|Preanalytic Variable Leading to False Thrombocytopenia|
The platelet count is only as good as the sample collected. Blood samples that are collected in EDTA or sodium citrate tubes for coagulation purposes should be inverted 5 - 10 times for proper mixing of the anticoagulant and the blood. If the tube is not mixed, small fibrin clots may form, causing a falsely decreased platelet count.
|Pseudo-thrombocytopenia: Platelet Satellitism and Platelet Clumping|
Platelet satellitism and platelet clumping can cause pseudo-thrombocytopenia. Platelet satellitism was first reported in the early 1960's. It is a rare condition that occurs when an IgG antibody forms in the presence of EDTA, the anticoagulant that is used for the collection of hematology blood specimens. The IgG antibody is directed against the glycoprotein IIb/IIIa complex on the platelet membrane. As the antibody coats the platelets, the platelets rosette around segmented neutrophils, bands, and sometimes around monocytes. Antibody-coated platelets that are huddled around white blood cells (WBCs) will not be counted as platelets by automated equipment and the platelet count will be falsely decreased. If a peripheral blood smear is reviewed, platelets will be observed attached to WBCs. The image on the right illustrates platelet satellitism with platelets adhering to a neutrophil. Platelet clumping can also occur in the presence of EDTA and the platelet count again will be falsely decreased. The count will probably be flagged by the analyzer for platelet clumps or giant platelets. If either platelet satellitism or platelet clumping is observed on the peripheral smear, the sample could be recollected using sodium citrate as the anticoagulant. Platelets can then be counted using the automated method. The platelet count from a tube that contains liquid sodium citrate will need to be corrected for the dilutional effect of the citrate. This can be accomplished by multiplying the platelet count that is obtained from the automated analyzer by 1.1.
|Which of these conditions is associated with a pseudo-thrombocytopenia?||View Page|
|Additional Complications Related to Heparin-Induced Thrombocytopenia|
Further complications may occur in some patients who experience HIT if a thrombotic component develops. This is a serious condition that may result in pulmonary emboli, venous thrombosis, and gangrene. For patients with HIT, the immediate treatment is to cease heparin therapy and provide an alternative anticoagulant agent if one is still necessary. In less severe cases of HIT, the platelet count will begin to rise in a few days once heparin is withdrawn.
|Properly Filling and Mixing a Microcollection Container|
It is important that the container is filled to the appropriate level when blood is collected into a microcollection container that has an anticoagulant. The device should then be capped and the blood mixed well immediately following collection of the specimen. The manufacturer of the containers that are used specifies what is considered adequate mixing and the laboratory's collection procedure should be based on these recommendations. Mixing involves a gentle tapping on a hard surface to move the blood further down into the tube during collection and then capping the tube upon completion of the collection so that the tube can be mixed end-over-end for the specified number of times as shown in the image on the right. The correct fill is also important. A container that is overfilled will not be properly anticoagulated and clots may form that will affect the laboratory test results. A container that is underfilled may not contain sufficient specimen to perform the test(s) or the excess anticoagulant may interfere with the test. For example, excess anticoagulant could cause morphologic changes in blood cells.
|Order of Draw|
The order of draw for a capillary blood collection is slightly different than the order of draw for a venous blood collection.If capillary blood gases are ordered, they are drawn first to avoid introduction of room air as much as possible. A specimen for blood count is collected before tubes containing other anticoagulants and additives. This is to ensure that the blood will not begin to clot before this specimen is collected; clots will affect the accuracy of the blood count. The following order of draw is commonly used: ContainerAdditiveUseLavender topEDTAFor hematology blood countsGreen top Lithium heparinTests that require a heparinized plasma sample__Other tubes containing anticoagulantsVariedRed or gold topClot activatorTests that require a serum sampleRed top No additiveTests that require a serum sample but clot activator and/or gel may affect test
|A lavender top microcollection container that has EDTA as an anticoagulant is used to collect a capillary hematology specimen for a complete blood count (CBC). If additional specimens are also collected with the same dermal puncture into a green top and a red top container, at what point should the lavender top for the CBC specimen be collected?||View Page|
