Physicians used NR results to determine the level of coumadin therapy usually after the patient has been on anticoagulant therapy for at least two weeks. The dosages are adjusted to keep the patient’s prothrombin time to a INR value of 2.0 to 3.0 or possibly 3.5 if the patient has a mechanical heart valve. INR values greater than 4.0 tend to place the patient at risk of hemorrhaging.

42     DESCRIBE COMMERCIAL THROMBOPLASTIN.

Also known as tissue factor III, thromboplastin is an organic extract of rabbit brain or lung tissue suspended in a buffered solution of citrate. This solution may or may not have calcium added. If the ISI value of thromboplastin is close to 1.0 then the thromboplastin is sensitive. The closer the ISI value is to 2.0 the less sensitive the thromboplastin. Commercial thromboplastin is insensitive to factor IX due to the concentration of reagents but if it is diluted, the sensitivity to factor IX increases but the reagent becomes less sensitive to factors II, VII, and X.

43    DESCRIBE WHAT FACTORS ARE EVALUATED IN THE FOLLOWING COAGULATION SCREENING TESTS.

            bleeding      clot        PT     APTT        TT      urea
              time      retraction                                  solubility
XIII           no             no         no       no          no        yes
XII            no             no         no       yes         no        no
XI             no             no         no       yes         no         no
X              no             no         yes      yes         no        no
IX             no             no         no       yes         no         no
VIII           no             no          no       yes        no          no
VII            no             no         yes*      no         no         no
V              no             no         yes       yes        no         no
II              no             no         yes        yes       no         no
I               no             yes        yes       yes        yes        no
Fletcher    no             no         no         yes        no          no
HMWK       no             no         no        yes         no          no
Platelets     yes          yes        no         no         no          no
*most sensitive

44    DISCUSS THE PROTHROMBIN TIME (PT) AS A SCREENING PROCEDURE.

This test is also called “pro-time”. This is the most often ordered test to monitor Coumadin oral anticoagulant therapy. This is the preferred screening method for extrinsic pathway abnormalities, detecting deficiencies in factors II, VII, IX, and X. If the factor concentration is decreased to 20% to 65% of normal, the PT is prolonged for one to three seconds. In cases of dysfibrinogenemia, where the fibrinogen concentration drops to ≥80 mg/dL, the PT will be prolonged. If factor II drops to 10% of normal, the PT will be prolonged. Other disorders that result in a prolonged PT are:
    [1]   dicumarol therapy          [6]   heparin therapy
    [2]   factor V deficiency       [7]   factor VII deficiency
    [3]   obstructive jaundice     [8]  hemolytic disease of the newborn
    [4]   Vitamin K deficiency     [9]  circulating anticoagulants
    [5]   factor X deficiency      [10]  liver diseases

The PT is a comparison of the patient’s prothrombin time to that of a normal control. If the normal control is 13 seconds and the patients PT is also 13 seconds, then there is 100% prothrombin activity. If the patient’s PT extends to 18 seconds, then the PT activity will be about 50%. If the PT should extend to 36 seconds, then the PT activity is less than 12%. The physician’s goal in coumadin therapy is to attain an INR value of 2.0 to 3.5 or a prothrombin time that is 1.5 to 2 times that of the normal control plasma.. See Objective #41.

45   LIST TEN ERRORS THAT CAN OCCUR IN PERFORMING THE PROTHROMBIN TIME.

[1]    Using the incorrect anticoagulant
[2]    Using the incorrect amount of anticoagulant
[3]    Using expired reagents.
[4]    Using an incorrect amount of blood to anticoagulant.
[5]    Using old or hemolyzed patient’s plasma
[6]    Using wet or dirty plastic ware or glassware in performing the tests.
[7]    Pipetting errors.
[8]    Failure to follow the manufacturer’s directions.
[9]    Changing from one manufacturer to another and not maintaining
        careful quality control.
[10]   Changing from one lot number to another without verifying the
        control results.

46    BRIEFLY DISCUSS THE PRINCIPLE OF COUMADIN THERAPY.

Coumadin is a warfarin compound and competes with vitamin K in the liver preventing synthesis of the vitamin-K dependent coagulation factors. Coumadin is also known as Warfarin sodium, Coufarin, Panwarfin, Warfilone, and Warnerin sodium. Coumadin is 4-hydroxycoumarin and inhibits the γ-carboxylation of vitamin K dependent inactive clotting factors (II, VII, IX, and X). The absence of these γ-carboxyglutamic acid units leaves these four clotting factors unable to bind calcium ions. The clotting factors cannot congregate on the phospholipid surface of the platelet nor to participate in clotting reactions.

When a patient is started on coumadin therapy, it become effective in about 10 hours. It takes about two weeks to stabilize the patient and for maximum therapy effect. The physician’s intent is to increase the PT to about 1.5 to 2.0 times that of normal. Each coumadin dose will be reflected from 36 to 48 hours later. The PT measures VII activity, but the activity level of factors II, IX, and X has the potential to affect the test.

Clinicians will prescribe oral anticoagulants to prolong the clotting time, inhibit clotting, and facilitate clot lysis without placing the patient at risk of a hemorrhage. Disorders in which coumadin may be prescribed are:
[1]  intravascular clotting     [6] post-operative thrombophlebitis
[2]  pulmonary embolism       [7] acute coronary thrombosis
[3]  atrial fibrillation              [8] recurring idiopathic thrombophlebitis
[4]  tissue heart valves          [9] recurring systemic embolism
[5] acute embolic and thrombotic occlusions of the peripheral arteries

Coumadin type drugs should not be prescribed for menstruating women, pregnant females, nursing mothers, during postpartum, and following cerebrovascular accidents.. The chief problem with coumadin therapy is hemorrhagic accidents.

47   DESCRIBE THE THREE TYPES OF ORAL ANTICOAGULANTS USED BY PHYSICIANS.

These are dicumarols, coumarins or coumadins, and indanediones (in-dan-e-di-onez). Coumarins are most commonly used. The dicumarols are very slow acting and indanediones have the most side effects. Because of the side effects indanediones are avoided as therapy strategies. The antidote for coumarin is vitamin K, but it may take up to three days to reach it full therapeutic effect. Fresh frozen plasma can immediately correct an emergency bleeding due to mis-dosage of coumarin. Caution: There is some degree of risk for hepatitis or AIDS if using fresh frozen plasma.

48    LIST MEDICATIONS / DRUGS THAT ARE KNOWN AFFECT THE PATIENT’S RESPONSE TO COUMADIN THERAPY.

When a physician prescribes coumadin therapy, a review of what medications the patient is currently taking should occur and a prescription written to withdraw and/or add medications. Patients on coumadin therapy must be monitored.

