| Which of the following is not a variable in the effectiveness of hemostasis? | View Page |
| Which of the following statements about anticoagulant therapy is false? | View Page |
| What laboratory test result is commonly used to monitor oral anticoagulant therapy? | View Page |
| An Introduction to the Fundamentals of Coagulation Blood flow is arrested by way of a complex series of interrelated physiological and biochemical processes. There are a wide variety of factors that influence the effectiveness of hemostatic processes including the following: Type of, and degree of, vessel damage Ability of vasoconstriction to occur Availability of platelets & their functionality Availability of clotting factors & their functionality Absence of inhibitors & anticoagulants | View Page |
| Secondary Hemostasis – The Extrinsic Pathway Functional control of the extrinsic pathway is mediated by Tissue Factor Pathway Inhibitor (TFPI) which binds to and inhibits factor X. Remember, for hemostatic processes to continue, factor VIIa must be able to promote the chemical conversion of factor X into factor Xa. TFPI effectively blocks this action, thereby controlling the initiation of the common pathway. The Prothrombin time (PT) is used to monitor the extrinsic pathway, and the activity of oral anticoagulants such as Coumarin. | View Page |
| Laboratory Tests of Hemostatic Function Coagulation tests provide information that is used in diagnosing coagulation disorders, evaluating hemostatic function prior to surgery, and monitoring the effectiveness of anticoagulant therapy. | View Page |
| Collecting Blood Specimens for Coagulation Testing The specimen of choice for coagulation testing is plasma. Venous blood is drawn into a 3.2% buffered sodium citrate tube (blue top tube), yielding a whole blood sample with a 9:1 blood to anticoagulant ratio. Inadequate filling of the collection tube will decrease this ratio, and may affect test results. A blue top tube used for coagulation testing should be drawn before any other tubes containing additives. This includes tubes containing other anticoagulants and/or plastic serum tubes containing clot activators. A serum tube that does not contain an additive can be collected before the blue top tube. If a winged blood collection set is used in drawing a specimen for coagulation testing, a discard tube should be drawn first. The discard tube must be used to fill the blood collection tubing dead space to assure that the proper anticoagulant/blood ratio is maintained, but the discard tube does not need to be completely filled. The discard tube should be a nonadditive or a coagulation tube. If a blood specimen used for coagulation testing must be collected from an indwelling line that may contain heparin, the line should be flushed with 5 mL of saline, and the first 5 mL of blood or 6-times the line volume (dead space volume of the catheter) be drawn off and discarded before the coagulation tube is filled. | View Page |
| Laboratory Tests of Hemostatic Function – Prothrombin Time The prothrombin time is a screening test that helps to assess the functionality of both the extrinsic and common pathways. The effectiveness and presence of factors I, II, V, VII, and X are assayed in this diagnostic test, as they are all found in the aforementioned pathways. The results of the prothrombin time are used in conjunction with other diagnostic tests, as well as the clinical picture of the patient, to determine any hemostatic abnormalities which may be present. In addition to being an integral part of the coagulation disorder assessment process, the PT is also used to determine therapeutic effectiveness of oral anticoagulants, by monitoring drugs such as Warfarin, Coumarin, and Dicoumarol. Prothrombin time test results are reported as the number of seconds needed for a clot to form in the patient specimen using the laboratory's instrument/reagent system, and as the International Normalized Ratio (INR). | View Page |
| Laboratory Tests of Hemostatic Function – Prothrombin Time The INR component of the laboratory result is a calculated value that is used by the clinician to monitor anticoagulant therapy and adjust dosage as dictated by clinical status. An INR of 2.0 - 3.0 is often desired as the therapeutic range. The following formula is used by the clinical laboratory to derive an INR value. The INR must be adjusted for every new lot of PT reagent. INR= (PT of patient/PT of geometric mean of the normal population)ISI The International Sensitivity Index, or ISI value, is provided by the reagent manufacturer as the relative sensitivity of the reagent itself. The INR is used to standardize PT results, and in turn, anticoagulant therapy, across laboratory instrumentation, methodologies, and locale. Be sure to frequently check that ISI values match those of the lot currently in use as erroneous results may otherwise occur . | View Page |
| Which of the following statements is incorrect? | View Page |
