What may occur if the needle is removed from the arm before removing the tourniquet

3.05.5.3 Tourniquet

A tourniquet allows for pressure to be applied to the arm so that venous blood returning to the heart can be slowed down. As a result, the blood vessel walls become temporarily occluded and the veins distend due to the pooling of blood. This allows veins to become more visible and easier to palpate.

Some tourniquets are made out of Velcro (Figure 12) but many tourniquets used are made out of a stretchy material (more tourniquets are now being produced without latex due to the increasing number of allergies). In a healthcare setting, these stretchable tourniquets are usually meant for a single use. A blood pressure cuff can also be used as a tourniquet, which is commonly used when a large volume of blood is collected (such as for transfusion purposes).

The phlebotomist should not leave the tourniquet on the patient’s arm for longer than a minute. This increased pressure against the vessel walls allows plasma and small molecules to flow through capillary walls and into the tissue. This process is known as hemoconcentration; it results in a relative increase in the number of red blood cells as well as higher-molecular-weight compounds in the sample drawn. With prolonged tourniquet application time, test results such as albumin, cholesterol, coagulation proteins, and red cell count are falsely increased (Section 3.05.8.4).

Clinical Management

The management of venomous snakebites is based on supportive care and appropriate timely antivenom therapy. Supportive care such as local wound care and pain treatment are important but potentially harmful interventions such as tourniquets, cutting, suction, and application of electricity should be avoided. Antivenom is the definitive treatment. Antivenom is produced by injecting a host animal, such as a horse or sheep, with diluted venom. The host animal then mounts an immune response to the venom and IgG are produced. The IgG are harvested and purified. In some cases the IgG are further treated with enzymes, which create Fab fragments. After further purification, these Fab fragments have the advantage of being much less antigenic than whole IgG preparations. Hypersensitivity reactions are a concern with the administration of any antivenom, especially equine-derived whole IgG products. These reactions can range from urticaria to life-threatening anaphylaxis. When using whole IgG antivenom, skin testing prior to administration is recommended. However, hypersensitivity reactions can still occur with a normal skin test result and standard anaphylaxis therapy (epinephrine, antihistamines, corticosteroids) should be available. Antivenom can be monovalent and directed toward only one species of snake or polyvalent and directed toward multiple snake species. Most of the antivenoms produced for use in the developing world are polyvalent. Some examples include the South African Institute for Medical Research Polyvent Snake Venom, which is directed against 10 different snake species or FAV-Afrique, which is made from venom of 11 different snake species. Additionally, antivenom frequently shows a high degree of cross reactivity. For instance, in North America the Crotalidae polyvalent immune Fab (ovine) antivenom is made from the venom of four pit viper species yet is effective in neutralizing the venom from over a dozen North American pit vipers species. The exact dose of antivenom will depend on the specific product and the clinical picture. Typically antivenom is administered until clinical signs of envenomation are controlled. It is important to administer antivenom as quickly as possible. Antivenom is effective at halting further damage from venom but does not reverse preexisting venom effects. The availability of antivenom will vary from country to country. The WHO website has an extensive searchable database of all medically important venomous snakes and their corresponding antivenom.

Latex

Natural rubber latex (NRL) is the product derived from the milky fluid produced by the tropical rubber tree, which secretes latex to seal and protect the wounded sites. NRL is widely used in the production of everyday articles, including household gloves, toys, balloons, condoms, baby pacifiers, sports equipment, elastic straps, mattresses, tires, and adhesives. Many medical devices also use NRL as material, such as gloves, catheters, drainage tubes, anesthetic masks, and tourniquets.

NRL can cause a wide spectrum of allergic reactions, ranging from urticaria, rhinoconjunctivitis, and asthma to extensive angioedema and life-threatening anaphylaxis. Sensitization and allergic reaction to NRL can occur from direct contact of NRL material with the skin, and mucosal and serosal membranes.

Latex-induced asthma usually results from inhalation of airborne NRL allergens bound to powder particles of gloves. Health-care workers wearing NRL-free gloves may be exposed to disseminated airborne NRL allergens from NRL gloves used by coworkers. Other occupations at risk of respiratory allergy to NRL are usually NRL glove users, such as hairdressers, food processors, pharmaceutical workers, and laboratory workers. In health-care workers, NRL-associated proteins Hev b 5 (acidic protein), Hev b 6 (hevein), and Hev b 7 (patatin) are the most common sensitizers identified by skin prick test. Among individuals sensitized to NRL, about a half have cross-reactions with fruits, such as banana, mango, kiwi fruit, walnut, and avocado. The cross-reactions can induce latex-fruit syndrome with symptoms ranging from itching of the throat to oral or facial swelling, rhinoconjunctivitis, and anaphylactic shock. Reduction of NRL exposure in health-care institutions by using NRL-free gloves can prevent NRL allergy in health-care workers.

3.01.4.2 Blood

Blood is the most common sample type used in laboratory diagnostics since it is easily accessible and can provide a wealth of information on the physiological and biochemical state of an individual, such as disease, mineral content, drug effectiveness, and organ function. Aspects that should be considered for blood collection are the type of sample (venipuncture, arterial puncture, and skin puncture), the site of collection and its preparation, the position and health of the patient, the tourniquet technique, the needle bore size, and the type of collection tube.

