|Positioning the Puncture Device for a Fingerstick|
The fingerstick device should be held firmly against the puncture site. To obtain the best capillary specimen using the finger, align the puncture device perpendicular (horizontal) to the whirls of the fingerprint. This cross-cut of the fingerprint whirls causes the blood to bead at the puncture site, allowing the phlebotomist to efficiently collect the drops of blood into the container. This image illustrates the correct position of the cut in relation to the fingerprint lines.If the puncture is made parallel to the fingerprint whirls (as shown below), the blood will not bead but rather it will travel down the channels between the lines of the fingerprint. This makes it difficult to collect the blood into the container. The phlebotomist may inadvertently "scrape" the blood from the skin while filling the container, resulting in hemolysis and/or clotting of the specimen.The tip of the finger should be avoided. Puncturing the fingertip may cause unnecessary discomfort to the patient.
|Dermal Puncture vs Venipuncture|
In some situations, the phlebotomist will make the decision if a blood specimen will be obtained by dermal puncture or venipuncture. The patient's condition, the age of the patient, the amount of blood needed for testing, and the risks associated with the procedure will help the phlebotomist determine the best method for collection.A dermal puncture requires less precision, therefore it is less critical for the patient to be still or immobilized. However, if the puncture is not performed correctly, or an approved site is not used, the puncture may cause more discomfort, or even injury to the patient.The risk of accidental needlestick injury to the patient and phlebotomist is minimal since the puncture device is designed to retract the needle once the puncture is made. The puncture is quick and standardized for puncture depth. However, the procedure takes longer to complete. This delay in collection of the blood specimen could result in hemolysis or clotting of the blood or tissue fluid contamination of the specimen and specimen rejection by the laboratory.The dermal puncture minimizes the amount of blood taken from the patient. This will be important to consider, especially with infants in an intensive care nursery. However, some laboratory tests require larger amounts of blood for testing; in these cases, capillary collection is not an option.If a patient is dehydrated or has poor peripheral circulation, an adequate blood collection from a dermal puncture may not be possible.
|Capillary Blood Collection for Metabolic Testing|
The collection of these specimens requires the same attention to detail as with any phlebotomy procedure. Gather all necessary equipment Be certain to choose a device that punctures the heel to a depth appropriate to the size of the infant. Only use the filter cards provided by your state to collect the specimen. These cards are calibrated to the exact specifications needed for testing of metabolic disorders. An alternate or homemade card must not be used. Put on all necessary personal protective equipment Gloves are always required. Gowns and eye protection may also be required. Positively identify the patient Use two identifiers. The infant who is in the nursery should have an identification band attached to the ankle or wrist. In special care nurseries an alternate form of identification may be used. However, a crib card should never be used as a form of identification. Follow the practice for your facility. Position the infant Be certain that the heel can be easily accessed. Follow all nursery requirements that apply to safe handling of newborns. Warm the heel using an approved warming device Clean the site with alcohol or the approved disinfectant. Allow the site to air dry before proceeding with collection of the specimen. Grasp the heel firmly but not tightly, activate the puncture device, wipe away the first drop of blood, and begin collection of the specimen.Allow the blood to wick onto the card. Completely saturate the circle with one continuous drop of blood. Avoid touching the card to the skin. Apply the blood only to one side of the card. Do not layer the blood by applying a second drop on top of the first. Repeat the procedure to completely fill each circle on the card. Each circle should be completely and uniformly saturated as shown in the bottom image on the right. Follow the policy of your institution or state to determine how many circles must be completely filled. Apply pressure to the puncture site using a sterile gauze Gently raising the infant's leg above the level of the heart will also aid in clotting the puncture site. Bandage according to site-specific policy.
|Capillary Blood Gases|
In some instances, the healthcare 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|
|Analyzing the Mixing Study Results|
A "corrected" mixing study result is defined as a PT or aPTT result that is now in normal range of the pooled normal plama control, or within approximately 10% of the normal range. A PT or aPTT test that was greatly prolonged at baseline, may not completely correct to within the normal reference interval on a 1:1 mix."No correction" would then mean that the patient sample/pooled normal plasma mixture had no significant decrease in clotting time for the PT or aPTT compared to the intial PT/aPTT results.
|Analyzing the Mixing Study Results (cont.)|
Various tools have been developed that identify whether a sample is "corrected" or "not corrected" by the addition of pooled normal plasma. One tool is the Rosner Index.The Rosner Index subtracts the clotting time of the pooled normal plasma (PNP) from the clotting time of the 1:1 mix. This result is then divided by the clotting time of the patient sample. The equation is as follows:Rosner Index = (1:1 mix clotting time result - PNP clotting time result) / initial prolonged clotting time of patient sampleWith this method, a high index value represents the possibility of an inhibitor. A low index value would represent a possible factor deficiency. For example, an index of 10 or lower indicates correction, 15 and above indicates no correction. If after the calculation is performed and a value of 10-15 is obtained, it is recommended that your test be repeated.Each laboratory must determine its own reference interval for the Rosner Index.