In addition to the puncture device, additional equipment is required to perform a safe and successful dermal puncture and to collect an acceptable specimen. This may include any of the items discussed below.Plastic microcollection devices: Plastic microcollection devices are small plastic tubes designed to collect capillary blood from a dermal puncture wound. Each small collection tube is color-coded in the same manner as blood collection tubes used for venipuncture. The color of the cap of each container tube corresponds to the type of additive inside the tube, most often an anticoagulant. The additive coats the inside of the tube. Examples of microcollection devices are shown below. Heel warmer: It is best practice to warm the heel of an infant with a warming device known as a heel warmer. The heel warmer, when activated, is designed to warm its contents to a standardized temperature. This temperature will be hot enough to effectively warm the heel and facilitate blood flow to the area without causing heat injury to the patient. It is unacceptable to warm a cloth using a microwave. There may be "hot spots" on the cloth that could potentially burn the patient. Keep in mind, what may feel warm to you, the phlebotomist, may feel hot to your patient!Plastic or Mylar-wrapped capillary tube: In some facilities blood from a capillary puncture is collected directly into a capillary tube. These tubes are very delicate and must be used with great caution. As soon as the tube is two thirds to three-fourths filled, one end is sealed to prevent blood from leaking out.Glass microscope slides: In some facilities, the person collecting the capillary specimen may also be required to prepare a blood smear for laboratory examination. A drop of blood is placed directly on a glass slide and spread to create an area for cell examination. If you are required to prepare blood smears, remember that the slide is considered infectious until fixed or stained. It is also important to remember that glass is a sharps hazard. If not used correctly, the glass may cause injury to both the patient and the phlebotomist. Be as cautious with a glass slide containing blood as you are with a contaminated needle. Dispose of glass slides that will not be used for testing in approved sharps containers.Alcohol and gauze pads: Alcohol is the disinfectant of choice for dermal puncture. The alcohol must be allowed to air dry, which will prevent hemolysis of the specimen and discomfort for the patient. A piece of clean or sterile gauze is used to wipe away the first drop of blood. Gauze is also used to apply pressure to the wound after the specimen collection is complete to stop the wound from bleeding.Iodine or other approved cleaning agents may be used as an alternative to alcohol.Bandage: It may be necessary to apply a bandage to the puncture wound on a finger or heel if the site continues to bleed. However, it is NOT recommended to bandage the finger of a child who is 2-years-old or younger since the bandage may become a choking hazard if the child puts that finger in his/her mouth.Personal protective equipment (PPE): All health care professionals that may come in contact with blood and/or body fluids while performing a laboratory procedure are required to wear intact gloves. It is against safety guidelines to alter gloves in any way that may compromise the integrity of the gloves. Eye protection, such as safety goggles, is recommended if there is the possibility of a splash of blood while collecting a capillary blood specimen. In many facilities, special gowns are required in some patient areas such as special-care nurseries. Always follow the policies of your facility in regard to PPE.
|Capillary Blood Gases|
In some instances, the health care provider may request an analysis of the capillary blood for blood gases. This is most often requested on infants. Collection of this specimen requires a skilled phlebotomist and specialized equipment. The patient must be positively identified. All appropriate PPE must be used. The procedure for site selection, preparation and puncture is identical to other infant dermal punctures, however, capillary blood gases are always drawn first if other capillary blood specimens will be collected.Blood specimens for capillary gases are always collected in long, large-bore heparinized glass tubes. Blood should be drawn into the tube using capillary action. The phlebotomist should start filling the tube using a large well-formed drop of blood, drawing continuously as the blood flows. Each tube must be filled completely end to end as shown in the image on the right. Every effort must be made to avoid drawing air bubbles or air gaps into the tubes as these could adversely affect the results of the test. Before sealing both ends of the tube, the phlebotomist will insert a tiny metal "flea" into the blood-filled tube and slide a magnet lengthwise back and forth on the outside of the tube. The magnet will cause the flea to move back and forth inside the tube mixing the specimen with the anticoagulant coated on the inside of the tube. This technique should also prevent the blood from clotting, which could result in specimen rejection by the laboratory.The properly filled glass tubes must be delivered to the analyzing laboratory in a timely manner. Delay in specimen delivery may adversely affect the quality of the patient results.