Drugs known to potentiate coumadin action are:
[1]  ethyl alcohol                    [8]   allopurinol
[2]  anabolic steroids                  [9]   aminoglycoside antibiotics
[3]  tricyclic antidepressants    [10]  cephalosporins
[4]  iodine based x-ray dyes     [11]  heparin
[5]  oral hypoglycemics            [12]  penicillin
[6]  sulfonamides                     [13]  choral hydrate
[7]  glucagon                                 [14]  thyroid hormone

Drugs known to depress coumadin effects are:
[1]  barbiturates         [5]  corticosteroids
[2]  estrogens             [6]  rifampin
[3]  xanthines             [7]  oral contraceptives
[4]  carbamazepine     [8]  phenobarbital

49   DESCRIBE THE BASIC PROCEDURE FOR THE ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) TEST.

The APTT procedure requires placing a designated volume of platelet poor plasma (PPP) into a reaction well followed by a designated volume of cephaloplastin reagent and allow the mixture to come to 37 ^oC. Three minutes is ample time for step. The final step is to add a designated volume of calcium chloride reagent. The ensuing reaction is watched and the time recorded when the fibrin clot develops. This test may miss mild factor deficiencies. Normal reaction time ranges from a low of 25 seconds to a high of 42 seconds.

Cephaloplastin is phospholipid substance obtained by petroleum ether extraction of acetone dried brain tissue. The APTT is called partial because it must have the assistance of the plasma procoagulants (exceptions are factor VII and XIII). It is considered to be a platelet phospholipoprotein substitute. The testing procedure requires addition of an activator to assist the activation of the contact factors.

50    DISCUSS THE PRINCIPLE OF THE ACTIVATED PARTIAL THROMBOPLASTIN TEST.

The activated partial thromboplastin test (APTT) is also simply called the partial thromboplastin time (PTT) test. This test evaluates the intrinsic clotting system. The APTT test monitors Fletcher, Fitzgerald, XII, VIII, IX and XI coagulation factors. This reagent contains a contact factor (Kaolin, ellagic acid, or celite) which induces conformational changes in Hageman (XII) factor and initiates the coagulation cascade mechanism. Most coagulation factor deficiencies and circulating anticoagulants involve these factors, therefore coagulation investigations should include the APTT as a testing strategy. APTT reagent are calibrated to provide prolonged values when the patient’s plasma has < 0.3 units/mL of factors VIII, IX, and XI. The APTT is also prolonged when lupus anti-coagulant antibody is present. The APTT is insensitive to factors VII and XIII.

51   LIST THE CIRCUMSTANCES IN WHICH A PHYSICIAN WOULD ORDER AN ACTIVATED PARTIAL THROMBOPLASTIN TIME.

When the patient is suspected of having a hemorrhagic disorder or the physician suspects a lupus anticoagulant antibody. The following are known to cause prolonged APTT’s:
[1]  deficiencies of factors II, V, VIII, IX, X, XI, or XII.
[2]  fibrinogen level <1.0 mg/dL
[3]  anti-factor VIII
[4]  fibrin degradation products (FDP)
[5]  disseminated intravascular coagulation
[6]  vitamin K deficiency (The APTT detects only Factors II, IX, and X)
[7]  heparin therapy.

What kind of an APTT test result is the physician looking for in heparin therapy? Since the normal range is from 25 to 42 seconds with a median time of 39 seconds, the physician is looking for a therapeutic time of about 58 seconds. This is abut 1.5 times that of the median time.

52   DESCRIBE THE LUPUS ANTI-COAGULANT ANTIBODY.

This antibody is found in up to 10% of patients with systemic lupus erythematosus (SLE), hence its name. It is also found in viral infections in children and HIV. This antibody has an affinity for phospholipid, anti-factor VIII antibody, and heparin. It will combine with the test reagents for APTT. The “lupus” antibody may cause false positive serological tests for syphilis. Its presence causes prolonged PT and APTT time results, but rarely causes hemostatic problems. The antibody is more often associated with thrombosis problems rather than those of bleeding. It is not known to activate clotting factors. The antibody is either IgM or IgG or both. It binds to the phospholipids blocking interaction in the coagulation scheme.

This antibody is difficult to identify in the laboratory. The following are a few steps to follow in order to identify the antibody.
[1]  Carefully collect the blood specimen and centrifuge to remove all platelets because they will inactivate the antibody.
[2]  Rule out heparin contamination. The thrombin time or reptilase test will accomplish this.
[3]  Mix one part of the patients plasma with one part of normal plasma. This will correct the problem if it is a one factor deficiency. There will be no correction for the presence of the antibody. Refer to Objectives 72 and 82.
[4]  A Russell viper venom time (see Objective 81) or kaolin clotting time will help to screen for the antibody. If these antibodies are present these tests will be prolonged.

53    DESCRIBE HEPARIN AND ITS MODE OF ACTION.

Heparin (a sulfated polysaccharide) is a proteoglycan (a acidic mucopolysaccharide) found inside mast cells and basophils, lung, liver, skin, and in circulation. It is a heterogenous molecule that consist of several components that can be separated by molecular size and weight. It is a unique molecule containing many sulfate groups. One of its known functions is to activate antithrombin III and another function is to interact with factor X, prothrombin, and platelet aggregation. Caution: Heparin should not be used to collect blood for platelet counts. It can be isolated from the liver, lungs, and spleen. It is poorly absorbed from the GI tract and is usually administered intravenously or subcutaneously as a sodium salt. Once in the blood stream, its action is immediate. The activated clotting time and APTT are used to monitor heparin therapy. The APTT test is sensitive to heparin and is the test of choice. Physicians try to establish a therapeutic range with the APTT test that is about 1.0 to 1.5 times that of the patient’s baseline or control value. As a rule this puts the heparin concentration to about 0.3 to 0.5 units/mL. Side effects of heparin therapy are hemorrhage and development of thrombocytopenia.

Heparin is thought to have binding sites for thrombin and antithrombin-III. By complexing with thrombin and antithrombin-III, heparin can induce a conformational change in antithrombin-III and facilitate the formation of covalent, one-on-one thrombin-antithrombin-III complex, thus speeding up the rate at which antithrombin III reacts with thrombin. One possible mode of action is listed as follows:
   
54    DISCUSS THE ROLE OF PROTAMINE SULFATE IN COAGULATION.

It does not have a direct role. It is a simple protein that has been sulfated to form an antagonist to heparin. One mg of protamine sulfate will neutralize 100 units of heparin. It is also a test procedure to evaluate the action of thrombin on fibrinogen. See Objective 87.

55    DISCUSS ANTITHROMBIN-III AS A NATURAL INHIBITOR OF COAGULATION.

Antithrombin-III (AT-III) is a single-chain, α2 - globulin, produced in the liver, and is a major inhibitor of coagulation. It is responsible for about 80% of coagulation inactivation. Its an anti-serine protease, whose primary target is inactivation of thrombin. It also inactivates any activated serine protease clotting factor (IX, X, XI, and XII). Also inactivated are protein C and Fletcher factor. AT-III contains an arginine residue that interacts with the active serine molecule of the coagulation enzyme.

It is decreased in thrombotic disease, disseminated intravascular coagulation (DIC) syndromes, cirrhosis of the liver, certain liver diseases, following surgery, septicemia, and women on oral contraceptives or estrogen therapy. It is assayed using electro-immunoassay, radial immunodiffusion, or enzyme-linked immunosorbent assay. Normal ranges (17 to 30 mg/dL) are listed as low as 70% and a high of 125%. Normal ranges are established by individual laboratories and will vary from lab to lab. If AT-III levels drop to <60% that of normal, the patient is at risk of a thrombosis.

