M B ‘ r".Indamental The F” iagnostic i Bleeding ‘ ematologyiv and - Clotting Disorders ** **** FUNDAMENTAL DIAGNOSTIC HEMATOLOGY THE BLEEDING AND CLOTI'ING DISORDERS (Second Edition) Bruce L. Evatt, M.D.* William N. Gibbs, M.D., F.R.C. Path.“ SM. Lewis, M.D., F.R.C. Path. James R. McArthur, M.D.**** Chief, Hematologic Diseases Branch, Division of HIV/AIDS, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia, USA \ Chief Medical Officer, Health Laboratory Technology and Blood Safety Unit, World Health Organization, Geneva, Switzerland Consultant Hematologist and Emeritus Reader in Hematology, Royal Postgraduate Medical School and Hammersmith Hospital, London, United Kingdom Professor of Medicine, Attending Physician and Hematology Consult- ant, University of Washington School of Medicine, Seattle, Washington, USA PLAbl U.S. Department of Health and Human Services Public Health Service Centers for Disease Control Atlanta, Georgia 30333 Published jointly by: and World Health Organization Geneva, Switzerland 1 9 9 2 ii a PREFACE ? P; "a. < The first edition of this manual was published in 1985. In preparing this 8 (.0 second edition, we have taken into account the rapid expansion of the field of hemostasis and the attendant advances in knowledge of the pathophysiology of bleeding and thrombosis. New and updated methods have been included where necessary, while retaining the essential requirement that the manual be designed for use in intermediate laboratories whose facilities are restricted to basic tests. The manual is also intended to help both laboratory staff and physicians deal with patients with bleeding and thrombotic disorders. Sec- tions dealing with diagnosis and management of thromboembolic disease have been included and the title has been changed to reflect this. The importance of quality assurance to ensure reliable results has been emphasized. The chapter on this topic has been extensively revised, and procedures are described for internal quality control. These procedures are easily practiced in any laboratory. Increased awareness of the danger of blood—borne infectious diseases and other hazards has led to the inclusion of a chapter on laboratory safety. As in the last edition, we have received much appreciated comments and encouragement from various colleagues, including members of the Intema— tional Council for Standardization of Haematology, the International Society of Haematology, and the World Health Organization Regional Offices. We are especially grateful to Drs. J .F. Davidson, J.C. Giddings, R.A. Hutton, and L. Poller, who critically reviewed the draft and gave helpful advice on correcting errors and omissions. The authors, however, accept responsibility for the final publication. For this edition, we also received valuable technical assistance from Ron Nuse, Jennifer Cyphers, and the Technical Information Activity, Division of HIV/AIDS, National Center for Infectious Diseases, CDC. In writing this manual the authors have included descriptions of selected laboratory methods that have appeared in standard reference books and in journals. These have not been given specific references in the text and readers are referred to the following sources of information: Coleman RW, Hirsh J, Marder VJ, et al. Hemostasis and thrombosis: basic principles and clinical practice. 2nd ed. Philadelphia: JB Lippincott Company, 1987. Sirridge MS, Shannon R. Laboratory evaluation of hemostasis and thrombosis. 3rd ed. Philadelphia: Lea & Febiger, 1983. Triplett DA, Harms CD. Procedures for the coagulation laboratory. Chicago: American Society of Clinical Pathologists, 1981. Triplett DA. Hemostasis: a case oriented approach. Tokyo: Igaku-Shoin Ltd, 1985. Bloom AL, Thomas DP. Haemostasis and thrombosis. 2nd ed. Edinburgh, New York: Churchill Livingstone, 1987. iii WW/ Contents PREFACE ............................................................................................................................... iii 1 INTRODUCTION TO HEMOSTASIS ................................................................................. 1 Normal Hemostasis, 1 The vascular component of hemostasis, 1 The platelet component of hemostasis: formation of the primary platelet plug, 3 The coagulation factor component Of hemostasis, 5 The extrinsic system, 5 The intrinsic system, 6 Normal Control of the Clotting Process and Fibrinolysis, 8 Pathologic Hemostasis, 8 Abnormal vascular component of hemostasis, 8 Abnormal coagulation factor component and inhibitors Of hemostasis, 13 Abnormal platelet component of hemostasis, 9 Abnormal coagulation factor component and inhibitors of hemostasis, 12 2 CLINICAL ASPECTS OF BLEEDING AND THROMBOTIC DISORDERS ............ 19 Bleeding Disorders, 19 Approach to the patient, 19 Thrombotic Disorders, 20 3 LABORATORY ASPECTS OF BLEEDING AND THROMBIC DISORDERS ......... 25 Introduction, 25 Approach tO the Patient with Thrombotic Disorders , 29 Disseminated Intravascular Coagulation (DIC), 31 Hemostatic Problems Associated with Surgery, 31 Hematologic Findings in AIDS Patients, 32 Hemostasis, 33 4 QUALITY ASSURANCE .................................................................................................. 35 General Considerations, 36 Reference and Calibration Preparations , 36 The Use of Control Specimens, 36 Standard deviation, 37 Calculation Of the standard deviation, 38 Coefficient Of variation (CV), 39 Preparation of a control chart, 39 Accuracy, 40 External Quality Assessment (EQA), 40 Calibration, Care, and Maintenance of Laboratory Instruments, 41 Special Aspects of Quality Control for Coagulation Studies, 44 Controls, 49 Reference Values (Normal Values), 49 Summary, 50 5 LABORATORY SAFETY .................................................................................................. 51 Microbiologic Safety, 51 Exposure to Toxic Chemicals, 55 Injuries, 55 Electrical Hazards and Fires, 55 Management of Injuries and Exposures, 56 6 GENERAL PROCEDURES ............................................................................................... 57 Blood Sampling, 57 Collecting Capillary Blood for Blood Films, 57 Collection of Venous Blood for Coagulation Studies, 57 Blood Films, 58 Observation of the Clot, 64 Platelet Counts, 65 Bleeding Time, 70 Measurement of the Extrinsic System, 76 Prothrombin time (one-stage), 76 Measurement of the Intrinsic System, 79 Activated partial thromboplastin time (APTT), 79 Inhibitors, 80 Fibrinogen (Clauss method), 81 Erythrocyte Sedimentation Rate (ESR), 86 7 SPECIALIZED PROCEDURES ......................................................................................... 89 One-stage Factor VIII Assay, 89 Factor Assays in the Extrinsic Pathway, 92 Euglobulin Lysis Test, 92 Factor XIII, Qualitative, 94 Vitamin K1 Test, 95 y 8 CONTROL OF ANTICOAGULANT THERAPY ........................................................... 