Venipuncture via the median cubital vein is the preferred location since it is easily accessible and results in the least amount of discomfort and stress for the patient. Blood taken from fragile veins can result in the leakage of hemoglobin, and other intracellular erythrocyte components into the blood sample, which can interfere with a variety of analytical reactions, cause increases in absorbance at 415 and 540 nm, and can be detected on PAGE, MALDI, and SELDI.2 Factors such as time elapsed since venipuncture before separation of plasma or serum from cells and processing temperature may also have a significant impact on the results.43,61

To prepare the collection site for puncture, alcohol must first be applied to sterilize the site. The alcohol must be allowed to evaporate before puncture to prevent residual alcohol from mixing with the sample, which can result in hemolysis and increased levels of certain analytes. The posture of the patient can alter the concentration of plasma proteins by as much as 10% due to variations in blood volumes when standing versus sitting or lying down. Improper tourniquet technique, such as duration, can lead to increased concentrations of proteins such as fibrinogen, potassium, and lactic acid.

Hemolysis can affect the specimen in a number of ways that may affect downstream analysis. Hemolysis changes the intracellular and extracellular concentrations of components such as lactic dehydrogenase, alkaline phosphatase, haptoglobin, bilirubin, potassium, and phosphorus within the specimen. This may result in changes in the concentration of the analyte if it was one of the components also present within the hemolyzed blood cells or if the cellular components dilute the sample. Colored compounds within the cell may considerably increase the low absorbance wavelength range (300–500 nm), which can affect the reported concentrations of colored analytes. The pH of the specimen may also be altered, which can interfere with chemical measurements such as enzyme activity kinetics. Hemolysis and clotting due to problems drawing blood from a patient have been reported to account for approximately 80% of the errors that occur in the sampling phase.62

Factors such as needle bore size, type of collection tube, and centrifugation speed and duration must be carefully controlled to prevent hemolysis.53,63 These factors may not be the same for all patients. For example, the bore size of the needle may depend on the disease state of the patient, since certain patient conditions may cause the blood to undergo hemolysis more easily than a normal healthy patient.52 Without due care, the blood sample may be contaminated.

One of the issues to be aware of in the process of blood collection is that, during coagulation, the cellular components, especially platelets, can secrete a variety of compounds into the plasma. Removal of cellular components must be done immediately after collection to minimize these effects. This can be accomplished by filtration through a 0.2-μm membrane filter, double centrifugation of the sample, or use of additives to minimize platelet activation such as a mixture of citrate, theophylline, adenosine, and dipyridamole.8

The storage of whole blood at cooler temperatures has its own associated problems. Low temperatures are generally required to preserve sample proteins; however, erythrocytes are less stable at lower temperatures, and storage of blood may result in hemolysis and the release of intracellular components into the sample.2

The tube used for collection and the additives within the tube will depend on the next step in the preparation of blood, which is the separation into serum or plasma. The separation of blood into plasma or serum has considerable implications for proteomic research.

Typical Clinical Epidemiologic Investigations

Typical clinical epidemiological investigations include:

Comparing patients' symptoms and signs with the results of “reference standard” diagnostic tests. High quality studies of this sort sample patients in whom it is clinically sensible to suspect a specific diagnosis (e.g., patients suspected of chronic airflow limitation), carry out specific bits of the clinical examination (e.g., the position of the thyroid cartilage relative to the suprasternal notch), and then carry out an independent, “reference standard” test (e.g., spirometry) while “blind” to the results of the clinical examination. The results of these studies, commonly expressed as likelihood ratios, improve both the accuracy and efficiency of the clinical examination. In doing so, they have confirmed the usefulness of some traditional signs and symptoms (e.g., the presence of an S3 gallop on cardiac auscultation of a patient with suspected heart failure), added new signs and symptoms (e.g., clinical prediction rules for deep vein thrombosis and the “Ottawa ankle rule” for ruling out the need for ankle radiographs) and, equally important, shown that other signs and symptoms are useless (e.g., the tourniquet test for carpal tunnel syndrome).

Relating patients' later outcomes (“prognoses”) to the results of earlier (“baseline”) clinical and laboratory findings. High quality studies of this sort sample patients at the very start of their illness (an “inception cohort”), perform baseline clinical and laboratory examinations on them, and then follow them to the conclusion of their disease. The results of these studies, often referred to now as “clinical prediction guides”, provide more accurate predictions and advice to patients at the start of an illness.

Randomized clinical trials in which consenting patients are assigned, by a system analogous to tossing a coin, to receive or not receive a new (“experimental”) treatment and then closely followed for the occurrence or prevention of unfavourable outcomes. High quality studies of this sort have been applied to medications (e.g., caffeine for premature babies), operations (e.g., carotid endarterectomy for threatened stroke), behavioral and educational interventions (e.g., behavioral maneuvers for improving compliance with medications and exercise), health professionals (e.g., the nurse practitioner), and the like. The results of these studies determine whether new treatments or other health care interventions do more good than harm. For example, the preventive and therapeutic interventions for coronary heart disease that have been validated in randomized trials are credited with halving both the incidence and case-fatality of myocardial infarction in high-income countries.