56    DISCUSS PROTEIN C AS AN INHIBITOR.

Protein C (a glycoprotein) is vitamin K dependent and produced in the liver. Plasma levels average from 3 to 5 mg/L. It is activated by thrombin and also a potent inhibitor of Factors VIII and X. The activation of protein C is enhanced and accelerated by the presence of thrombomodulin and Ca⁺⁺. Endothelial cells contain thrombomodulin molecules which interact with thrombin forming a 1:1 complex. The complex forms a modified thrombin molecule that is not free to cleave fibrinogen, but is available to activate protein C. In this way thrombin becomes an anticoagulant. Activated protein C (complexed with protein S) destroys activated factor V, which limits the formation of thrombin. This protein C activity make activated factor X more vulnerable to deactivation by antithrombin-III. Protein C also enhances the release of tissue plasminogen activator by inactivating a plasma inhibitor of tissue plasminogen activator.

57    DISCUSS THE ROLE OF PROTEIN S IN THE INHIBITION OF HEMOSTASIS.

Protein S is a vitamin K dependent glycoprotein and exists in plasma in two forms. Forty percent of Protein S exists in the functional free form and the remainder exists in a form complexed to C4b-binding protein (linked to the compliment system) which is non functional. The amount of Protein S in plasma is from 20 to 25 mg/dL. Normal laboratory ranges for Protein S ranges from 63% to 160%. If protein S levels fall to ≤ 60%, then the patient is at risk for thrombotic complications. Protein S is synthesized by hepatocytes and megakaryocytes. This protein has a high affinity for phospholipid surfaces and epithelial cells. This affinity promotes the binding of Protein C to such surfaces and facilitates Protein C activation. Free protein S, in binding to Protein C, forms a complex with Protein C. It is this complexed form of Protein C that acts upon specific clotting factors.

58     DISCUSS α₂ - MACROGLOBULIN AS A COAGULATION INHIBITOR.

α₂ - Macroglobulin is a glycoprotein found throughout the body. This protein will form complexes with thrombin, plasmin, and Fletcher factor (kallikrein) to inhibit their activities. The α₂ - macroglobulin will bind slowly with thrombin, then undergo cleavage, leaving the thrombin molecule less effective in its proteolytic activity. It appears that the complexes formed by the α₂ - macroglobulin tend to be cleared from plasma by phagocytic cells of the Mononuclear Phagocytic System (MPS), formerly the reticuloendothelial system (RES). About 50% of the inhibition of Fletcher factor is conducted through the macroglobulin. α₂ - Macroglobulins are in the highest concentrations in infants, small children, and pregnant women. It is reported that this glycoprotein can form complexes with serine proteases.

59     BRIEFLY DESCRIBE THE PURPOSE OF C1-INACTIVATOR IN THE INHIBITION OF HEMOSTASIS.

C1-Inhibitor (also called C1-inactivator) is a neuraminoglycoprotein. It is the principle inactivator of activated factors XI and XII, plasmin, and activated Fletcher factor (kallikrein).

60   BRIEFLY DESCRIBE THE PURPOSE OF α1 - ANTITRYPSIN IN THE INHIBITION OF HEMOSTASIS.

α₁ - Antitrypsin is a glycoprotein, which is thought to have a secondary role in coagulation inactivation. It is considered to be a weak inhibitor of thrombin and activated factors X and XI.

61    BRIEFLY DESCRIBE THE PURPOSE OF HEPARIN COFACTOR II IN THE INHIBITION OF HEMOSTASIS.

Heparin cofactor II is a glycoprotein synthesized in the liver. Its activity is strongly augmented by heparin, indicating that it has limited activity by itself. It is limited in its substrates, suppressing thrombin and chymotrypsin. There is no known evidence of activity against activated factors IX, X, and XI.

62    DISCUSS THE ROLE OF VITAMIN K IN HEMOSTASIS.

Vitamin K is required by the hepatocytes to complete the alteration of factors II, VII, IX, and X. It converts the glutamic acid residue in these factors to a γ-carboxyglutamic acid residue that has the capability to bind calcium ions.
   
Vitamin K1, typical of plants, is found in green leafy vegetables. Bacterial flora (Escherichia coli, Bacteroides fragilis, etc.) in the intestinal tract synthesizes vitamin K2, a useable form characteristic to animals for absorption. If the patient has mal-absorptive syndrome of the GI tract, vitamin K deficiency can result. Other factors that lead to vitamin K deficiency are bile duct obstruction, use of broad spectrum antibiotics, and sprue. Vitamin K is depressed by the use of oral anticoagulants (coumadin) when given as an anticoagulant therapy strategy. The therapeutic use of vitamin K can reverse the deficiency within 24 hours.

Sprue is a tropical disease. Its actual cause is not known. Symptoms include anemia, weight loss, and steatorrhea.

63 GIVE A BRIEF OVERVIEW OF FIBRINOLYSIS WITHIN THE CONTEXT OF HEMOSTASIS.

The fibrinolytic system is activated when the coagulation system is activated. The purpose of this mechanism is to digest (dissolve) fibrin clots as they are formed to keep the vascular system free of emboli (clots). This process is initiated when plasminogen is converted to plasmin. This proteolytic activity produces fragments called fibrin degradation products (FDP). Plasmin is a degrading enzyme which converts the fibrin clot into basic units called dimers. Plasminogen activating factor (tPA) is attracted to the fibrin site where dimer cross-linking occurs. This means that plasminogen will degrade at this site. Cleavage of the fibrin clot produces the following products: first a YY/DXD complex, second . . . DED and DY/YD complexes, and third . . . E-fragment, D-D dimers, and DED complexes. The following degradation scheme illustrates the degrading process.
   

64    DISCUSS THE ROLE OF PLASMINOGEN IN THE FIBRINOLYTIC SYSTEM.

Plasminogen is a single chain β-globulin that circulates in the blood in an inactive state. The plasma concentration ranges form 20 to 40 mg/dL. It is produced in the liver. It is drawn into the clotting process in its inactive form and becomes absorbed into the clot. Activators of plasminogen (t-PA) can be found in the blood and most tissues. Active coagulation factor XII, Fletcher (kallikrein), and Fitzgerald (HMWK) convert plasminogen to plasmin. These are designated as intrinsic activation. Extrinsic activation occurs when the endothelial cells of the vascular system release tissue activator, when stimulated by certain activated clotting factors, exercise, hypotensive shock, pharmacologic stimulators, and venous stasis.

65   DISCUSS THE ACTIVATION OF PLASMINOGEN TO PLASMIN.

Plasminogen activator factor (tPA) has a strong affinity for fibrin and it is absorbed onto the fibrin polymers, then incorporated into the fibrin clot. The tPA transfers from the fibrin polymers to the plasminogen molecule, activating it. The active plasmin degrades the fibrin polymer by forming degraded fibrin fragments. These fibrin fragments are capable of activating plasminogen and the fibrinolysis process will continue. If plasmin is free in blood circulation, it will proteolytically degrade several of the coagulation protein factors (V, VIII, and XIII), the kinin system, and the complement system. If the plasminogen activator is in the blood, it is called an intrinsic activator. Fletcher factor (Kallikrein) is considered to be an intrinsic plasminogen activator. If the plasminogen activator is from the epithelium of the vascular system, it is known as an extrinsic activator or tissue activator. Activated factor X, thrombin, platelet activating factor, bradykinin, and protein C can stimulate the release of tissue activator. Tissue activator can also be found in the heart, kidney, and other body organs.