| Anticoagulation Therapy Anticoagulant therapy is employed in a number of clinical situations Some of these clinical situations include: After an episode of thrombosis, such as deep venous thrombosis (DVT) in the veins of the legs, to prevent reoccurrence. Prophylactically after some surgeries, especially those involving vascular repair such as coronary bypass surgery to prevent clots from blocking newly formed vasculature. In heart valve and chamber disorders where there is an increased risk of thrombosis occurring. | View Page |
| Anticoagulation Therapy - Heparin Therapy The use of heparin is prophylactic. It is used either to prevent thromboembolism (a condition in which a blood clot forms inside a vessel), or used to limit a previous thromboembolism. Heparin inhibits thrombin. The degree of inhibition is dosage dependant. Low doses of heparin inhibit initial thrombin formation in the coagulation cascade, and act to slow down overall thrombin generation. At higher doses, heparin can inhibit thrombin entirely, making blood coagulation impossible. Heparin is a potent anticoagulant. Accurate monitoring is essential. The activated partial thromboplastin time (APTT) and/or activated clotting time is used to monitor unfractionated heparin therapy. | View Page |
| Anticoagulation Therapy - Oral Anticoagulant Therapy The therapeutic use of oral anticoagulants is typically the long-term solution for the patient in terms of managing situations of thrombosis. Warfarin, a dicumarol derivative, is one of the most popular oral anticoagulants used today. While heparin is administered intravenously and acts to inhibit thrombin, warfarin is given orally, taken in pill form, and functions as a Vitamin K antagonist. In earlier discussions, it was mentioned that certain clotting factors are considered to be vitamin K dependant. They require vitamin K molecules for their action to occur. Vitamin K dependant factors include factor II, VII, IX, and X. Vitamin K dependant metabolic processes involved with these coagulation factors are inhibited by drugs such as warfarin. The chemical structure of warfarin and similar anticoagulants enables them to bind competitively with free vitamin K. The prothrombin time (PT/INR) is used to monitor oral anticoagulant therapy. | View Page |
| Pre-analytical Variables Pre-analytical variables are those that affect the specimen before the actual testing begins. Some of the pre-analytical variables to consider with molecular testing include those that are applicable to all clinical specimens but should be emphasized when discussing molecular methodologies; some of these include but are not limited to: Receipt of valid order Proper patient identification Proper venipuncture procedure for blood collection Use of correct anticoagulant Collection of correct specimen type (i.e.- plasma, serum, whole blood) Order of draw Proper storage Proper transport Procedures if there is a delay in testing and/or transport | View Page |
| Specimen Collection and Handling Some global specimen collection and handling issues to consider include: Specimens that contain nucleated cells will be of interest in DNA methodologies while specimens lacking nucleated cells are more useful in RNA methodologies. rRNA is more stable than mRNA, which is labile and sensitive to contamination. DNA is relatively stable and can be obtained from nonviable sources. Serum or plasma obtained by standard routine venipuncture procedures can be used as long as proper site selection and decontamination occur. Standard anticoagulants such as Ethylenediaminetetraacetic Acid (EDTA) and Acid Citrate Dextrose (ACD) can be used; however avoid the use of heparin as an anticoagulant as it interferes with some polymerase chain reaction (PCR) methodologies. When using fluorescence, fasting serum or whole blood specimens should be used to decrease the interference by lipids. | View Page |
| Which of the following are examples of pre-analytical variables that affect molecular methodologies? | View Page |
| When collecting blood samples, one anticoagulant to avoid, especially when performing PCR is: | View Page |
| Overview All of these peripheral blood cells have different characteristics. In order to accurately identify each of them, a peripheral blood film must be made, preferably from capillary blood or blood anticoagulated with EDTA (Ethylenediaminotetracetic Acid). EDTA, in contrast to many other anticoagulants, preserves cellular morphology. The individual characteristics of each cell type are made visible by staining the blood films with the Wright stain, and observing them under the microscope. | View Page |
| Glossary of Terms A through M. Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus. | View Page |
| Warfarin Metabolism The first specific PGx testing application most labs are likely to encounter is that used in patients taking warfarin. Recent studies have revealed that the variations seen in patients taking the anticoagulant warfarin are due to PGx factors. The consequences of incorrect warfarin dosing are obviously serious, with inadequate doses predisposing patients to thrombosis and higher doses placing them at risk for hemorrhage. The United States' Food and Drug Administration (FDA) recently approved updated labeling for Coumadin (warfarin sold by Bristol-Myers Squibb). The new labeling suggests that physicians incorporate PGx information into warfarin-dosing regimens for patients. Manufacturers of generic warfarin products are now adding similar labeling. | View Page |
