the tube to form an aneurysm with an outside radius of 3.0 cm results in a decrease in wall thickness to 0.06 cm. The increased radius and decreased wall thickness increase the wall stress to 98.0 × 105 dynes/cm2, assuming that the pressure remains constant. In this example, the diameter has been enlarged by a factor of 3, and the wall stress has increased by a factor of 12.

Although the tensile strength of collagen is very high, it constitutes only about 15% of the aneurysm wall.31 Furthermore, the collagen fibers in an aneurysm are sparsely distributed and subject to fragmentation. The tendency of larger aneurysms to rupture is readily explained by the effect of increased radius on tangential stress (Eq. 8.6) and degenerative changes in the arterial wall. The relationship between tangential stress and blood pressure accounts for the contribution of hypertension to the risk of rupture. Another factor is that in about 55% of ruptured abdominal aortic aneurysms, the site of rupture is in the posterolateral aspect of the aneurysm wall.32 The posterior wall of the aorta is relatively fixed against the spine, and repeated flexion in that area could result in structural fatigue and a localized area of weakness that might predispose to rupture.

Body Fluids

Whole blood is collected in blood collection tubes or syringes that contain the appropriate anticoagulant to inhibit coagulation. Routine hematology tests require a blood collection tube containing EDTA, a chelating anticoagulant. EDTA preserves the morphology of the cellular elements of blood, making it a satisfactory anticoagulant for hematologic studies. Blood collection tubes containing heparin, which stabilizes the red blood cell membranes, are used for specialized hematology studies, such as red cell fragility tests and several specialized chemistry tests. Blood collection tubes containing sodium citrate are used for coagulation studies.

Clotted blood is obtained in blood collection tubes that contain a silicon coating or a serum separator. The blood collected into these tubes is allowed to clot for 15 to 30 minutes prior to centrifugation. Once centrifuged, the serum is separated from the clotted red blood cell mass. Serum specimens are required for routine chemistry, serology, and blood bank tests.

Blood for culture and sensitivity must be collected by aseptic technique into a sterile culture bottle. Alternatively, blood may be collected by aseptic technique into two blood collection tubes that contain transport growth media. The blood culture bottles or transport growth media tubes must be delivered promptly to the microbiology laboratory. See Table 8-3 for a summary of blood collection tubes.

Many other types of blood collection tubes are available for an array of special tests. The PA is advised to consult the laboratory before collecting any blood test specimen that may require specialized collection procedures.

Urine is collected in a clean specimen container for routine urinalysis, either by having the patient void into the container or by pouring the urine from a bedpan or catheter collection bag into the specimen container. Urine for culture and sensitivity must be collected by aseptic technique. The patient is instructed to cleanse the genital area around the urethral meatus and then to collect the midstream portion of the urine. The first morning specimen is always preferred for any urine test. Urine specimens are collected for 24 hours for a variety of chemical analyses. For most 24-hour urine specimens, some type of preservative is added to the specimen container. The laboratory should be consulted for specific preservative requirements. All urine specimens must be refrigerated until delivered to the laboratory, to prevent bacterial overgrowth. As with any specimen, prompt delivery to the laboratory is essential for maintaining the quality of the specimen.

Cerebrospinal fluid is collected by standard lumbar puncture, under sterile conditions, into sterile specimen containers provided in lumbar puncture kits. The CSF must be transported to the laboratory within 5 to 10 minutes of collection for immediate analysis. A CSF specimen should never be allowed to sit at the patient's bedside. Likewise, it should not be refrigerated prior to microbiologic culture and staining for microorganisms because many of the causative organisms of meningitis are fastidious and fragile in suboptimal conditions outside the body. Protein, glucose, cell counts, and other enzymatic or serologic tests of CSF are not adversely affected by refrigeration.

Semen is collected by the patient into a sterile specimen container. The patient must be advised to deliver the semen specimen to the laboratory within 20 minutes of collection to allow accurate evaluation of motility, viscosity, and viability of the sperm. The specimen should be kept as close to body temperature as possible during transport and is never refrigerated.

Stool specimens are collected into wide-mouth containers appropriate to the volume of stool required for testing. Specimens for ova and parasites and for stool culture are collected into standard-sized, sterile specimen containers. These specimens are best delivered to the laboratory within an hour of collection and should not be refrigerated unless lengthy delay is anticipated. Stool specimens collected for 24 hours for fecal fat evaluation are put in wide-mouth, clean, gallon-sized metal containers with tight-fitting lids; refrigeration is recommended.