98 Oral Anticoagulants, 97 Heparin Anticoagulation, 101 APPENDIX ........................................................................................................................... 105 GLOSSARY OF TERMS .................................................................................................... 109 INDEx ................................................................................................................................... 1 15 V CHAPTER 1 INTRODUCTION TO HEMOSTASIS The purpose of this manual is to help laboratory staff, physicians, and other health—care workers diagnose hemostatic disorders. This introductory chapter presents a simple outline of normal hemostasis and the causes of abnormal bleeding or clotting. Hemostasis is the maintenance of blood flow within the vascular system and involves the interaction of vessels, platelets, and coagulation factors. Bleeding and clotting disorders are the result of the failure of hemostatic mechanisms. The vessel wall is important in maintaining hemostasis. In small vessels, vasoconstriction plays an early role in achieving hemostasis. When the vascular endothelium is lost, platelets will adhere to the exposed collagen, microfibrils, and basement membrane. Once platelets adhere to the subendo- thelial tissue, they release a variety of mediators including adenosine diphos— phate (ADP), serotonin, epinephrine, and prostaglandin derivatives - espe- cially thromboxane A2. Some of these mediators promote vasoconstriction, and all attract other platelets to form an aggregated mass called the primary platelet plug. Platelet factor 3, a surface phospholipid, is exposed during platelet plug formation. It potentiates clotting by accelerating the formation of thrombin. The coagulation of blood is a series of enzymatic reactions involving plasma proteins, phospholipids, and calcium ions that transform the circulat- ing whole blood into an insoluble gel by trapping it within a network of fibrin. This fibrin network extends and anchors the evolving thrombus. The proteins involved in the coagulation process act as an amplification system that allows the very small initiator product of a few molecules to induce a whole series of proteolytic reactions. Coagulation factors are listed in Table 1. The sequence of steps by which clotting proceeds is described below (p. 6). NORMAL HEMOSTASIS The vascular component of hemostasis The blood vessel wall is the first line of defense for normal hemostasis. The vessel is supported by subendothelial tissue, which maintains the vascular integrity of the structure. Vessels are lined with endothelial cells, which form a tight, selective membrane that keeps blood cells and plasma inside the vessel while simultaneously allowing needed gases (e.g., 02 and C02), nutrients, and selected cells to enter or leave the system. In the normal state this lining also produces prostacyclin and other substances that inhibit platelet function. Nerve and muscular tissue in the supporting subendothelial tissue allow 2 The Bleeding and Clotting Disorders Table 1 - Coagulation factors and their synonyms Factor Synonyms I Fibrinogen II Prothrombin III Thromboplastin, Thrombokinase, Tissue Extract IV Calcium V Labile Factor, Proaccelerin, Plasma Accelerator Globulin, Plasma Ac-Globulin VI Not assigned VII Stable Factor, Proconvertin, Autoprothrombin l, Serum Prothrombin VIII Antihemophilic Factor, Antihemophilic Globulin Platelet Cofactor I, Thromboplastinogen, Antihemophilic Factor A IX Plasma Thromboplastin Component, Christmas Factor, Platelet Cofactor ll, Autoprothrombin ll, Antihemophilic Factor B X Stuart Factor, Prower Factor, Stuart-Prower Factor, Autoprothrombin Ill XI Plasma Thromboplastin Antecedent, Antihemophilic Factor C XII Hageman Factor, Surface Factor, Contact Factor, Clot-Promoting Factor XIII Fibrin-Stabilizing Factor, Fibrin-Stabilizing Enzyme, Fibrinase, Laki-Lorand Factor Prekallikrein Fletcher Factor High Molecular Weight Kininogen Fitzgerald Factor 3 constriction of the vessel when injured. The constriction slows or stops the blood flow from an acute wound. This effect is temporary and needs to be supplemented by platelets and blood coagulation factors to maintain perma— nent hemostasis of the injury. The platelet component of hemostasis: formation of the primary platelet plug Platelets are cytoplasmic fragments of megakaryocyte mother cells. They range in size from less than 5 femtoliters (fl) to more than 12 fl; however, the average platelet is about 7.3 fl. To exert normal functions, platelets have: - receptors on their surface to bind substances like ADP, collagen, von Willebrand factor, fibrinogen, and other coagulation factors; - dense granules in the cytoplasm, which contain ADP and serotonin (storage pool); - microtubules and a canalicular system to allow release of ADP and serotonin from the storage pool; - a membrane component called “platelet factor 3.” In vivo the platelet is shaped like a porous disc and resembles a microscopic sponge (Figure 1). The invaginations of the surface membrane into the center of the platelet provide canals through which components within granules of the platelet escape into the surrounding plasma during the release reaction. The platelet is covered by a plasma protein coat that is the mediator for the platelet membrane’s adhesion to surfaces and allows coagulation proteins or factors to adhere to it. Neither adhesion of platelets to surfaces nor to proteins requires energy, but each requires fibrinogen. The membrane beneath the surface coat contains phospholipids and selectively absorbs certain coagulation factors. Below the coat and membrane are submembranous filaments made of actomyosin that cause the platelet to contract. The equator of the disc-shaped platelet contains a prominent structure composed of microtubules that maintain the platelet’s disc—like shape. After the release reaction begins, the microtubules constrict concentri- cally. This process moves the clusters of organelles and granules toward the center of the platelet. The granules are important because they contain several constituents that take part in the release reaction, i.e., ADP, catecholamine, and serotonin. Energy for these reactions is derived primarily from glycogen storage granules, and the platelet is capable of both aerobic and anaerobic metabolism. Platelet production is regulated to meet the demand for circulating platelets by growth factors that are as yet not clearly defined. One of these growth factors is thought to be “thrombopoietin” but several others may play a crucial role. The growth factors promote the development of megakaryocytes within the bone marrow. As the megakaryocytes mature, the cytoplasm becomes 4 The Bleeding and Clotting Disorders Surlace coat Plasma membrane Submembranous filaments Microtubules Dense tubular system Specific granule Glycogen Electron dense granule nu .. .. - Km Figure 1 - Diagram of platelet structure demarcated into platelet subunits. Platelets are released into