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- 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.
|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.
|Introduction: Factor Deficiencies|
A deficiency in one or more coagulation factor will also cause abnormalities in hemostasis. The image to the right depicts the coagulation cascade. Notice how one factor acts upon another to eventually form a stabilized fibrin clot, the end product of the coagulation cascade. Having an abnormally low level, or a complete lack, of a coagulation factor can cause the extrinsic, intrinsic, or common pathways to malfunction, resulting in dangerous hemorrhagic issues including spontaneous bleeding. Two of the most common factor deficiencies are factor VIII (hemophilia A) and factor IX (hemophilia B). Hemophilia A comes in two forms: congenital (inherited) or acquired. Congenital hemophilia A represents the condition where an individual is born defecient (to various degrees) of factor VIII. Acquired hemophilia A is a condition in which an individual spontaneously produced an autoantibody to factor VIII, leaving the body unable to use the factor VIII that may be present. Hemophilia B is an inherited condition where the individual has a mutation of the factor IX gene and is unable to produce adequate levels of this coagulation factor.In some cases, patients have multiple factor deficiencies that are secondary to a primary condition such as vitamin K deficiency, disseminated intravascular coagulation (DIC), and liver disease. With vitamin K deficiency, the liver is unable to produce the coagulation factors that are vitamin K-dependent. During liver disease, the liver may be unable to produce coagulation factors effectively. In DIC, the clotting processes are in overdrive and will consume the coagulation factors that are being produced, leading to low levels of circulating coagulation factors.
|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
|Which of the following is not a variable in the effectiveness of hemostasis?||View Page|
|What organ is associated with the production of the majority of clotting factors?||View Page|
|What laboratory test result is commonly used to monitor oral anticoagulant therapy?||View Page|
|Introduction to the Fundamentals of Coagulation|
The ability of the body to maintain a state of homeostasis, or physiological equilibrium, is absolutely essential for effective, efficient functionality of all body systems. Hemostasis is the cessation of free blood flow, external to the vascular system, when a vessel wall has been breached.With the maintenance of homeostasis in mind, it is vital that the body be able to rapidly repair vascular damage, arresting blood flow in the process, while simultaneously maintaining blood in a fluid state within the vascular compartment.
|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.
|An Introduction to the Fundamentals of Coagulation|
Vessel size as related to time required for clotting to occur, amount of products used (platelets and clotting factors), and size of the corresponding bleed.
|Primary Hemostasis: Characteristics of the Platelet|
Platelets play a significant role in primary hemostasis, as they are the "bricks", or building blocks of the developing primary platelet plug, the forerunner to the end stage fibrin clot. Platelets have inherent adhesive properties, which are essential for adherence to the site of vascular damage, and for binding to one another in aggregation activities. Platelets must be present in sufficient numbers, and be functionally active for optimal clotting to occur. Platelet functionality tends to be more crucial than the number of platelets available however, as patients with moderately decreased platelet counts can still form clots relatively effectively as compared to those patients with functional platelet defects. The image to the right shows a highly magnified cluster of platelets. Notice their spiny shapes, which allow them to adhere to one another and a damaged vessel wall.
The fibrinogen assay performed in the clinical laboratory is a quantitative measure of factor I. This assay is used to determine whether there is enough fibrinogen present to allow for normal clotting. It is performed in cases of an unexpected, prolonged bleeding event, or an unexpected abnormal PT and/or aPTT. Additionally, it is also used to aid in the diagnosis of disseminated intravascular coagulation (DIC).The fibrinogen reference range is typically around 200-400 mg/dL. Fibrinogen levels <100 mg/dL are often associated with free bleeding, or impaired hemostatic function.
|Coagulation Disorders - Acquired|
Disseminated Intravascular Coagulation (DIC) is best described as a disorder of consumption, because clotting factors are depleted from the blood. Basically, clotting occurs randomly throughout the body, as opposed to just in the localized areas where vascular damage has occurred, consuming clotting factors and other components such as platelets in the process. Symptoms may range from a mild bleed, to severe, profuse bleeding, primarily dependant upon the availability of clotting factors. As more and more coagulation factors and components are consumed, the disorder progresses and symptoms worsen. Most heavily impacted are the levels of factors I, V, and VIII as well as the number of available platelets. Clinically, DIC is detected via an elevated (positive) FDP, positive D-dimer test, a prolonged PT and APTT, plus the manifestation of hemorrhagic episodes. DIC is diagnosed as two primary types, acute and chronic. Acute DIC manifests in a few hours or a few days, has a high mortality rate, and is seen in infections, obstetric complications, liver disease, and tissue injury. Chronic DIC is a secondary condition to some other disease state. Once you treat the primary disease, this type of DIC will go away. Treatment is often factor replacement therapy through the use of fresh frozen plasma and/or cryoprecipitate.
|Coagulation Disorders and Liver Disease|
The liver is the site of production for the vast majority of our clotting factors. Therefore, impaired liver function could adversely affect these hemostatic proteins. Some early indicators of a potential liver problem include:An increase in factor VIII. It is not produced in the liver and will be present in elevated numbers as the body attempts to compensate. The PT is sensitive to liver function, so an unexpected, prolonged PT should be evaluated. A lack of fibrinogen is often indicative of severe liver disease. It is difficult to treat liver disease, so therapy typically centers around replacing the missing factors by way of administration of fresh frozen plasma.
|Which of the following statements regarding coagulation disorders is correct?||View Page|
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.
|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.
|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.
|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.
Numerous types of proteins are dispersed in the plasma. These include:
Coagulation proteins (blood clotting factors), which, if activated, will form a blood clot , and
Serum proteins, which are left dispersed in liquid after the clot is formed. Serum proteins include:
Albumin, a marker of nutrition, and
Globulins, or antibodies.
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.
|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.
|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