Synovial, thoracentesis, and paracentesis fluids are collected by sterile technique in sterile containers of appropriate size. If synovial fluid is submitted to the laboratory in the aspirating syringe; the needle must be removed and the syringe capped prior to transport of the specimen to prevent accidental percutaneous injury. These specimens may be refrigerated unless culture for anaerobic organisms is requested.

Errors Related to Sample Collection, Transport, and Storage

EDTA is the typical anticoagulant used in blood collection tubes. It can be in a dry format or as a solution. The amount and concentration of EDTA require that blood should be collected up to a specific mark on the tube. If too little blood is collected, dilution of the sample can become an issue with alteration of parameters. Relative excess EDTA in such cases also affects the morphology of blood cells. Transport of specimen should ensure that high temperatures are avoided. Red cell fragmentation is a feature of excess heat [28].

Prolonged storage will result in degenerative changes in WBCs. This is best illustrated in neutrophils, in which WBCs have a round pyknotic nucleus. To the casual observer, these cells may appear as nucleated red cells. Abnormal lobulation of the lymphocyte nuclei is another established phenomenon with prolonged storage of blood [29]. These cells may be considered as atypical lymphocytes, with an incorrect implication of an underlying lymphoproliferative disorder.

Table 19.1 summarizes sources of laboratory errors in hematology testings.

Table 19.1. Sources of Laboratory Errors in Hematology

MCV, mean corpuscular volume.

Aluminum

Aluminum in serum collected using a metal-free vacutainer is analyzed by ICP-MS methods. Because many commercially available vacutainers use rubber caps that contain considerable amounts of aluminum, check with your laboratory for specific instructions on which tubes have been shown to be aluminum free. Never use wooden applicator sticks to break up the clot: they contain large amounts of aluminum. The samples are stable for several days at all temperatures. The reference range for all ages, both genders, is less than 6 ng/mL. Patients undergoing renal dialysis have higher levels, and aluminum is not removed during dialysis. Levels in these patients are usually less than 60 ng/mL, with toxic symptoms being evident at levels greater than 100 ng/mL.

Isolation of primary human monocytes

1.

Collect blood samples from subjects into heparinized BD Vacutainer® plasma tube (BD, United States), invert several times gently and proceed to monocyte isolation.

Fill Greiner LeucoSep tubes (50-mL) (Millipore Sigma, United States) with 15 mL Ficoll-Paque PLUS (GE Healthcare, IL, United States) and centrifuge for 30 s at 1000 × g at room temperature.

Dilute blood sample in phosphate-buffered saline (PBS) (1:2 dilution) and distribute 30 mL of diluted blood on each LeucoSep tubes.

Using density gradient centrifugation at 1000 × g for 10 min (without deceleration), separate peripheral blood mononuclear cells (PBMCs), collect cells from the interphase and wash 3 times with PBS at 300 × g for 5 min at ambient temperature.

After last wash, remove the supernatant and add 10 mL ACK lysing buffer (Thermo Fisher Scientific, United States) and incubate for 10 min to lyse red blood cells.

Wash cells with PBS as above and resuspend in EasySep Bufffer™ (Stemcell Technologies, Canada). Count cells and adjust cell concentration to 5 × 107 cells per mL.

Isolate monocytes by immunomagnetic negative selection using the EasySep Human Monocyte Isolation Kit according to manufacturer's instructions and proceed to differentiation and activation of macrophages.

Equipment

The following equipment should be assembled:

▪

Tourniquet.

Alcohol pads.

Disposable latex gloves.

Vacutainer needle holder or syringe (5, 10, or 20 mL).

Vacutainer needle or a 20-gauge needle for the syringe. If a large amount of blood is to be drawn, it is best to use an 18-gauge needle. Needles smaller than 22-gauge should be avoided because the blood sample tends to hemolyze in the small bore. A butterfly needle may be needed for small veins.

Properly labeled specimen Vacutainer tubes.

Sterile gauze pads.

Self-adhesive bandage.

Needle disposal container.

Important: Know which specific tests are to be collected so that the proper tubes are available.