the circulation through a process of megakaryocyte fragmentation, and have a lifespan of 7- 10 days. Normally, two—thirds of the platelets released from the bone marrow stay in the general circulation. The remainder are in a pool in the spleen that is freely exchangeable with circulating platelets. With progressive spleno— megaly, a larger percentage of the body’s platelets are pooled in the spleen and, as a result, peripheral thrombocytopenia may develop unless the bone marrow can increase platelet production sufficiently to compensate. Spleno- megaly by itself does not lead to a shortened platelet survival. In general, there is a direct relationship between the megakaryocyte mass in the bone marrow and the rate at which platelets are added to the circulation. This relationship breaks down when megakaryocyte development is faulty or when megakaryocytes are destroyed within the bone marrow. In these circumstances platelet delivery to the circulation is impaired, and thrombo- poiesis is ineffective. Ineffective thrombopoiesis is a frequent finding in patients with vitamin B12 or folate deficiency and is analogous to the production problem of red cells that occurs in these conditions and gives rise to ineffective erythropoiesis. When maximally stimulated, the bone marrow can increase platelet production six-fold. However, when platelets are destroyed very rapidly increased delivery to the peripheral blood will not occur for approximately 5 days. This results in transient thrombocytopenia, which may persist if the rate of platelet production cannot keep up with the rate of platelet destruction. In hemostasis, platelets play three main roles: I. They maintain the endothelial surface. Loss of circulating platelets quickly results in morphologic Changes in the endothelial cells of the capillaries. These changes cause intravascular material to leak into the capillary bed. 2. They initially arrest bleeding in severed blood vessels. 5 3. They provide phospholipid, which acts as the catalytic surface for initiation of the coagulation process. When they encounter a break in the endothelial surface, several important actions of the platelets normally cause the bleeding to cease (Figure 2). 1. Adhesion occurs when they encounter collagen, membranes, or noncollagenous microfibrils beneath the basement membranes. 2. They then undergo a “release reaction”. This process involves change from a disc shape to a spherical shape, constriction of the microtubules toward the center of the platelet, and the release of contents of the granules (primarily ADP, catecholamine, and serotonin) into the open canalicular system. 3. Platelet aggregation occurs as platelets are “recruited” from the immediate area by the released contents, like ADP. When ADP release is minimal or the ADP concentration does not reach a high level locally, this aggregation may be reversible; with higher concentra- tions, aggregation is irreversible. 4. Associated with the change of shape of the platelet and the release reaction is the appearance of platelet factor 3 on the platelet mem- branes. Platelet factor 3 serves as a catalytic site for the clotting proteins and helps initiate the clotting mechanism (Figure 2). 5. Clot retraction occurs when platelets are trapped within the enlarging blood clot. The coagulation factor component of hemostasis The primary platelet plug, by itself, is only a temporary seal, and the formation of the clot is necessary to secure the repair of the damaged vessel. The sequence of steps by which clotting proceeds is shown in Figure 3. There are two pathways: the extrinsic system and the intrinsic system. These systems reflect how clotting occurs in the test tube during tests. Clotting in the body is initiated differently. However, it is clinically useful to think about the clotting process in terms of these two systems because the laboratory worker can direct investigation towards only a few tests. The extrinsic system The extrinsic coagulation system (measured by the prothrombin time) is initiated when factor VII is activated by a combination of calcium ions and tissue thromboplastin. Once activated in the presence of calcium, factor VII in turn activates factor X to Xa. When factor Xa has been formed, activation can proceed down the common pathway of both the intrinsic and extrinsic system as follows: factor V binds factor Xa and factor II in the presence of a phospholipid surface so that factor Xa can cleave factor II to form thrombin. The final step in this series of reactions is proteolytic conversion of fibrinogen to fibrin (measured clinically by the thrombin time). 6 The Bleeding and Clotting Disorders ENDOTHELIALCELL ‘— ENDOTHELIAL CELL Figure 2 - Platelet functions in vivo For this reaction, thrombin splits off fibrinopeptides A and B from fibrinogen. After the removal of the fibrinopeptides, fibrin monomers un- dergo spontaneous polymerization by hydrogen bonding to form a network. This network is then stabilized by factor XIII. The cross-linked fibrin is not only far more insoluble than the fibrin polymer, it is also more resistant to digestion by plasmin. Abnormalities of this system (i.e., absence or abnor- malities of factors VII, X, V, II, or fibrinogen, and inhibitors of the conversion of fibrinogen to fibrin) produce an abnormal prothrombin time. Similarly, abnormalities of fibrinogen and inhibitors of the conversion of fibrinogen to fibrin result in an abnormal thrombin time. The intrinsic system In the test tube, the initiation of clotting via the intrinsic system (measured by the partial thromboplastin time) begins with the activation of factor XII when it is exposed to the glass surface. This surface activation of factor XII —_ THE CLOTTING SEQUENCE Intrinsic System Extrinsic System Hogh Molecular Weight KIninogen Contact l Surface X” ' Xlla Kallikrein Prekallikrem l XI -—>Xla Ca”; IX m Tissue Factor (IIII I VII VIII ca” Phosphol-pId I (Platelets) Ca” ‘ PamaI Thromboplastin Tune X ——> Xa <————— X V Phospholipld (Platelets) Ca++ Prothrombin Time XIII Prorhrombin (ll) Thrombin FIbrInogen III __L_> FIbrin 3 Ca“ Xllla ThrombIn Time Stabilized Fibrin Figure 3 - The clotting sequence depends upon two other molecules found in plasma, high molecular weight kininogen (Fitzgerald factor) and prekallikrein (Fletcher factor). Of the two, prekallikrein appears to be more important. When the glass surface combines with factor XII, a conformational change in the molecule occurs that allows factor XII to be more susceptible to the proteolytic action of prekallikrein. High molecular weight kininogen attaches factor XI to the surface in close proximity to factor XII. Although factor XII, prekallikrein, and high molecular weight kininogen are all essential for normal clotting in the test tube, their biologic purpose for clotting is uncertain. Persons who are deficient in these factors have no significant bleeding disorders. In the body, numerous substances such as