| Warfarin cont. The genes involved in warfarin metabolism are CYP2C9 and vitamin K epoxide reductase complex subunit 1 (VKOR). Warfarin owes its anticoagulant action to its inhibition of VKOR. This enzyme recycles vitamin K, a critical element for the clotting factors II, VII, IX, and X, as well as for proteins C, S, and Z. There are six CYP2C9 alleles that are known to cause prolonged metabolism of warfarin: CYP2C9 *2, *3, *4, *5, *6, and *11. (Polymorphisms in CYP450 genes are denoted with asterisks.)One-third of the patients that receive warfarin metabolize it differently than expected and experience a higher risk of bleeding.Genetic testing for the two most common polymorphisms (CYP2C9*2 and *3) as well as for VKOR may be able to reduce the variability associated with warfarin dosing response. Labs performing PGx testing can provide general warfarin dosing recommendations based on the patient's genotype analysis. The lab report will indicate whether a patient has a normal, mild, moderate, high, or very high sensitivity to warfarin. For example, a patient who has one CYP2C9 normal wild-type allele (CYP2C9 *1), one polymorphism (CYP2C9*3), and also a VKOR polymorphism is predicted to have a moderate sensitivity to warfarin. This patient should have frequent INR monitoring and possible warfarin dose reduction. It is important to recognize that knowing a genotype does not necessarily guarantee accurate dose prediction; other drugs and/or environmental or disease factors can also alter CYP2C9 activity. Therefore, monitoring the INR is still very important. | View Page |
| What could have caused the clotting? | View Page |
| Discussion Clotting of blood specimens may be caused by several factors. Clotting usually occurs due to improper phlebotomy technique,and clotted specimens will generally be rejected for those tests that require the blood to be mixed with an anticoagulant. When a clot forms in a tube containing anticoagulant, it usually indicates that the blood and anticoagulant aren’t in proper balance. That is why it is crucial to invert tubes with anticoagulant almost immediately after collection to ensure proper mixing of blood and anticoagulant. Relevant topics: Lavender top tubes, Light blue top tubes, Unsatisfactory specimens: Clots, Causes of clotting | View Page |
| Discussion Tubes are drawn in a specific order to avoid the possibility of erroneous test results caused by carryover of an additive from one tube to the next. If a blood culture is ordered, it should be drawn as the first tube. Additional tubes should follow this order of draw. Sodium citrate - coagulation tube (light-blue top) Serum tube - with or without clot activator or gel. This tube is either a red top tube or a gold top tube depending on manufacturer and tube additive. Sodium or lithium heparin with or without gel plasma separator (green top) Potassium EDTA (lavender or pink top) Sodium fluoride, and sodium or potassium oxalate (gray top) | View Page |
| Lavender top tubes Contain anticoagulant Ethylendiaminetetraactic acid (EDTA) to prevent clotting.
Are used mostly for hematology studies.
Must be completely filled to assure a correct anticoagulant to blood ratio.
Must be inverted after filling to assure proper mixture of anticoagulant with blood.
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| Blood collection tubes: types Rubber stoppers of blood collection tubes are color coded.
Each type of stopper indicates a different chemical additive (usually an anticoagulant to prevent clotting), or a different tube type.
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| Blood collection tubes: introduction A blood collection tube generally consists of a glass or plastic tube with a rubber stopper. It has a vacuum so that blood will flow into the tube.
Blood collection tubes may contain anticoagulants and/or other chemical additives.
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| Blood collection tubes: inversion All tubes (except red top tubes which contain no additives) must be gently inverted 5 to 8 times immediately after filling, to ensure proper mixing of blood and anticoagulant, or other additives.
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| Light blue top tubes These tubes contain the anticoagulant sodium citrate.
They are used mostly for coagulation (clotting) studies.
They must be completely filled to assure proper ratio of anticoagulant to blood.They must be inverted immediately after filling to prevent clotting.
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| Gray top tubes Contain an inhibitor of glycolysis, such as sodium fluoride.May also contain an anticoagulant such as potassium oxalate.
Used for accurate determination of glucose levels.
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| Royal blue top tubes Contain either sodium heparin or sodium EDTA anticoagulants, or no anticoagulant.