66     BRIEFLY DESCRIBE HOW PLASMIN DEGRADATION OF FIBRINOGEN DIFFERS FROM FIBRIN.

Plasmin does not distinguish between fibrinogen or fibrin. The degradation process is essentially the same as outlined in Objective #63.

67     DESCRIBE HOW FIBRINOLYSIS IS CONTROLLED.

Fibrinolysis must proceed at a rate that does not interfere with the healing of the wound and the dissolving of the clot. The molecules that act to inhibit the process of fibrinolysis are as follows:
[1] α₂ - Antiplasmin is a single chain α₂ - glycoprotein synthesized in the liver. It is the principle inhibitor of fibrinolysis. α₂ - Antiplasmin forms a one-on-one stoichiometric complex with either plasmin or plasminogen. When plasminogen is inhibited, it cannot form plasmin. It is thought that plasmin’s enzyme sites are blocked, preventing binding or dissolution of the plasmin substrate molecules (fibrin, fibrinogen, factors V and VII, and thrombin). α₂ - Antiplasmin also inhibits plasma kallikrein and activated factors II, X, XI, and XII which are serine proteases. If α₂ - antiplasmin is deficient because of hereditary condition, hemorrhage may occur due to lysis of fibrinogen and degradation of factors V and VIII. If the problem is a pathology which involves excessive clotting as with disseminated intravascular coagulation (DIC), the patient’s α₂ - antiplasmin may be depleted due to excessive conversion of plasminogen to plasmin.
Stoichiometric describes a mass relationship of molecules to each other. It is the relative proportion of reacting molecules that forms a product. Two moles of hydrogen plus one mole of oxygen forms water as its product. One α₂ - Antiplasmin molecule plus one plasmin or one plasminogen forms one product, the antiplasmin - plasmin complex.

[2] α₂ - Macroglobulin is a plasma glycoprotein that combines slowly with thrombin, plasmin, and kallikrein. It is considered to be an important backup inhibitor when α₂ -antiplasmin levels are low. This is a very large molecule and it mode of action is to trap the plasmin molecule as it attempts to attach proteolytically to a substrate molecule. The level of inhibition is not as complete as for α₂ - antiplasmin since the bound molecule can exert small levels of activation. Deficiencies of this inhibitor are rare and medically these patients are asymptomatic.
[3] PAI-1 is produced in the hepatocytes, endothelial cells, and megakaryocytes. PAI-1 is found in the α-granules of platelets. PAI-1 inhibits tissue plasminogen activator (t-PA) and two-chain type glycoprotein (urokinase-like) plasminogen activator (tcu-PA) by neutralizing the molecule and preventing it from forming plasmin.

The extrinsic plasminogen activators designated as urokinase-type plasminogen activator exists as two types [1] scu-PA and [2] tcu-PA. The scu-PA (single chain type glycoprotein (urokinase-like) plasminogen activator) molecule does not activate plasminogen, but tcu-PA does.

[4] PAI-2 weakly inhibits t-PA and tcu-PA, but not scu-PA.
[5] PAI-3 inhibits tcu-PA and activated protein C.
    Note: PAI = plasminogen activator inhibitor.

68    DESCRIBE THE LEE-WHITE WHOLE BLOOD CLOTTING TIME.

This testing procedure is included for its historical interest. It will not be used for test questions. This is a whole blood clotting time test that has been used to monitor heparin therapy. Prepare three 8.0 mm diameter tests tubes for the test. Four mLs of blood is drawn with a 20 gauge needle via a clean, non-traumatized venipuncture. One mL of blood is deposited in each of the three test tubes and the last mL is discarded. The tubes are placed in the water bath for three minutes. Begin looking for clot formation as follows: [1] Tilt tube #1 at 30 second intervals until the blood can be completely inverted without spilling. [2] Repeat with tube #2, and [3] lastly with tube #3. Note that the handling and tilting of blood hastens the clotting process. The results obtained for tube #3 are reported. A prolonged clotting time is considered to be indicative of a coagulation deficiency. The normal range for the Lee-White clotting time is five to ten minutes. If the tube diameter is 9.0 mm, then the normal range increases to six to thirteen minutes. This clotting procedure is considered obsolete and is no longer performed. The prothrombin time (PT) and activated partial thromboplastin time (APTT) tests involve the same coagulation time principle and are more reliable. Comments . . . . Tube #1 may be left in the water bath over a 2 to 4 hour period and observed for clot retraction. The tilting of the tubes are to be gentle, always in the same direction, and at the same angle.

69    DISCUSS THE ACTIVATED COAGULATION TIME (ACT) TEST.

The activated coagulation time (ACT) test is a modification of the whole blood clotting time test. It is a test designed to be performed at the bedside of the patient. It is used to monitor heparin therapy. The test requires the use of a tube that contains an activator (example: diatomaceous earth) to which must be added a minimum of 2.0 mLs of whole blood. The blood is mixed to distribute the activator and timing begins as soon as the blood is collected. The tube is tilted and observed until a clot is formed. The mean normal time for the clot to form is 98 seconds. If a person is on heparin therapy for deep vein thrombosis, the expected clotting time ranges from 180 seconds (3 minutes) to 240 seconds (4 minutes). A person on heparin therapy for cardiopulmonary bypass operation should demonstrate a mean clotting time of 400 seconds (7 minutes).

70   BRIEFLY DESCRIBE THE ROLE OF UROKINASE AND STREPTOKINASE IN HEMOSTASIS THERAPY.

Urokinase and streptokinase are thrombolytic drugs used to treat pulmonary emboli and coronary thrombosis. These drugs will activate plasminogen to plasmin. These drugs are usually given via IV infusion and can induce a high degree of fibrinolysis.

71     DISCUSS THE BLEEDING TIME IN THE INVESTIGATION OF HEMOLYSIS.

The bleeding time is an in vitro testing procedure to detect platelet dysfunction and von Willebrand’s disease which will prolong the bleeding time. Because aspirin causes prolongation of the bleeding time in normal patients, this procedure should not be attempted on patients who have taken aspirin within the past eight days. Drug therapy may also prolong the bleeding time. Thrombocytopenia prolongs the bleeding time.

The Duke method (introduced in 1910) is the least reproducible of the bleeding time techniques. It is not longer performed in most laboratories. The Ivy method replaced the Duke procedure in 1941, which employs a blood pressure cuff on the arm of the patient. The “cuff” is inflated to 40 mm Hg. Two or three lancet incisions (about 1 mm deep and 0.3 mm long) is made on the inside forearm and the bleeding time of the two or three cuts averaged. This method greatly improved the bleeding technique with more reproducible results. Severed capillaries tend to collapse (vaso-spasm phenomenon) which shortens the clotting time and is independent of platelet function. The Duke method had three variables and the Ivy method eliminated the capillary variable. This left the platelet function variable and the human variable of the capillary puncture. In 1969, the template method was introduced in which a mechanical device produces a standardized length and depth of cut (1 mm deep and 3 mm long), removing the human variable. The template bleeding time is reproducible and the preferred technique. Normal template bleeding time values range from 2.3 minutes to 9.5 minutes. The direction of the cut on the forearm should be consistent for all patients, either perpendicular (vertical) or horizontal to the bend of the elbow. Horizontal incisions tend to yield longer bleeding times. Caution: The template method can cause mild to significant scarring in some patients.