collagen, fatty acids, or joint cartilage can activate factor XH. Factor X1 is activated by factor XIIa and, in turn, activates factor IX. Factors IXa and VIII form a complex that leads to the activation of factor X. Factor VIH probably serves as a regulatory or organizing protein so that factor IXa can become optimally bound to factor X for proper proteolysis to occur. In vivo a phospholipid requirement is probably provided by platelets. The product of this reaction (IXa, VIII, and X) is Xa. Factor Xa forms the crossroads of the two clotting systems by participating in both the extrinsic and the intrinsic 8 The Bleeding and Clotting Disorders pathways. Once factor Xa is formed, activation can proceed down the common pathway as described above. Abnormalities of any of the following clotting factors produce an abnormal partial thromboplastin time: high molecular weight kininogen, prekallikrein, factors XII, XI, X, IX, VIII, V, II, or fibrinogen. NORMAL CONTROL OF THE CLOTTING PROCESS AND FIBRINOLYSIS If the clotting process is not self-limited, abnormal clotting is produced. During the normal hemostatic mechanism, the process of limiting the clot occurs through several mechanisms. The most important are (1) removal of activated clotting factors by blood flow past the clot, (2) inactivation of clotting factors by circulating inhibitors (e. g., antithrombin III and protein C), (3) consumption of platelets and clotting factors by the clotting process, and (4) degrading the clot by the fibrinolytic enzyme, plasmin. Of these processes, the plasminogen-plasmin system and the circulating inhibitors are recognized as being important clinically. During the clotting process, the precursor (zymogen) of plasmin (plas- minogen) is bound to the forming fibrin network by attaching to specific receptor sites on the forming fibrin strands. Circulating tissue plasminogen activator and other plasminogen activators cleave the plasminogen to form plasmin. This activated plasmin begins degrading the fibrin strands and causes the clot to dissolve. The fibrinolytic process is illustrated in Figure 4. Recently, protein C has been identified as an important factor in the regulation of hemostasis. Protein C is a vitamin K-dependent factor that acts as an anticoagulant when activated. Its mechanism of action is the inactivation of factors V and VIII--b0th in their native and active forms. Before protein C can exhibit its anticoagulant effect, it must be activated. It is activated by thrombin, but only after the thrombin has been modified by a protein called thrombomodulin, found on the surface of endothelial cells. Once thrombin has been modified, it can activate protein C, but can no longer convert fibrino gen to fibrin. Protein S is an additional vitamin K-dependent factor that serves as a cofactor for activated protein C in its capacity as an inactivator of factors V and VIII. Protein S does not require activation to serve as a cofactor. PATHOLOGIC HEMOSTASIS Abnormal vascular component of hemostasis The integrity of the vessel wall is important for maintaining hemostasis. Bleeding may be due either to a weakness in the structure of the vessel or abnormalities in the surrounding structures. When either of these conditions exist, the vessel may be unable to contract in response to injury. Processes that can affect the vessel wall include: ascorbic acid deficiency, inflammation, A PLASMINOGEN B. 0- FIBRlN PLASMIN Figure 4 - Fibrinolytic system immune damage, certain toxins, aging, and congenital defects. In these conditions, bleeding into the skin produces ecchymoses called “vascular purpura.” Abnormal platelet component of hemostasis Bleeding may occur if A) the platelet count is low (thrombocytopenia) or B) if the circulating platelets do not function normally. Both of these conditions, which may be either acquired or congenital, are described below. A. Decreased number of platelets (Thrombocytopenia) Low platelet counts develop because of decreased production, ineffective maturation, increased destruction or utilization, or pooling of platelets. These conditions are as follows: 1. Decreased production of platelets may be due to marrow depression (hypoplasia); marrow replacement by tumor or malignant blood disease; immune damage from toxins, drugs, and bacterial and viral infections; and unidentified (idiopathic) causes. 2. Ineffective maturation of platelets in the cytoplasm of developing megakaryocytes occurs in megaloblastic anemia, and in myeloprolif— erative and myelodysplastic disorders. 10 The Bleeding and Clotting Disorders 3. Increased destruction or increased utilization of platelets. In this situation the production of platelets from the marrow is normal or even increased, but the marrow is unable to compensate adequately for the increased rate of destruction. This increased rate may occur as a direct toxic effect, by an autoimmune process, by the presence of infection, or in disseminated intravascular coagulation, a disorder in which there is increased utilization of platelets. In severe hemorrhage, platelets are actually lost from the circulation, and the problem can be compounded by massive transfusion with stored banked blood, which contains no viable platelets. The spleen plays an important role in platelet destruc- tion. It may be responsible for increased production of antiplatelet antibodies, and it is also the site where platelets are pooled. Heparin therapy can induce thrombocytopenia. In many patients the thrombocytopenia appears to be immunologically mediated, while in others it is not. Heparin-induced thrombocytopenia appears to occur less frequently with heparin of hog mucosa] origin than with heparin derived from beef lung. Severe heparin-induced thrombocytopenia may be associated with acute arterial thrombosis. Thrombotic thrombocytopenic purpura presents acutely with throm- bocytopenia, microangiopathic hemolytic anemia, transient fluctuating neurologic signs and symptoms, renal insufficiency, and fever. This disorder may result from antibody-mediated damage to the vascular endothelium or the lack of a plasma factor that inhibits platelet aggregation. It can be fatal despite a variety of therapies. 4. Pooling of platelets. When the spleen is distinctly enlarged, it can pool without destroying a disproportionate amount of the circulating plate- lets. This pooling results in a lowering of the platelet count in the peripheral blood. B. Disorders of platelet function These occur when one or more of the five normal platelet functions is impaired. A problem with platelet function is usually suggested when the bleeding time is prolonged to a greater extent than expected from the patient’s platelet count. The dysfunction may be either acquired or inherited. 