Are used for trace element, toxicology, and nutritional studies. | View Page |
| Syringe - Transferring blood to collection tubes contd It is important to transfer the blood to appropriate tubes immediately because a syringe contains no anticoagulant, and the transfer must be complete before blood starts to clot.Do not push the plunger while transferring blood into a collection tube.
This may cause hemolysis, ruining the specimen. | View Page |
| Insufficient volume Insufficient blood volume (short draws) within a collection tube containing anticoagulant will result in an incorrect ratio of blood to anticoagulant, and yield incorrect test results.Short draws can be caused by: A vein collapsing during phlebotomy.The needle coming out of the vein before the collection tube is full.Loss of collection tube vacuum before the tube is full. (Always keep extra tubes on hand.) | View Page |
| Clots Blood clots when the coagulation factor proteins within the plasma are activated.Blood starts to clot almost immediately after it is drawn unless it is exposed to an anticoagulant.Clots within the blood specimen, even if not visible to the naked eye, will yield inaccurate results. | View Page |
| Causes of clotting Clotting can be caused by: Inadequate mixing of blood and anticoagulant within the collection tube.Delay in expelling blood within a syringe (which contains no anticoagulant), into a collection tube with anticoagulant. | View Page |
| Partial collection tubes Filling a light blue-topped tube to its recommended volume is especially critical; if it is filled incompletely, coagulation results will be incorrectly reported as abnormal.If a short draw is anticipated, a “partial collection” tube which contains less anticoagulant and requires less blood may be used.The light blue topped collection tube shown on the left requires reduced blood volume, and is filled only to the line.
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| Blood clots When a blood sample is left standing without anticoagulant, it forms a coagulum or blood clot.
The clot contains coagulation proteins, platelets, and entrapped red and white blood cells.
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| Pre-analytical Errors Preanalytical Error What is it? How does it happen? What is the result? Hemolysis Red blood cells (RBCs) break and release contents of cell into plasma. Needle incorrectly positioned in vein; cells forced to squeeze through opening. Needle gauge too small; slow blood return into tube. Vigorous mixing or shaking of tube. Alcohol on skin that has not had sufficient time to dry. Some test results may be falsely elevated. (Potassium is especially affected by hemolysis.) Patient may have to be re-drawn. Clotted specimen Clumped or clotted cells in specimen that requires anticoagulated or whole blood Insufficient mixing of blood with anticoagulant in tube. Delay in mixing tube. Slow filling tube. Inaccurate test results for cell counts and clotting studies. Patient may have to be re-drawn. Tube filled to incorrect volume Too little or too much blood in tube. Tube removed from needle too quickly. Vacuum in tube has been compromised due to use of tube past the expiration date (Results in a short fill). Manual fill of tube may lead to over-fill. Test results may be unreliable due to dilution errors. Patient may have to be re-drawn. | View Page |
| What is a Hidden Error? Hidden errors are those that cannot be detected or corrected by the laboratory analyst prior to testing. Most often these errors can be prevented by the phlebotomist following correct venipuncture procedure for every procedure, every time.Hidden errors include hemoconcentration, incorrect order of draw, and (the most serious of all errors) misidentification of patient or specimens. Because these errors often are unknown, the analyst may inadvertently report erroneous patient results which could be harmful to the safety and well-being of the patient. Condition What is it? How does it happen? What is the Result? Hemoconcentration Blood pools at site of venipuncture Tourniquet is applied for a prolonged period of time Test results may be inaccurate because blood components move between blood and tissues Pouring Blood between tubes Mixing contents of two or more tubes Removing top of tube to combine contents of one tube with another Inaccurate test results due to over or under dilution or incorrect anticoagulant Clots form due to lack of mixing Patient may have to be redrawn Incorrect patient identification and incorrect specimen labeling Using the wrong name to label a specimen Failure to positively identify EVERY patient using 2 unique identifiers BEFORE beginning venipuncture Failure to label EVERY specimen in the presence of the patient Failure to concentrate fully on the task Results reported to caregiver for wrong patient Compromises patient care; may be life-threatening | View Page |
| Correct Fill Fill blood collection tubes completely (until vacuum is exhausted) to ensure the correct blood to anticoagulant ratio necessary for accurate patient results. Specimens may be rejected by the laboratory if the tube is short-filled or over-filled. To avoid short-filling of tubes, the phlebotomist must ensure that the blood flow stops completely before removing the tube from the needle. When using a winged device (butterfly) to collect blood for coagulation studies (e.g., protime, aPTT), the phlebotomist must draw a light blue top "waste" tube before attaching another light blue top tube for testing. If the air in the tubing of the winged device is not displaced into a waste tube and is drawn into the tube used for testing, the tube used for testing will short-fill. The laboratory may reject the specimen because of invalid blood to anticoagulant ratio. | View Page |
