Clotting Information and Courses from MediaLab, Inc.

The most common cause of thrombocytopenia is increased destruction of platelets. Platelets are eliminated from peripheral circulation faster than the bone marrow can produce new platelets.Increased platelet destruction may be the result of immune or nonimmune mechanisms. Immune platelet destruction begins when antibodies coat platelets. These sensitized platelets are then destroyed by macrophages, mostly from the spleen but also from the liver. Disorders that are associated with immune mechanisms of destruction include: Idiopathic (or immune) thrombocytopenic purpura (ITP) Heparin-induced thrombocytopenia (HIT) Neonatal alloimmune thrombocytopenia (NAIT)Increased destruction of platelets is not always caused by the immune system. Platelet destruction can occur as a result of abnormal platelet aggregation or endothelial cell injury. Both of these occurrences can cause fibrin to form in arterioles and capillaries. This leads to platelet activation and consumption. Conditions associated with nonimmune destruction and consumptive thrombocytopenia include: Thrombotic thrombocytopenic purpura (TTP) Hemolytic uremic syndrome (HUS) Disseminated intravascular coagulation (DIC) All of these conditions are associated with significantly decreased platelet counts that may become life threatening. Restoration of platelet numbers is essential to promote clotting and vascular patency.

Some pathologists prefer their bone marrow smears to be made fresh at bedside without the use of any anticoagulants. This however limits the number of smears that can be made before the sample clots. Using a syringe, that has been rinsed with preservative-free heparin, to pull the marrow during the procedure will prevent clotting but will introduce morphology changes and staining artifact.It is preferable to make smears as soon as possible after sample collection. However, when stored in the refrigerator, acceptable smears can be made from an EDTA tube as long as 8-10 hours after sample aspiration without introducing excessive amounts of artifacts. This is useful when marrows are collected at times when staffing trained in marrow smear preparation may not be available.

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.

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.

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.

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.

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.

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

Wear disposable latex gloves if available.Place a thick, clean compress (consisting of gauze or soft clean cloth) directly over the wound. The compress will absorb blood and help the clotting process.Apply pressure to the victim's wound by placing your palm directly over the compress and pressing firmly.If blood soaks through, do not remove the compress. Instead, add more cloth pads over it as needed. Removing the compress may reopen the wound and result in further bleeding.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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