1. The acquired platelet disorders a. Many drugs induce platelet dysfunction. Aspirin, which causes this more frequently than other drugs, interferes with the function of all the platelets that were exposed to it at any time during their entire lifespan. Other nonsteroidal anti-inflammatory drugs also inhibit platelet activ— ity; these include indomethacin, butazolidin, and sulfinpyrazone. Aspirin and the other nonsteroidal anti-inflammatory drugs inhibit platelet function by inhibiting platelet prostaglandin synthesis. Aspi- rin inhibits the enzyme cyclooxygenase, which converts arachidonic acid to a cyclic endoperoxide, which is then converted to thromboxane A2, a mediator of the platelet release reaction (Figure 2). Aspirin 11 permanently inhibits this enzyme by acetylating it; the other nonsteroi- dal anti-inflammatory compounds reversibly inhibit it. Miscellaneous platelet function inhibitors include carbenicillin, dipyridamole, clofibrate, phenothiazines, tricyclic antidepressants, and certain gen— eral anesthetics. Plasma expanders, such as dextran or hydroxyethyl starch, also interfere with platelet function, probably by coating the platelet. b. Acquired platelet dysfunction has been frequently associated with dysproteinemias such as Waldenstrom’s macroglobulinemia and multiple myeloma, which result in platelet coating, and with uremia. A variety of metabolic products accumulate in the circulation of patients with uremia. Many of these have been associated with platelet dysfunction. One of the more important compounds appears to be guanidinosuccinic acid. c. Acquired platelet dysfunction may also occur in association with some disorders in which there are high platelet counts (thrombocyto- sis). Thrombocytosis is defined as platelet counts above 450 X 109/L. When the thrombocytosis represents a primary disorder of the bone marrow, the problem is called thrombocythemia. Patients with thrombocythemia usually have a platelet count in excess of 1000 X 109/L. The platelets usually appear large and abnormal on the blood film. The bleeding time is frequently prolonged, and platelet aggregation may be abnormal. Platelet production in this disorder is autonomous, and thrombocythemia may be a part of other myelopro- liferative disorders, including chronic myelogenous leukemia, primary proliferative polycythemia (polycythemia vera), and myelofibrosis (agnogenic myeloid metaplasia). Patients with thrombocythemia have both arterial and venous thrombotic problems as well as hemorrhagic complications. The serum potassium may be artificially elevated because of the large amount of potassium released during clotting from the increased numbers of platelets. However, plasma potassium concentration is normal. It is important to differentiate thrombocythe- mia from secondary thrombocytosis. Patients with secondary thrombocytosis usually have platelet counts of greater than 500 X 109/L and less than 1000 X 109/L, and typically platelet morphology is normal on the blood film. Increased numbers of megakaryocytes are usually seen in the bone marrow, but they are decreased in size. Secondary thrombocytosis is associated with a wide variety of conditions, including acute and Chronic bleeding, acute and chronic inflammatory disorders, iron deficiency anemia, the anemia of chronic disease, hemolytic anemia, lymphomas (including Hodgkin’s disease), and carcinomas. Thrombocytosis can transiently result after a splenectomy and as a rebound phenomenon after myelosuppression induced by cancer chemotherapy or radiation therapy. Thrombotic 12 The Bleeding and Clotting Disorders problems are not common, although they do occur. Hemorrhagic complications and abnormal platelet aggregation are distinctly atypi- cal. Generally, patients with secondary thrombocytosis require no specific therapy except for that directed at their primary disorder. 2. Forms of hereditary platelet disorders a. A defect similar to that caused by aspirin occurs in a heterogeneous group of inherited disorders of platelet function. Although the platelets have apparently normal electron—dense1 granules, patients with this group of disorders have platelet aggregation patterns similar to those observed in patients with dense granule storage—pool disease. Their platelets behave as if they had been exposed to aspirin. b. Storage-pool disease constitutes a heterogeneous group of inherited disorders characterized by an absence or deficiency of electron-dense platelet granules. Their platelets aggregate very poorly with collagen, and the second wave of aggregation with ADP and epinephrine is lacking. c.Thrombasthenia is an autosomally inherited recessive disorder with a defect in primary platelet aggregation. This disorder is characterized by an absence or marked reduction of clot retraction and an inability of the platelets to aggregate with ADP, epinephrine, or collagen. Patients with this disorder have a defect in the platelet membrane glycoproteins Hb and IIIa, which normally act as receptors. (1. Bemard-Soulier syndrome is an autosomally inherited recessive disorder associated with a defect in platelet adhesion. The platelets are large and aggregate normally with ADP, epinephrine, and collagen but fail to aggregate with ristocetin. Patients with this disorder have a defect in platelet membrane glycoprotein Tb: their platelets lack the receptor portion of the factor VIII molecule, which contains the von Willebrand factor activity. Such binding is necessary for normal platelet adhesion and aggregation with ristocetin. Abnormal coagulation factor component and inhibitors of hemostasis Excessive bleeding or clotting can occur when coagulation factors have one or more of the following: deficiency or malfunction; excessive consump- tion or destruction, enhanced fibrinolysis; or inhibition. These conditions are discussed below. A. Coagulation factor deficiency or abnormal function The most important congenital deficiency is that of factor VIII or hemo- philia A (Table 2). This disorder occurs almost exclusively in males, and affects one in 10,000 of the general population (1 in 3000 male births). Factor 1 This describes their appearance when examined by electron microscopy. 