| Do Not Tamper With the Specimens A phlebotomist should not uncap a blood tube and pour blood between tubes or combine two partially filled tubes of blood into one. This may lead to over-fill of tubes and more importantly, invalid patient results. Combining two tubes with the same additive into one tube will alter the blood to anticoagulant ratio by doubling the amount of anticoagulant in the tube. When blood is being transferred from a syringe to a tube, the phlebotomist must not apply pressure to the plunger to force blood into the tube. This may cause over-filling of the tube and hemolysis of blood cells. With the aid of a transfer device, the tube will draw the amount of blood required to fill the tube based on the amount of vacuum in the tube. | View Page |
| Importance of Using the Correct Blood Collection Tube Specific anticoagulants must be used for each test that requires plasma or whole blood. If the blood is drawn into a tube with the wrong additive, patient results may be adversely affected. For example, the test for lithium usually requires a serum sample. If instead of a serum tube, the phlebotomist used a tube that contained lithium heparin, the lithium result for the patient would be falsely elevated. It is imperative that the phlebotomist use the tube with the correct additive to avoid erroneous patient results. | View Page |
| Pre-analytic and hidden errors can greatly affect a laboratory result.Match the error listed below with the cause from the drop-down box. | View Page |
| Blood Tube Labeling Information Each tube used for blood collection is labeled by the manufacturer with important information. This information includes: tube volume in milliliters (mL), expiration date, lot number and, if applicable, the type of additive that is in the tube. Tube volume: Each tube contains a vacuum that allows a specific amount of blood to enter the tube. In a tube that contains an anticoagulant, the amount of blood that is drawn into the tube will establish the correct blood to anticoagulant ratio. Tubes not filled to the correct volume (over-filled or under-filled) may cause inaccurate test results. Expiration Date: An expiration date is stamped on all blood collection tubes. The tube manufacturer determines this date based on its studies of vacuum maintenance and anticoagulant effectiveness. The expiration date should be checked routinely; tubes that are past the expiration date should be discarded.If a blood collection tube is used past its expiration date, the vacuum may not draw the amount of blood needed to fill the tube completely. Short-filled tubes may not be acceptable for testing and the specimen would have to be recollected. If the tube contains an anticoagulant, it may not work effectively (may not prevent the blood from clotting). Lot Number: A lot number listed on the tube identifies a specific group of tubes that were manufactured at the same time. This information is important to know if a problem is identified with several collection tubes. If the defective tubes are all part of the same lot number, the manufacturer should be notified for replacement of the tubes. Additive: Most blood collection tubes contain a type of additive or chemical that, when mixed with the blood, will yield a specimen acceptable for testing. The various types of additives that are contained in blood collection tubes are discussed on the following page. | View Page |
| Blood Collection Tubes Most blood collection tubes contain an additive that either accelerates clotting of the blood (clot activator) or prevents the blood from clotting (anticoagulant). A tube that contains a clot activator will produce a serum sample when the blood is separated by centrifugation and a tube that contains an anticoagulant will produce a plasma sample after centrifugation. Some tests require the use of serum, some require plasma, and other tests require anticoagulated whole blood. The table below lists the most commonly used blood collection tubes. Tube cap color Additive Function of Additive Common laboratory tests Light-blue 3.2% Sodium citrate Prevents blood from clotting by binding calcium Coagulation Red or gold (mottled or "tiger" top used with some tubes is not shown) Serum tube with or without clot activator or gel Clot activator promotes blood clotting with glass or silica particles. Gel separates serum from cells. Chemistry, serology, immunology Green Sodium or lithium heparin with or without gel Prevents clotting by inhibiting thrombin and thromboplastin Stat and routine chemistry Lavender or pink Potassium EDTA Prevents clotting by binding calcium Hematology and blood bank Gray Sodium fluoride, and sodium or potassium oxalate Fluoride inhibits glycolysis, and oxalate prevents clotting by precipitating calcium. Glucose (especially when testing will be delayed), blood alcohol, lactic acid | View Page |
| A blood collection tube that has a light-blue top contains which of these anticoagulants? | View Page |