72    EXPLAIN WHY A PHYSICIAN WOULD ORDER A THROMBIN TIME (TT) TEST.

This test (synonym: thrombin clotting time) measures the availability of functional fibrinogen. The prothrombin time will be prolonged when [1] the fibrinogen level drops to 75 mg/dL to 100 mg/dL, [2] if heparin is present, [3] if fibrin or fibrinogen degradation products are present, and [4] a thrombolytic agent (as streptokinase) is present. The prothrombin time is not specific, but can detect a Factor VII deficiency. If the physician suspects heparin inhibition, then the thrombin time (TT) test will help to confirm this problem. The procedure requires citrated plasma (one part 0.109 M sodium citrate in 9 parts of whole blood). The specimen must be centrifuged to obtain platelet poor plasma. The plasma must be kept cold and the test performed within four hours after collection. Normal values are determined by the thrombin concentration used. Reference ranges may be [1] 8 - 9 seconds, [2] 15 to 20 seconds, [3] less than 24 seconds, or [4] 20 to 25 seconds. Caution: Thrombin is not a stable reagent, once it is made up and warmed to 37 oC (takes about three minutes), it must be used immediately (usually within seven minutes).

73    DISCUSS THE CLOT RETRACTION TEST FOR HEMOSTASIS STUDIES.

This is a test that evaluates the ability of the clot to shrink, becoming denser, and expressing serum from the clot. This phenomenon was associated with platelet function. Normal clot retraction is dependent upon a normal number of functional platelets, calcium, ATP, and minimum of 200.0 mg of fibrinogen/L.   Normally clot retraction is initiated 30 seconds after the blood has clotted (does not flow) in the tube. At 60 minutes, there should be obvious/visible retraction. At four hours, most of the retraction that will take place has occurred. The clot retraction tube is held for 24 hours, at which time, the retraction is considered to be complete. In the retraction process, the clot will pull away from the sides of the tube and some “free” erythrocytes may be observed at the bottom of the tube. In a normal retraction, the clot should make up 45% to 50% of the total blood volume in the tube. If there is an abnormality in the clot retraction process, the clot will occupy more than 50% of the total volume in the tube. There may be no evidence of a discernable clot or its retraction. One feature of abnormal clot retraction is the increased fallout of RBC’s. See illustrations of normal and abnormal clot retraction. Clot retraction is abnormal when para-proteins are present (as in multiple myeloma), thrombocytopenia, Glanzmann’s thrombasthenia, Bernard-Soulier disease, dysfibrinogenemia, and disseminated intravascular coagulation (DIC). Observe the following illustrations of a normal and abnormal clot retraction. This test is seldom performed in the laboratory. It is included to describe how blood behaves in the clotting process.
       
Comment: The clot retraction test is generally considered to be of little clinical use. The more sophisticated platelet function test have made this procedure a seldom requested procedure.

74    EXPLAIN THE CONCEPT OF AGGREGOMETRY AS A PLATELET FUNCTION TEST.

This testing procedure requires special “aggregation” reagents and a special photometer with a stirring device. Blood must be collected with a plastic syringe and stored in plastic tubes. The blood is centrifuged at as speed equivalent to 150 X g for 30 minutes to prepare a platelet-rich plasma (PRP). If RBC’s appear to be present in the PRP, repeat this centrifugation step again for 5 minutes. A platelet poor plasma (PPP) must also be prepared by spinning at 1500 X g for 15 minutes. Cap the specimens in a plastic tube to prevent CO₂ loss and pH changes. Perform a platelet count to determine the platelet concentration in the PRP. If the count is between 200 X 10⁹/L and 300 X 10⁹/L, proceed with the aggregometry studies. If the count is greater than 300 X 10⁹/L, adjust the sample by diluting it with PPP. Repeat the platelet count for verification. A designated volume (usually 0.5 mL of PRP) is placed in the aggregometer well or cuvette and appropriate aggregating reagent is added. The optical density of the specimen is monitored and as aggregation takes place the specimen becomes clear and the light transmittance increases. All changes in optical density over time are recorded in a graph format as a curve. If the curve deviates from the normal expected curve, the slope of the curve changes and forms the basis for determining the amount or percent of platelet aggregation. Examine the following illustration of the features of the platelet aggregation curve.

The principle of platelet aggregometry is to deliberately add a platelet aggregating agent to PRP, induce shape change and then cause the platelets to aggregate or clump together. When the test is initially set up, the aggregometer (type of photometer), the suspended platelets form a turbid suspension. As aggregation takes place the test suspension clears and a change in the light transmission is measured and recorded. The test procedure requires that the patient be in a fasting state and that their last meal be fat free. The patient should not be taking aspirin or non-steroid anti-inflammatory medications. The patient’s blood is to be drawn with a plastic syringe and the blood added to 3.8% sodium citrate. The blood to citrate ratio is nine parts blood to one part anticoagulant. Testing for platelet aggregation should be completed within two hours after collection. See the following illustration  for platelet aggregation scheme.


75    DISCUSS SIX PLATELET AGONISTS USED IN AGGREGOMETRY STUDIES.

EPINEPHRINE. Also called adrenalin, it produces a biphasic curve of irreversible platelet aggregation. A small percentage of patients will demonstrate a monophasic curve. Abnormal platelet curves are observed in thrombosthenin, uremia, and storage pool disease.
Storage Pool Disease.   This is a platelet disorder characterized by the absence of dense bodies and/or α-granules. Platelets cannot release ADP, calcium, platelet factor-4, etc. This is observed in Wiskott-Aldrich syndrome, thrombocytopenia (with absent radius bone in both arms) known as TAR, and Chediak-Higashi syndrome. There is no treatment for this platelet disorder.
ARACHIDONIC ACID. When this reagent is added to the patient’s specimen, a single monophasic curve is produced, resembling that produced by collagen. If there is a platelet deficiency of thromboxane and/or cyclooxygenase, the curve is suppressed. Aspirin inhibits this reagent resulting in the absence of a curve. The patient must be aspirin free for eight days before testing. Abnormal curve results are observed in thrombasthenia.  Note: Arachidonic acid is heat and light labile and must be stored in the frozen state away from light. It is permissible to refreeze arachidonic acid aliquots three times.
COLLAGEN. When collagen is added to a patient’s specimen the reagent initiates a monophasic curve after 30 to 60 seconds. This aggregation phenomenon is dependent upon an intact platelet membrane and membrane receptors, normal phospholipase pathway, and normal cyclooxygenase and thromboxane functions. If an abnormal response occurs, this may suggest disorders such as platelet membrane defect, storage pool disorders, aspirin-like defects, thrombasthenia, and uremia. NOTE: Collagen should not be frozen. Store at refrigerator temperature.

Aspirin-like defects is an anomaly that describes platelets with normal appearing granules which do not have the ability to release their contents. Deficiencies in cyclooxygenase or thromboxane synthetase may be the cause.