Cluster randomized trials in which groups of patients, clinicians, clinics, hospitals or communities are randomly allocated to receive or not receive an intervention to improve the application of validated health interventions or services. This approach has become the mainstay of knowledge translation research and implementation science and is used, for example, to assess the value of computerized clinical decision support, continuing education, and quality improvement interventions.

Biochemical analysis

All procedures regarding manipulation of zinc samples were performed according to international standards. Venipuncture was performed using plastic syringes without a tourniquet. All tubes and pipette tips were trace metal-free. All material used for zinc collection, separation, and storage was plastic trace metal-free, and procedures were performed according to guidelines for trace elements [16]. Blood samples, immediately after collection, were maintained in trace metal-free tubes without anticoagulants and kept 120 min in the stainless steel incubator (FANEM 502, São Paulo, Brazil) until clot formation. Next, 500 μL of serum were collected with plastic trace metal-free pipettes and transferred to plastic tubes containing 2000 μL of ultra pure water (Milli-Q plus, Millipore, USA) to dilute the serum. Samples showing hemolysis were discarded because erythrocytes are rich in zinc [17]. Urine was collected in a plastic trace metal-free recipient, the volume was measured in a cylinder and then 500 μL was collected and diluted with 2000 μL of ultra pure water. Serum and urine samples were frozen and stored at −20 °C, for up to 2 months until analysis. Serum and urine zinc samples from each individual were analyzed in duplicate within the same assay using an atomic absorption spectrophotometer (SpectrAA-200, Varian, Victoria, Australia) in accordance with the manufacturer's instructions. Assay sensitivity was 0.01 μg/mL, the intra-assay coefficient of variation was 2.6%, and the normal reference values were 0.7–1.20 μg/mL. Zinc concentration of the samples was determined using a standard solution from our laboratory as quality control, in order to check reproducibility and accuracy of the measurements. The standard zinc solution (0.5 μg/mL) was obtained by diluting the stock zinc solution (500 μg/mL) prepared from 0.5 g of zinc powder purchased from Merck (Darmstadt, Germany) and dissolved in a small volume of hydrochloric acid (HCl, Merck, Darmstadt, Germany), which was later reconstituted to 1 L, with 1% HCl (v/v). Zinc concentration of the samples was determined with a widely used standard solution from our laboratory as quality control [11]. Wavelength was 213.9, lamp current was 5 mA and all other procedures, such as calibrations and measurements were carried out in accordance with the manufacturer's instructions.

Clinical laboratory parameters were also evaluated at the beginning of the study and after 3 months using standard clinical laboratory methods: hematologic (Horiba ABX Diagnostics, Micros 60, Montpellier, France), biochemical (Dade Behring, Dimension AR, IL, USA). The analyses were performed by the Multidisciplinary Laboratory of Chronic Degenerative Diseases.

Tourniquet and skin-grafting

Pre-hospital treatment is crucial to prognosis in snake bites. Traditional methods of first aid such as sucking, burning, and tourniquet can prolong the absorption of venom (Meggs et al., 2010). It is traditionally believed that binding with a tourniquet can prevent the spread of toxins. Under normal circumstances, only substances with molecular weight less than 5 kDa can pass through the walls of blood vessels. Chinese cobra venom, with a molecular weight of 11 kDa, does not easily enter the blood circulation (Li et al., 2004). Therefore, binding with a tourniquet tends to cause the venom to accumulate in the loose connective tissue near the bite site, further aggravating local necrosis (Saul et al., 2011). Other reports have suggested that many traditional techniques, including tourniquets, incision, suction, cryotherapy, and/or electric shock are not helpful for snakebite (Tricoci et al., 2009). In the latest guidelines for venomous snakebites, both the American Academy of Clinical Toxicology (AACT) and the European Association of Poison Centers and Clinical Toxicologists (EAPCCT) do not recommend binding with a tourniquet as pre-hospital care for non-neurotoxic snakebites (American College of Medical Toxicology (ACMT), 2011).

2.3 Blood collection for in vitro platelet assays (binding, activation, phagocytosis)

For the platelet activation assay, blood was collected from normal healthy human and cynomolgus monkey donors that had not been exposed to aspirin, ibuprofen, or other anti-inflammatory analgesics in the preceding 7 days. Phlebotomy was performed using methods to reduce clotting, (i.e., using 21G needle, light tourniquet technique, slow flow into Vacutainer tubes containing anticoagulant sodium citrate, and discarding the first 2–5 ml blood collected). Eight ml of blood was collected for the assay. Blood samples were used on the day of collection.

For the binding and phagocytosis assays, human whole blood was collected via venipuncture in ACD tubes (Acid Citrate Dextrose tubes, BD Biosciences) from healthy volunteers and approved by the Amgen Blood Donation Program. Cynomolgus monkey whole blood was obtained from the University of Washington Primate Center. Blood was collected in ACD tubes. All study protocols and animal housing were approved by the Institutional Animal Care and Use Committee (IACUC).