13 VIII is a large molecule (molecular weight [MW] approx 360,000) that has procoagulant activity (factor VIII:C), which acts as a catalytic co-factor in the activation of factor X by factor IXa. If factor VIII is measured immunologi- .cally, it is referred to as factor VIII:Ag. In the circulating blood, factor VIII is associated with the von Willebrand factor (vWF, formerly called factor VIII-related antigen or VIIIRzAg). The vWF is thought to “protect” the factor VIII against proteolysis. Thus, when vWF is deficient (von Willebrand’s disease) factor VIII:C is also frequently reduced. The functional activity of vWF is usually measured using ristocetin- induced platelet aggregation and is referred to as vWF:RCO. When assayed immunologically it is known as VWF:Ag. Although the inheritance of the coagulant activity (factor VIII:C) is controlled by an X-linked gene, inheritance of the vWF activity and antigenic properties is under the control of an autosomal gene. Patients with hemophilia A (factor VIII deficiency) have a greatly diminished coagulant activity but possess both the vWF activity and the vWF antigen in normal or increased amounts. The severity of disease in patients who have hemophilia A parallels the factor VIII coagulant activity; that is, patients are clinically divided into three groups: severe, moderate, or mild, corresponding to factor VIII coagulant activity of less than 1%, l%-5%, and 6%- 30%, respectively. Patients with severe hemophilia develop spontaneous bleeding in infancy. Hemarthrosis is the major clinical problem and a frequent cause of arthropathy in these patients. On the other hand, patients with moderately severe factor VIII deficiency have only occasional hemarthrosis and often reach adulthood without showing signs of crippling joint disease. Patients with mild hemo- philia may lead active lives and have abnormal bleeding only with major surgery or major trauma. Von Willebrand’s disease is an inherited hemorrhagic disorder character- ized by a defect in platelet adhesion. People with this disorder have a diminished plasma concentration of vWF. Normal concentrations of this protein are required for proper platelet adhesion. Several varieties have been described. In the common type, Type I, the defect in platelet adhesion can best be demonstrated by prolongation of the template bleeding time. Factor IX deficiency, called hemophilia B, resembles factor VIII defi- ciency in that it has an x-linked inheritance and is practically identical to factor VIII deficiency in its clinical manifestations. However, factor IX deficiency occurs much less frequently than factor VIII deficiency and tends to be milder in its clinical presentation. These are several variants of the antigenic portion of the molecule. An example of an abnormally functioning factor is seen in vitamin K deficiency. The binding of calcium to factors 11, VII, IX, and X is required for normal clotting. Without the attached calcium, these factors will not bind phospholipid, and the rate of factor activation will be sharply reduced. 14 The Bleeding and Clotting Disorders Table 2 - Coagulation factor details Clinical Inheritance Incidence Production Affects Therapy Half-life of deficiency site in body infused factor Factor I Autosomal Rare Liver Both sexes Cryoprecipitate, Fresh 96-144 hours (Fibrinogen) recessive Frozen Plasma, Fibrinogen Factor V Autosomal Rare Liver Both sexes Fresh Frozen Plasma 24 hours (Proaccelerin) recessive Factor VIII 1 in 3,000 ?Throughout Male Factor VIII 12 hours (Antihemophilic recessive males only R.E. system, (female Concentrate. factor) ?Liver, Spleen, carrier) Cryoprecipitate, Fresh Hemophilia A Kidneys Plasma Factor X Autosomal Iver. (V min II-Vll-lX-X Concentrate. 25 - 60 hours (Stuart-Prower) recessive ' K-dependent) Plasma Factor XII Autosomal Rare Unknown Both sexes Treatment 50 hours (Hageman) recessive unnecessa von WIIIebrand Autosomal Unknown Endothelial cells Both sexes DDAVP, 24 hours Factor (Factor VWF dominant Cryoprecipitate. Fresh Antigen Assayed) Plasma. Fresh Whole Blood “Assumes that the patient is in a steady state and has no inhibitor. 15 Calcium binds to certain glutamic acid residues, which have been modified by the posttranslational addition of a carboxyl group to the gamma carbon of each amino acid. If this carboxyl group is not added to each glutamic acid residue, calcium cannot bind and normal clotting will not proceed. Vitamin K in the presence of an epoxidase enzyme is required for this gamma carboxylation reaction to take place (Figure 5). If vitamin K deficiency exists, functional clotting factors will not be reduced. During the carboxylation reaction, vitamin K is converted into an inactive epoxide. Ordinarily, the liver contains a reductase enzyme that converts the inactive epoxide back to active vitamin K. Oral anticoagulants, such as warfan'n, inhibit the reductase enzyme and prevent the reversion of Vitamin K epoxide to its active form. Through this mechanism, active vitamin K is depleted, and thus it is not available to participate in the gamma carboxylation reaction necessary to make factors II, VII, IX, and X functional. Humans have two types of vitamin K. Vitamin K], which is absorbed from the diet, appears to be the more important. However, vitamin K2, which is produced by intestinal bacteria, enables a person to maintain sufficient levels of the vitamin in the absence of dietary vitamin K. Thus a vitamin Kl—free diet by itself normally does not lead to changes in the prothrombin time, unless intestinal bacteria that produce vitamin K2 are simultaneously reduced by oral antibiotics. Combinations of the vitamin Kl-free diet and oral antibiotics may be enough to prolong the prothrombin time because of the suppression of vitamin K—dependent factors. Similarly, malabsorption can produce increased re- sponsiveness to oral anticoagulants. Numerous other drugs alter the effect of oral anticoagulants. Examples of these drugs and their effects are shown in Table 6, page 101. Other vitamin K-dependent factors that are inhibitors of clotting are protein C and protein S. Protein C deficiency can be either inherited or acquired. Hereditary deficiency is associated with an increased incidence of venous thrombosis and pulmonary embolism. Clinically, patients with protein C deficiency appear identical to patients with antithrombin III deficiency. Both disorders have an autosomal dominant pattern of inheritance. Thus, a 50% reduction in the plasma concentration of protein C results in a clinically identifiable disorder. Acquired protein C deficiency is seen in patients with severe liver disease, in which the synthetic capabilities of the liver to maintain production of this high tumover-rate protein are compromised, and in patients with disseminated intravascular coagulation (DIC), in whom the protein is rapidly consumed. A total absence of protein C is usually associated with death in utero. Occasionally, some infants are born with a total lack of protein C; they suffer from purpurafulmz'nans neonatalis. A few such children have been kept alive by repeated infusions of prothrombin complex. An increased frequency of venous thrombosis and pulmonary embolism has also been observed in patients with protein S deficiency. Protein S exists 16 The Bleeding and Clotting Disorders w PRECURSOR FACTOR _ H CO3 glutomyl $H2 CIZH resudues Cl: H2 Corboxylase | 2 H-C COOH / \ Coupled COOH Epoxidase COOH VIT K VIT K Epoxide Reductase Figure 5 - Vitamin K-dependent factors in the circulation both as a free protein and as a protein bound to C4b binding protein. Only free protein S can function as a cofactor for the anticoagulant activity of protein C. Determining how much free protein S a patient has complicates the evaluation of protein S deficiency. Protein C may also have a role in the fibrinolytic system by inactivating an inhibitor of tissue