|A patient has an order for microhematocrit testing. Which of the following should be collected for this testing?||View Page|
|Elevations in PT or aPTT assays|
PT and/or aPTT may be prolonged for a number of reasons. Prolonged PT causes include: Warfarin therapy Liver disease Disseminated intravascular coagulation (DIC) Vitamin K deficiency Liver conditions such as cirrhosis or hepatitis Inadequate level of Factors I, II, V, VII, and/or XProlonged aPTT causes include: Presence of heparin Liver disease, other liver conditions Vitamin K deficiency Hemophilias DIC von Willebrand disease Lupus anticoagulant Inadequate levels of Factors I, II, V, VIII, IX, X, XI, and/or XII
|Preanalytical Variables That Can Cause Falsely Elevated PT or aPTT Results|
Improper collection of the blood specimen that is used for testing can cause false prolongation of PT or aPTT results. The following table covers several preanalytical variables that may affect PT or aPTT test results Preanalytical Variable Cause of False Elevation of PT and or aPTT Test Result Corrective Action Blood collection tube is inadequately filled. Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Recollect specimen ensuring proper fill to achieve a blood to anticoagulant ratio of 9:1. Patient has a hematocrit level above 55% Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Prepare a specimen collection tube that contains less anticoagulant. Refer to your laboratory's procedure for the proper amount of anticoagulant. Specimen is clotted. Coagulation factors have been activated; insufficient levels left in the plasma. PT and aPTT results will be affected. Recollect the specimen. Specimen collected from an arm with a heparin lock or from a heparinized vascular access device (VAD). Heparin contamination will prolong the aPTT. Collect the blood from a vein rather than a VAD. If blood must be drawn from the VAD, flush it first with 5 mL of saline, and discard the first 5 mL of blood before collecting the specimen. Patient is receiving heparin therapy. Heparin will prolong the aPTT If the patient is being evaluated for possible factor deficiencies or coagulation inhibitors, use a heparin digesting enzyme as a pretreatment before testing the PT or aPTT. .
|Which of the following may produce a falsely prolonged aPTT test result?||View Page|
|When To Perform a Mixing Study|
A mixing study should be considered when a patient has a prolonged PT and/or aPTT along with: no history of heparin or warfarin therapy no history of liver disease It is also essential to first verify that a correctly collected sample (free from clots) with a proper blood to anticoagulant ratio has been obtained.
|Mixing studies may help to determine the presence of which of the following?||View Page|
|Interpreting the Mixing Study Results|
Interpretation patterns for mixing study results include the following scenarios: If the results of the mixing study show correction for both the immediate and incubated PT/aPTT tests, the patient most likely has a factor deficiency (or multiple factor deficiencies). If the results of the mixing study show no correction in either the immediate or incubated PT/aPTT, the patient may have a coagulation inhibitor, most likely a lupus anticoagulant. If the results of the mixing study show correction for the immediate PT/aPTT results, but no correction in the incubated PT/aPTT, the patient may have a slow-acting inhibitor such as anti-factor VIII.Note: if the control tubes also show prolongation after incubation, there may have been a problem with the heat-labile factors in the sample losing their activity. If this is the case, the mixing study should be repeated.
No single screening test can detect all lupus anticoagulant-positive (LA-positive) patients. Several tests are available and at least two should be employed to verify the presence of LA. Before any LA screening test is done, a thrombin time (TT) should be performed to rule out therapeutic heparin or the presence of a thrombin (factor-II) inhibitor.These are some of the LA screening procedures that can then be used: Dilute Russell's Viper Venom time (DRVVT). This test utilizes a reagent containing venom from the viper Vipera russelli (which activate factor V and X), low levels of phospholipids, and calcium ions in a clotting time test. The DRVVT test principle is based on the idea that the reagents can help to identify the antibody's dependence on phospholipids . Platelet neutralization procedure. This assay will show the dependence on phospholipids for the lupus anticoagulant to take effect. This can be performed using the aPTT based technique, with the DRVVT test, or using Taipan snake venom time tests. Kaolin clotting time or silica clotting time Hexagonal Phospholipid test (HPP). This is a similar assay to the platelet neutralization procedure, but thought to be more sensitive.
|Further Analyses for Coagulation Inhibitors (cont.)|
When considering the presence of a coagulation factor inhibitor other than the common lupus anticoagulant, specific inhibitors such as anti-Factor VIII should be considered. Titers can be performed for the antibody in question to quantify the inhibitor and determine the extent of the antibody proliferation. These tests are called Bethesda titer assays. These tests use a universal inhibition unit in measuring specific-factor antibody activity. The appropriate dosing of medications may be measured based on these titer results.
|Further Analyses for Coagulation Inhibitors- Lupus Anticoagulant|
For the diagnosis of lupus anticoagulant, the International Society on Thrombosis and Hemostasis has set a protocol of diagnostic criteria that should be met. This includes the following requirements: The patient sample must show abnormal phospholipid-dependent reactions in the coagulation lab. The patient sample must show inhibition of clotting after the mixing study test has been performed. The patient sample must be proven to have an inhibitor and not a factor deficiency. The patient must have a definitive phospholipid-dependent antibody and not a specific factor inhibitor.