RISTOCETIN.   Ristocetin is an antibiotic obtained from Nocardia lurida. The action induced by this agonist produces a monophasic curve. A normal curve is dependent upon intact platelet membranes, a functional von Willebrand factor (vWF) receptor site, and ristocetin cofactor in the patient’s serum. Abnormal curves may indicate von Willebrand’s disease (curves vary with the type of vWF disease) or Bernard-Soulier syndrome. 
THROMBIN. Thrombin reacts with several receptor sites on the platelet membrane. A monophasic curve is produced in normal individuals. Abnormal curves may indicate a storage pool defect disorder.
ADENOSINE DIPHOSPHATE (ADP). ADP produces a monophasic curve if used in low concentrations, but a biphasic curve in high concentrations. Abnormal curves are observed when aspirin is present, aspirin-like release defects, afibrinogenemia, uremia, storage pool disease, and thrombasthenia.

76    ILLUSTRATE AND/OR RECOGNIZE AGGREGATION CURVES WITH DIFFERENT AGGREGATING REAGENTS.


77    EXPLAIN THE PRINCIPLE OF THE PLATELET ADHESIVENESS TEST.

This is an in vitro procedure to evaluate the ability of platelets to adhere to each other and to walls of damaged vessels. Two samples of blood is collected, one specimen collected under standard procedures in an EDTA tube and the other into a similar tube but requiring the blood to pass through a tubing packed with glass beads. If the platelets are normal, the second tube will contain from 40 to 75% of the platelets counted in the first tube. The adhesive nature of the platelets will cause them to adhere to glass beads. The results reported reflect the number of platelets that adhered to the glass beads. Determine the difference between the two values and divide the difference by the count without beads and multiply the result by 1000 to obtain the percentage. Normal values reported are variable; from a low of 26% to a high of 95% platelet adhesion. This test is difficult to interpret. Decreased values are observed in [1] Glanzmann’s thrombasthenia, [2] von Willebrand disease, [3] Chediak-Higashi syndrome, [4] certain myeloproliferative disorders, [5] uremia, and [6] ingestion of drugs and/or aspirin. Increased values are observed in [1] venous thrombosis, [2] pulmonary embolism, [3] carcinoma, [4] pregnancy, [5] splenectomy, and [6] patient’s taking oral contraceptives.

78    DISCUSS THE CIRCULATING ANTICOAGULANT TEST.

Some coagulation problems are due to the presence of inhibitors, something that interferes with the coagulation mechanism, and are not due to a deficiency of a coagulation component. These may develop because of some disease state or an idiopathic problem. Such inhibitors are IgG antibodies (some antibodies are reported of the IgM class). There are two groups of circulating inhibitors:
[1] Specific inhibitors . . . . This group are antibodies and are directed against certain coagulation factors. These often react in a progressive manner and tend to be irreversible. The inhibitors that are seen the most are those directed against factors VIII and IX.
[2] Non-specific inhibitors . . . . These are not directed against any specific coagulation factor. Non-specific inhibitors may not be associated with bleeding. These are immediate in action and will form a protein-protein complex with a specific factor or group of factors. Such complexes are reversible and generally do not destroy the factor(s). The lupus-like group of circulating inhibitors are specific against phospholipids and do not cause bleeding. Another group of inhibitors seen in this category are seen in patients with multiple myeloma or benign paraprotein disorders. Liver disease may result in the development of non-specific inhibitors.

These inhibitors can be detected by the activated partial thromboplastin time (APTT) test and sometimes by the prothrombin time (PT) test. To determine if the abnormal result is due to a coagulation factor deficiency or a circulating anticoagulant or inhibitor, repeat the test by mixing one volume of the patient’s citrated plasma with an equal volume of normal plasma. If the cause is due to a coagulation factor deficiency, the APTT test result will be normal. If circulating inhibitors are present the patient’s plasma will not be corrected and the results will be prolonged. The reason that the patient’s plasma would be corrected if the problem were a coagulation deficiency is that it requires about 50% of the coagulation factors to produce a normal test result.

79    EXPLAIN THE DIFFERENCE BETWEEN AGED AND ABSORBED PLASMA IN COAGULATION STUDIES AND HOW TO INTERPRET THE RESULTS OBTAINED.

Aged serum is obtained by allowing a tube of clotted normal blood to incubate at 37 ⁰C for three hours. One procedure requires that to this tube of blood, add one part of 0.109 M sodium citrate to nine parts of whole blood. Allow the blood to incubate an additional two hours. Centrifuge for ten minutes and remove the serum. The serum is ready to be used. The second procedure requires that the clotted blood be centrifuged without the addition of additives. If stored, store at -20 ⁰C. When using aged serum, dilution may be required. If so, use 0.85% NaCl. Aged serum contains factors VII, IX, X, XI, and XII. Incubation destroys the labile factors.

Absorbed plasma is made by adding 100 mg of barium sulfate to each mL of fresh oxalated plasma (use sodium oxalate). NOTE: Aluminum hydroxide gel may be used but requires fresh citrated plasma, use sodium citrate. Mix the mixture for ten minutes at room temperature, then place in the refrigerator for an additional ten minutes. Centrifuge at 250 rpm for ten minutes and remove the supernatant plasma. Barium sulfate or aluminum hydroxide will absorb out factors II, VII, IX, and X. Absorbed plasma contains factors I, V, VIII, XI, and XII.

To use either absorbed plasma or aged serum, mix equal parts with the patient’s plasma and perform the APTT or PT test. Examine the following table to see how substitution studies might reveal the factor deficiencies.
 N = normal, A = abnormal, and * = testing not required.
                                                                                            Probable
                        ABSORBED PLASMA         AGED SERUM               factor
APTT         PT       APTT         PT          APTT             PT            deficiency

  A             N          N             *              N                *              XI or XII
  A             N          A             *              N                *                 IX
  A             N          N             *              A                *                VIII
  N             A          *              A              *               N                 VII
  A             A          A              A             N               N                 X
  A             A          N              N             A               A                 V
  A             A          *               *              *               *                  II
  N             N                                                                   No deficiency

80     BRIEFLY DESCRIBE THE UREA SOLUBILITY TEST.

The urea solubility test is specific for factor XIII deficiency. It is also called the Factor XIII Screening Test. Routine coagulation screening tests do not detect factor XIII deficiency. To perform this test, add calcium to citrated plasma to form a fibrin clot. Next add 5M urea to the clot and incubate at room temperature for 24 hours. If there is no deficiency, the clot will remain intact. If factor XIII is missing, the clot will dissolve in this period of time. This test will detect a factor XIII deficiency if 98% of the factor is defective or missing.

81    EXPLAIN WHY A PHYSICIAN WOULD ORDER A STYPVEN TIME TEST.

Stypven is Russell viper venom that has thromboplastin-like activity. If this reagent is added to plasma together with platelets and calcium chloride, the coagulation process will begin at the factor X step followed by conversion of prothrombin to thrombin, then fibrinogen to fibrin. This procedure will detect deficiencies in factors I, II, V, and X.