plasminogen activator. B. Consumption of coagulation factors Disseminated intravascular coagulation (DIC) is an acquired coagulation defect secondary to other pathologic processes, which results in accelerated consumption of platelets and several clotting factors, particularly fibrinogen. Typically, the patient with DIC bleeds from multiple sites. The pathogenesis of DIC is complex and far from being fully understood. The accelerated consumption of platelets and clotting factors undoubtedly results from the actions of a variety of initiating agents acting at different steps in the sequence of clot formation. These actions disturb the careful balance that exists between promotors and inhibitors of coagulation. DIC has been associated with a wide variety of disorders, including infections (gram— negative bacterial infection, other types of bacteremia, and viral, rickettsial, and malaria infections), complications of pregnancy (abruptio placentae, amniotic fluid embolism, retained dead fetus, and toxemia of pregnancy), 17 metastatic carcinomas and acute myeloblastic leukemia (especially promyelocytic), tissue damage (shock, heat stroke, burns), hemolytic trans- fusion reactions, and venomous snake bites. No matter how the consumptive process begins, the major event is 1.thrombin generation (except in the case of certain snake venoms) with the subsequent release of fibrinopeptides A and B from fibrinogen, which results in fibrin formation. Once accelerated platelet and fibrinogen consumption begins, secondary fibrinolysis follows. As fibrin is laid down in a clot, plasminogen is incorporated by adsorption. An activator of plasminogen enters the fibrin clot, the active fibrinolytic enzyme plasmin is formed and plasmin initiates local clot dissolution. This plasmin action is localized because of potent antiplasmins in the circulation. Plasmin degrades fibrin, along with some fibrinogen, into a series of degradation products (fibrin degradation products [FDP]). With the rapid consumption of clotting factors that occurs in patients with DIC, the ability of the reticuloendothelial system to clear activated clotting factors and FDP is overwhelmed, and they accumu- late in the circulation. The activated clotting factors promote further clotting, while the FDP interferes with platelet function and fibrin stabilization. Thus, patients with DIC can have both a thrombotic and a hemorrhagic diathesis. C. Inhibitor to a coagulation factor Two basic types of circulating anticoagulants have been described. The first is a circulating anticoagulant with inhibitory activity directed against a specific coagulation factor. Ten percent of patients with classical hemophilia will develop an alloantibody against exogenous factor VIII at some time. Similarly, patients with other congenital factor deficiencies may develop alloantibodies after factor replacement. Occasionally, factor autoantibodies arise spontaneously in previously healthy men and women of advancing years. Women sometimes develop factor autoantibody a few days to a few months after childbirth. Patients with systemic lupus erythematosus (SLE) and other immunologic disorders may develop a factor autoantibody. By far the most common specific factor antibody observed clinically is one directed against therapeutically administered factor VIII. The other most common variety of circulating anticoagulants observed is the one directed against the phospholipids needed for factor interaction. This type of inhibitor is often referred to as the lupus anticoagulant. It has been found in patients with SLE and other related immunologic disorders. Patients with the lupus anticoagulant rarely have any hemorrhagic difficulty. Rather, they have an increased incidence of both alterial and venous thrombosis. Pregnant patients with the lupus anticoagulant have a very high frequency of spontaneous abortion. 18 The Bleeding and Clotting Disorders CHAPTER 2 CLINICAL ASPECTS OF BLEEDING AND THROMBOTIC DISORDERS BLEEDING DISORDERS Introduction Investigation of hemostatic function is necessary if there are one or more of the following: 1) a history of recent excessive bleeding following injury, surgery, or dental extractions; 2) a past history or family history of excessive bleeding or bruising; 3) menorrhagia, hematuria, and/or gastrointestinal bleeding; 4) poor healing of superficial lacerations; 5) easy bruising; 6) petechiae; 7) hemarthrosis; 8) bleeding into muscles or other organs; and 9) the incidental finding of an abnormal hemostatic test. It is important to take account of any local pathology. Approach to the patient The four steps in a complete hemostatic investigation are these: 1. A complete history (perhaps the most important single component) 2. Thorough physical examination 3. Screening tests of hemostasis 4. Special tests to define the precise nature of the defect. The history and physical examination will be discussed in this chapter. An overall approach to the problem and some major diagnostic possibilities will also be described. A. History 1. Chief complaint/chief features This includes the patient’s presenting problem in his or her own words; the duration of the chief complaint, especially if it has been present since childhood; and the age and sex of the patient. This last point may have an important bearing on the diagnosis. 2. Present illness The patient may complain of easy bruising; the development of purpura; nosebleeds; excessive bleeding after dental extraction, which may even require transfusions; heavy menses, frequently accompanied by the passage of clots; onset of abdominal pain; dark or bloody stools; red urine; swollen, painful joints or muscles; excessive bleeding after minor scratches; bleeding that recurs hours to days after the original trauma; poor wound healing; or fatigue. A careful dietary history is essential. In addition, the patient may report symptoms of a coexisting disease or process. Specifically, there may be jaundice and/or symptoms of hepatic 20 The Bleeding and Clotting Disorders failure, symptoms of localized or general infection, and a history of weight loss or other manifestations of cancer. Pregnancy must also be noted. 3. Past history The past history should be taken carefully in an attempt to identify other coexisting conditions, as well as to establish whether the patient ever had a surgical procedure or injury complicated by bleeding. 4. Exposure to drugs or toxins A thorough list of all current or past drugs used by the patient must be made. In addition, any exposure to chemicals or potential toxins at work or in the home should be documented, including the use of tobacco, alcohol, and narcotics. 