|Treatment for Coagulation Inhibitors|
Currently, there are several treatment options for patients suffering from coagulation inhibitors. Treat the patient by administering recombinant factor replacements. For example, using a recombinant factor VIII or factor IX for the treatment of acquired hemophilia due to coagulation inhibitors. Treat the patient with immunosuppresants, such as prednisone, to prevent large amounts of coagulation antibodies from forming. Then factor replacement can be given to the patient. If lupus anticoagulant is suspected, anticoagulants may be ordered to prevent thrombotic episodes. Often for treatment purposes, a patient is given a very high level of the coagulation factor that the antibody is targeting. The goal is to overwhelm the antibody with excess factor so that the antibody is neutralized and the residual factor can participate in the normal coagulation process.
|Introduction: Coagulation Inhibitors|
As the name implies, coagulation inhibitors (also called circulating anticoagulants) interfere with normal blood coagulation. Coagulation inhibitors may be congenital or acquired (developing in patients during the course of a disease) and are almost always immunoglobulins, either IgG or IgM. There are two types of inhibitors: those directed toward a coagulation factor (or multiple factors) and the lupus anticoagulant. Lupus anticoagulant is one of the more commonly encountered coagulation inhibitors. It is also known as antiphospholipid antibody because it is directed toward phospholipids. Lupus anticoagulant is usually an IgG antibody. It differs from factor-specific inhibitors in that lupus anticoagulant causes thrombosis and abnormal clotting while factor-specific inhibitors cause serious bleeding.
|Coagulation Inhibitors and Coagulation Screening Tests|
Patients with factor-specific coagulation inhibitors will have prolonged prothrombin time (PT) and/or aPTT test results (depending on the coagulation factor that is targeted by the inhibitor). Clinically, this is associated with abnormal clotting and bleeding complications. A prolonged aPTT, and sometimes PT, is seen with lupus anticoagulant. The antibody combines with the phospholipids on the surfaces of test reagents that are used in the aPTT test, and sometimes in the PT test, prolonging the test result(s). Clinically, lupus anticoagulant is associated with thrombosis and not with bleeding symptoms.Click here to read an important note regarding lupus anticoagulant
1. Aniara Learning Center. Coagulation Corner. Mixing Studies: To correct or not correct-that is the question. June 2009. http://www.aniara.com/learning-center/Coagulation-Corner/articles/2009/01/mixing-studiesto-correct-or-not-correct.aspx.2. Bethel, M and Adcock, D: Laboratory evaluation of a prolonged APTT and PT. Lab Med, 285, May 2004.3. Devreese KM. Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants. Thromb Res 2007;119:369-76.4. Harmening, D. Clinical Hematology and Fundamentals of Hemostasis. 5th edition. F.A. Davis, 2009.5. Katrien M.J. Devreese, Interpretation of normal plasma mixing studiesin the laboratory diagnosis of lupus anticoagulants, ThrombosisResearch, Volume 119, Issue 3, 2007, Pages 369-376, ISSN 0049-3848,DOI: 10.1016/j.thromres.2006.03.012.(http://www.sciencedirect.com/science/article/B6T1C-4JYKP68-1/2/12550b597f6b88b11e09b26e74963d4f)Keywords: Lupus anticoagulants; Mixing tests; Percent correction formula; Rosner index6. McKenzie, S. Clinical Laboratory Hematology. 2nd edition. Pearson, 2010.7. National Committee for Clinical Laboratory Standards. Determination of Factor Coagulant Activities, H48A. NCCLS, 1997.8. Santora SA, Eby CS, Chapter 106: Laboratory evaluation of hemostatic disorders. Pages 1841-1844. In: Hoffman R, Benz, EJ, Jr et. al Hematology. Basic Prinicples and Practice. 3rd edition. Churchill Livingstone. 2000.9. Vancott, E and Laposata, M: Coagulation, Fibrinolysis and Hypercoagulation. 2001.
|Interpreting the Mixing Study Results: A Case Study|
Case StudyA young patient is admitted from the emergency room with petechial bruising. The attending physician orders a battery of tests including a PT and aPTT. The laboratory performs the requested testing and the result of the aPTT is normal; however the PT is prolonged:PT: 38 seconds (normal range 11-13 seconds)aPTT: 32 seconds (normal range 21-34 seconds)The physician then decides to order a mixing study for further analysis since the patient is not taking any oral anticoagulants, nor does she meet the known patient histories associated with prolonged PT results.
|Interpreting the Mixing Study Results: Case Study 3|
Case Study 3A third patient has had no prior bleeding complications to date. He is in good health, but a recently performed aPTT test is prolonged. He is not receiving heparin or other anticoagulant therapy and the patient's physician orders a mixing study. The results of the mixing study are as follows: Initial aPTT Immediate aPTT mixing study Incubated aPTT mixing study 63 sec. (normal range 21-34 seconds) 26 sec. 65 sec.