82     EXPLAIN WHY A PHYSICIAN WOULD ORDER A REPTILASE TEST.

Reptilase is an enzyme from the venom of the Bothrops atrox viper and will convert fibrinogen to fibrin. It is not affected by heparin. If a thrombin time is prolonged because of heparin, this test can determine if fibrinogen is functional/normal. The reptilase time and thrombin time will be prolonged in hypofibrinogenemia, dysfibrinogenemia, streptokinase therapy, or when fibrinogen degradation products are present.

83    EXPLAIN WHY A PHYSICIAN WOULD ORDER A FACTOR VIII:C INHIBITOR ASSAY.

There are antibodies that can develop in patients with a factor VIII deficiency. If they develop in such patients, they can provoke serious bleeding episodes. They develop after sensitization by a factor VIII protein transfusion. Some patients will develop high antibody titer (designated as high responders) with repeated transfusions. This is an anamnestic response. Other patients are almost non-responsive when other will demonstrate little or no response in titer. These patients are designated as low responders. Factor VIII antibodies can arise in patients without hemophilia for reasons not understood. Such antibodies can arise in collagen vascular disease, dermatologic disease, drug reactions, multiple myeloma, and Waldenstrom’s macroglobulinemia.

The presence of factor VIII antibodies in non-hemophilic patients can induce a state of bleeding that mimics hemophilia. The clinical course of bleeding is variable, from being very mild to a fatal episode. Bleeding is manifested by dissecting hematomas, epistaxis, hematuria, post-traumatic hemorrhage, post-surgical hemorrhage, and hemarthrosis.

Laboratory findings usually begin with a prolonged APTT (measures all factors except VII and XIII) and an normal PT (measures factors I, II, V, VII, and X). The PT is prolonged in Factor V deficiency.

Treatment is dependent upon the titer of the antibody and it avidity for the factor VIII antigen. Human, porcine, and bovine factor VIII concentrates and vitamin K-dependent factors have been used as treatment strategies. There are risks in using this approach.

84    EXPLAIN WHY A PHYSICIAN WOULD ORDER A EUGLOBULIN CLOT LYSIS TIME.

He probably would not order this test. If he did, it would be to measure fibrinolytic activity. Normal fibrinolysis occurs at a slow rate in the euglobulin system. If a firm clot is present after 90 minutes, the fibrinolysis process is normal. If the clot is lysing and deteriorating in the first 90 minutes, then fibrinolysis is increased and abnormal.

The procedure is simple. Plasma is acidified with cold dilute acetic acid until the solution attains a pH of 5.35 to 5.40. The next step is to refrigerate the mixture for 30 minutes, in which time a precipitate should form that contains factor I, plasminogen, plasmin, and plasminogen activators. This precipitate is the euglobulin fraction. Centrifuge the tubes and decant the supernatant. Resuspend the residue in borate buffer, then add thrombin. Begin timing. Incubate at 37 oC. The clot will form in a short time. Check the clot every ten minutes for lysis. If no lysis after 90 minutes, the test is complete. Report as normal. If the time is shorter, then this indicates increased plasminogen activator activity.

85    EXPLAIN WHY A PHYSICIAN WOULD REQUEST A TEST TO DEMONSTRATE THE PRESENCE OR ABSENCE OF FIBRINOGEN DEGRADATION PRODUCTS.

Fibrinogen degradation products (FDP), also called Fibrin Split Products (FSP) are observed in patients with acute and chronic disseminated intravascular coagulation, alcoholic cirrhosis of the liver, surgical complications, primary fibrinolysis, late pregnancy, deep vein thrombosis, and pulmonary embolism. The very presence of FSP in these examples indicate that a plasmin release has occurred or is occurring. Pathologic degradation of fibrinogen and fibrin are the result of increased plasmin activity and sets the state for abnormal secondary bleeding. FDP’s are significant because that they have hemostatic effects (includes antithrombin activity and interference with platelet activity and fibrin monomer polymerization) and can be life threatening. Fibrinogen fragments into X, Y, D, and E fragments and other low-molecular weight products. It is the X and Y fragments that exert the anticoagulant effects by polymerizing with fibrinogen. The Y and D fragments obstruct the polymerization of fibrin which results in soluble fibrin formation. The E fragments is inhibitory to thrombin. In addition, all the fragments are capable of adhering to platelet surface membranes and causing platelet dysfunction with poor aggregation properties. These complexes can be detected by the protamine sulfate test, ethanol gel test, and latex FDP assay.

86    DIFFERENTIATE BETWEEN PRIMARY AND SECONDARY FIBRINOLYSIS.

Primary fibrinolysis (PF), a rare event, is the degradation of fibrinogen. This is a pathological state characterized by increased amounts of plasminogen activators, increasing the amount of plasmin in circulation. This results also in the splitting apart of factors V and VIII. The fibrinogen degradation products form fibrin like threads slowly and require about 24 hours for the evidence of “clotting” to appear. This is not a common occurrence but appears when damaged cells appear or malignant cells (as in prostate cancer) are present, surgery, shock, acute leukemia, or cirrhosis of the liver.

Secondary fibrinolysis (SF) occurs in conditions like disseminated intravascular coagulation (DIC) where there is deposition of fibrin. Secondary fibrin degradation products tend to polymerize earlier, requiring about 30 minutes to become observable.

There are four tests that will differentiate between these two types of fibrinolysis.
[1 ]  The Euglobulin Clot Lysis Time. It is significantly decreased in primary fibrinolysis.
[2]   Platelet count. It is increased in PF (>100 X 109/L). In SF the platelet count is usually less.
[3]   Antithrombin III assay. Levels will be normal in PF, but decreased in SF.
[4]   D-dimer test will be negative in PF, but positive in SF.

87    EXPLAIN THE PRINCIPLE BEHIND THE PROTAMINE SULFATE DILUTION (PSD) TEST.

Fibrin degradation products (FDP) are soluble entities that interfere with the normal coagulation process. If protamine sulfate is added to plasma containing FDP’s, then a variation of a phenomenon known as paracoagulation takes place. This means that the various FDP’s will begin to dissociate from fibrin and other sites and will polymerize in the presence of protamine sulfate to form a gelatinous button. The button is comprised of X and YY fragments. Firbrin monomers will also join this odd polymerization phenomenon. Note if FDP are present in large quantities, the test will not work well. The protamine sulfate (also true for ethanol) will displace the small FDP’s resulting in spontaneous polymerization. For this reason the protamine sulfate dilution test and the ethanol gelatin test can be used to tests for FDP’s. The better testing technique is Agarose gel chromatography. Caution: the gel is sometimes difficult to observe. Laboratorians who read the PSD test require experience. False positives occur in females before and after menses and in patients with advanced cirrhosis. This test does not detect just fibrinogen degradation products. If fibrinogen is present in large quantities, it tends to precipitate as an amorphous mass which occurs as large FDP complexes with fibrinogen. This is not a gelatinous clot.

By definition, paracoagulation occurs when thrombin splits fibrinogen into fibrin monomers. The fibrin monomers will start form a fibrin mesh (development of the clot). The fibrin monomers will bind to fibrinogen and or fibrin split products to form a precipitate. The end product is not a typical clot but an amorphous, gelatinous-like mass.