|Which of the following is not a variable in the effectiveness of hemostasis?||View Page|
|Which of the following tests is used to monitor therapy with unfractionated heparin?||View Page|
|What laboratory test result is commonly used to monitor oral anticoagulant therapy?||View Page|
|Introduction to the Fundamentals of Coagulation, continued|
When a vessel wall is damaged, blood flow out of the vessel 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 damageAbility of vasoconstriction to occurAvailability of platelets & their functionalityAvailability of clotting factors & their functionalityAbsence of inhibitors and anticoagulantsThe image on the right illustrates vessel size as related to time required for clotting to occur, amount of coagulation products used (platelets and clotting factors), and size of the corresponding bleed.
|Secondary Hemostasis: The Extrinsic Pathway, continued|
Functional control of the extrinsic pathway is mediated by tissue factor pathway inhibitor (TFPI), which binds to and inhibits factor X. 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) test is used to monitor the extrinsic pathway and the activity of the oral anticoagulant warfarin (also known as Coumadin®).
|Laboratory Testing of Hemostatic Function|
Coagulation tests provide critical information that is used in diagnosing coagulation disorders, evaluating hemostatic function prior to surgery, and monitoring the effectiveness of anticoagulant therapy.
|Collecting Blood Specimens for Coagulation Testing|
Venous blood specimens for coagulation assays should be collected into a tube containing 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.
|Prothrombin Time (PT)/INR|
PT 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 PT test are used in conjunction with other diagnostic tests, as well as the clinical picture of the patient, to determine any hemostatic abnormalities that 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 the oral anticoagulant, warfarin. PT 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).
The INR component of the laboratory PT/INR result is a calculated value that is used by the clinician to monitor warfarin 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 (thromboplastin).INR= (PT of patient/PT of geometric mean of the normal population)ISIThe International Sensitivity Index (ISI) value, is provided by the reagent manufacturer as the relative sensitivity of the reagent itself. When opening a new lot of PT reagent (thromboplastin), it is essential to verify that the ISI value provided by the reagent manufacturer is being used with that reagent lot to prevent the reporting of erroneous INR results, which may have serious consequences for the patient.The INR is used to standardize PT results, and in turn, anticoagulant therapy, across laboratory instrumentation, methodologies, and locale.
|Which of the following statements is NOT correct?||View Page|
Anticoagulant therapy is employed in a number of clinical situations, including: 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.
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 dose-dependent. 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), activated clotting time (ACT), and/or anti-Xa assays are used to monitor unfractionated heparin 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 dependent. They require vitamin K molecules for their action to occur. Vitamin K dependent factors include factor II, VII, IX, and X. Vitamin K dependent 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 test is used to monitor oral anticoagulant therapy.
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.
|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.
The first specific pharmacogenomics (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.
|Warfarin Metabolism, continued|
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.
|What could have caused the clotting?||View Page|
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
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)
|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.
|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.
|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.
|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.
|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.
|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.
|Royal blue top tubes|
Contain either sodium heparin or sodium EDTA anticoagulants, or no anticoagulant.
Are used for trace element, toxicology, and nutritional studies.
|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.
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.)
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.
|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.
|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.
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.
Preanalytical ErrorWhat is it?How does it happen?What is the result?HemolysisRed 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 specimenClumped or clotted cells in specimen that requires anticoagulated or whole bloodInsufficient 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 volumeToo 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.
|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
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.
|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.
|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.
|Preanalytic 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, as shown in the image on the right. 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.
|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 colorAdditiveFunction of AdditiveCommon laboratory testsLight-blue3.2% Sodium citratePrevents blood from clotting by binding calciumCoagulationRed or gold (mottled or "tiger" top used with some tubes is not shown)Serum tube with or without clot activator or gelClot activator promotes blood clotting with glass or silica particles. Gel separates serum from cells.Chemistry, serology, immunologyGreenSodium or lithium heparin with or without gelPrevents clotting by inhibiting thrombin and thromboplastinStat and routine chemistryLavender or pinkPotassium EDTAPrevents clotting by binding calciumHematology and blood bank GraySodium fluoride, and sodium or potassium oxalateFluoride inhibits glycolysis, and oxalate prevents clotting by precipitating calcium.Glucose (especially when testing will be delayed), blood alcohol, lactic acid
|A blood collection tube that has a light-blue top contains which of these anticoagulants?||View Page|