88   EXPLAIN THE PRINCIPLE BEHIND THE ETHANOL GELATION (GEL) TEST.

Fibrin monomers can be precipitated in plasma by the addition of 0.15 mLs (3 drops) of 50% ethanol to 12 drops of platelet poor plasma (PPP) that has been pretreated with one drop of 0.1 N NaOH (to raise the pH to 7.7 or higher). The ethanol is layered over the plasma (do not mix) and allowed to sit at room temperature. Look for a line of precipitation or gel at the interface. If either of these are present, it is a positive test for the presence of soluble fibrin monomers. This is an indicator of a disseminated intravascular clotting (DIC) problem, pulmonary emboli, deep vein thrombosis, acute myocardial infarction, eclampsia, postpartum hemorrhage, malignant tumors, pre-eclampsia, renal failure, liver cirrhosis, surgical complications, etc. If you observe an amorphous (without form) flocculation, the test is negative. If nothing happens after one minute, the test should be allowed to sit for an additional nine minutes. If strands or gel appears after ten minutes, add one drop of 0.1 N NaOH. If a precipitate appears and disappears, ignore it . . . . this particular phenomenon is of questionable significance and should be considered as a negative result. If the precipitate or gel persists, then the test is positive. The presence of the precipitate or gel is called paracoagulation. The procedure is considered to be more sensitive that the protamine sulfate dilution test and it is equally difficult to interpret. COMMENT: [1] If the fibrinogen level is >400 mg/dL, the test will be a false positive. [2] Do not use oxalate anticoagulants as they adversely affect the pH. [3] The presence of heparin and/or erythrocytes will not affect the test results.

89    DESCRIBE THE PRINCIPLE OF OPERATION OF THE FIBROMETER.

It is a modified manual technique that uses electromechanical means of detecting the clot. It uses two wire electrodes on a probe arm, one of which has a back-and-forth (0.5 second) sweeping action through the reaction mixture. When the fibrin clot forms, it is engaged by the electrodes, and electrical contact occurs and the timer is stopped. Timing is initiated automatically via its pipet or a manual switch. Its precision is ± 0.5 seconds. It is no longer a primary instrument in the laboratory, giving way to the optical detection instruments. Its role in the laboratory is that of a back-up instrument. It does have one advantage over the optical instruments, it can detect fibrinogen levels <50 mg/dL

90   LIST FIVE SOURCES OF ERROR THAT OCCUR WITH THE ELECTROMECHANICAL METHOD OF CLOT DETECTION.

[1]   Possibility of transferring activated coagulation factors on the probe’s electrode into the next patient’s sample and shortening of the clotting time.
[2]   Use of incorrect reaction wells or cups.
[3]   Technical error on the part of the laboratorian.
[4]   Incorrect collection of the specimen.
[5]   Expired reagents.

91   DISCUSS THE OPERATION PRINCIPLE OF THE PHOTO-OPTICAL COAGULATION INSTRUMENT, SUCH AS THE COAG-A-MATE, MDA, OR MLA.

These instruments come in a variety of models and use a photocell to detect the fibrin clot. They have an advantage over the electromechanical detection systems in that the photo-optical detector can detect a rapid and significant increase in light transmission. Accuracy is expected to be ± 0.1 seconds. The specimen and reagents are pipetted into the reactions wells in such a way that maximum mixing is obtained. There is no moving probe to mix the system as required in the fibrometer. Accuracy of reagent and specimen delivery is maintained by sensor systems that cause the probe(s) to make minimal contact with the fluids. The technology and sensitivity of these instruments permits detecting clots in chylous and icteric patient specimens.

In detecting the forming clot, at the onset of the test, the light is transmitted through the reaction mixture with little scattering. As the reaction proceeds and more and more fibrin is incorporated into polymers, then into the clot, the amount of light scattering increases proportionally. This creates an electrical signal that is analyzed by algorithms. At some point in the reaction, the signal becomes such that the instrument determines that the clot has formed. A timer is linked into the reaction which is turned on when the plasma is added to the reagents. A detection system also turns on at the same time and it will turn off the timer when the formation of the clot occurs.

An algorithm is a series of algebraic equations. This means that it is an ordered sequence of steps. Each step must be completed before the next step can proceed. This mathematical strategy is programmed into a computer as a series of mathematical progressions to enable it to use the degree of light scattering as a means to determine the completion of the clot.

92    LIST SEVEN SOURCES OF ERROR THAT MAY OCCUR IN THE PHOTO-OPTICAL DETECTION COAGULATION SYSTEM.

[1]   The possibility of a rippling optic effect that occurs when reagents and specimen are added to the reaction chamber and causing the instrument to report a shortened clotting time. This effect is eliminated by a “guard interval” that delays the end-point detection that is built into the test procedure. During this time the instruments sets up a base line for transmitted light.
[2]   Some very abnormal specimens (those that are very lipemic, icteric, or hemolyzed) may cause erroneous clotting times.
[3]   Patient’s with fibrinogen levels < 50 mg/dL.
[4]   Patient specimens containing elevated heparin concentrations.
[5]   Precipitation of cryoglobulins if present in large amounts in plasma that is allowed to sit at room temperature.
[6]   Incorrect collection of the specimen.
[7]   Expired reagents.
[8]   Laboratorian error.

93   LIST FIVE SOURCES OF ERROR TO AVOID IN COAGULATION REAGENT USE.

[1]   Leaving caps/lids off of reagent bottles allowing evaporation to occur and changing the concentration of the reagent.
[2]   Ignoring expiration dates.
[3]   Changing reagent lots and not re-evaluating appropriate control and reference ranges.
[4]   Improper shipping, storage, and handling of reagents from the manufacturer to the vendor to the customer.
[5]   Failure to reconstitute reagents to manufacturer’s specifications.
[6]   Improper storage temperatures in the laboratory.

94   LIST NINE STEPS IN ESTABLISHING A POPULATION REFERENCE RANGE FOR COAGULATION TESTING.

The criterion for the establishment of a population reference range is the same for hematology and chemistry testing. Remember that commercial controls are not permitted in establishing this range. The purpose of establishing this specific range is to make accurate and unbiased generalizations about the sample taken from the patient. The nine steps are:
[1]  Use a minimum of forty subjects. One hundred is ideal.
[2]  Select healthy volunteers.
[3]  Volunteers must represent the demographics of the region in age, gender, race, etc.
[4]  Specimens collected from the volunteers must be handled the same identical way as that for a patient’s specimen.
[5]  Specimens should be collected and tested the same day.
[6]   Use the same reagents and instrumentation as used for patients.
[7]   Perform a frequency distribution histogram to determine if the sample population falls within the limits of the Gaussian curve.
[8]   Investigate the reason for any results designated as “outliers”.
[9]   Perform statistical calculations to establish standard deviations.

95. DISCUSS PROFICIENCY TESTING IN THE COAGULATION LABORATORY.

This is a quality control program for interlaboratory testing to evaluate the coagulation results. By comparing the test results between the patient and the controls, the laboratory can access their performance to that of other laboratories. This increased the reliability of the test results reported to the physician. When comparing results with other laboratories, use those laboratories that use the same testing instruments and reagents used in your laboratory. In coagulation testing, the prothrombin time (PT) test and the activated partial thromboplastin time (APTT) test are dependent upon the reagent system.

Reviewed and Revised.