5. Family history A review of the family history is important in all patients with bleeding problems. As much detail as possible should be obtained about all relatives. Focused questions should be asked about bleeding, and a family tree should be constructed if possible. At the very least, whether anybody else in the patient’s family has a similar condition should be determined. B. Physical examination The patient may appear pale or have evidence of bleeding such as petechial hemorrhages, purpura or ecchymoses, bleeding from mucous membranes, muscle hematomas, hemarthroses or deformed joints, gastrointestinal blood loss, hematuria or epistaxis. Bleeding into the bowel itself can produce physical signs that may be confused with acute appendicitis or other surgical abdominal problems. Other signs to look for include splenomegaly, hepatomegaly, jaundice, emaciation, fever, tenderness, lymphadenopathy and joint abnormalities. In some instances, it is possible to predict the type of defect from the presenting features (see Table 3). The skin manifestations of bleeding disorders (i.e., petechial hemorrhages or ecchymoses) are sometimes difficult to see in dark-skinned patients, and special attention must be given to thoroughly examining the mucous mem— branes, including the conjunctivae, oral mucosa, and optic fundi, for evidence of superficial bleeding. THROMBOTIC DISORDERS As discussed in Chapter 1, many factors are involved in maintaining the hemostatic balance. When these factors are shifted towards clotting, throm- bosis can occur. Abnormal thrombosis, i.e. thrombotic disease, causes pathology in tissues as a result of occluded blood supply. Thrombotic disease can be divided into two main categories: 21 Table 3 - Clinical summary of bleeding disorders A. From History Observations Diagnostic Possibilities History of bleeding/bruising since childhood Recent history of bleeding/bruising Sudden onset of bleeding/bruising, poorly healing cuts Congenital defect Acquired defect Acquired defect, probably drug-related B. From Physical Findings Observations Diagnostic Possibilites Skin and mucous membrane hemorrhages, usually small, relatively more common in females. Persistent bleeding from superficial lesions. Delayed bleeding, hemarthroses or hematomas; skin hemorrhages, large and few in number. Family history of bleeding disorders. Relatively more common in males. Joint deformities. Minimal bleeding from superficial lesions. Usually manifest after trauma. Bleeding from only one organ system Abnormalities of the vessel wall or platelet number or function Defects of coagualtion Probably not caused by hemostatic defect 1. Thrombosis associated with an underlying medical disorder is usually acquired. 2. Thrombosis due to a specific primary hemostatic abnormality is usually hereditary. The clinical evaluation is useful in the differential diagnosis of possible causes for thrombotic disease (Table 4). Most patients fall into one of the categories of acquired thrombotic disease secondary to an underlying disor- der. The evaluation should begin with a careful history and physical exami— nation supplemented by appropriate diagnostic tests. When problems cannot be classified as a secondary thrombotic disorder, primary hemostatic abnor- malities should be considered. Specific diagnosis in this category is usually beyond the resources of a district or regional laboratory. 22 The Bleeding and Clotting Disorders Table 4 - Clinical findings and diagnostic possibilities in thrombotic disease CLINICAL FINDINGS POSSIBLE CAUSES Age (> 45) Localized without recurrence; sudden Acquired thrombosis associated onset with infection/inflammation, trauma, venous stasis, drug reaction Other symptoms/signs of systemic disease - arterial occlusion underlying atherosclerosis, hyperlipidemia, diabetes, protein S deficiency - weight loss, anorexia, weakness, underlying malignancy tumor(s) - fever, localized or systemic underlying infection/inflammation infection - severe or massive trauma predisposed to thrombosis; DIC - deep vein thrombosis or thrombo- any thrombotic disease phlebitis, emboli Age (10 - 45) Migratory and/or recurrent thrombi; Acquired hypercoagulable state gradual onset or chronic; (lupus inhibitor) collagen-vascular or other systemic disease - multiple organ failure; massive DIC; hemolytic-uremic-syndrome purpura, severely ill Age (birth - 45) Family history; migratory and/or Hereditary hypercoagulable state recurrent thrombi (protein S or C deficiency; ATlll deficiency); homocystinuria Smoking, progesterone, estrogens, Aggravate all conditions and oral contraceptives 23 A. History and physical examination The basic approach to the history and physical examination has been described on page 19. Other important features associated with thrombotic disorders are as follows: 1. Chief complaint/chief features Older patients tend to have localized thrombosis for which there are definable local causes or secondary illness. Younger people (aged 10-45) can have an acquired hypercoagulable state due to the lupus inhibitor. Protein S and protein C deficiency often manifest themselves during the teens or early twenties, but can also be seen in the first decade, as can other abnormal clotting associated with other congenital disorders such as homocystinuria. 2. Present illness The type of thrombosis, i.e. whether arterial or venous, can help in suggesting a diagnosis. Migratory vascular occlusions, arterial or venous, may occur in an occult malignancy, whereas unilateral thrombophlebitis beginning in the same vein suggests localized venous insufficiency. Recur— rent venous thrombosis in multiple locations is often associated with pulmo- nary emboli; in a younger person this suggests congenital antithrombin III, protein C or protein S deficiency. Arterial thrombosis in multiple sites should suggest embolic episodes or arterial disease. Multiple organ failure with massive purpura and severe illness are hallmarks of DIC. 3. Past history The past history should carefully try to identify other coexisting conditions that might predispose to thrombosis. Many situations are associated with an increased risk of thrombosis. These include peripheral vascular disorders; pregnancy; atherosclerotic cardiovascular disease; smoking; vascular dis— ease; certain medications; and prolonged immobilization. Diseases associ- ated with thrombosis are listed in Table 4. These include atherosclerotic cardiovascular disease; malignancy; infections; trauma; inflammatory prob— lems; diabetes; collagen-vascular disease. 4. Family history As in the evaluation of bleeding disorders, a family history is very important in the evaluation of thrombosis. The health care worker should attempt to obtain as much data as possible about all relatives concerning the history of thrombosis. If possible, a family tree should be constructed and particular attention should be directed towards family members with similar problems. B. Physical examination The physical examination very frequently gives clues to primary underly- ing disease. Xanthomatous deposits in the skin or eye ground changes should suggest atherosclerosis or hyperlipidemia. Very tall, thin persons should be evaluated for homocystinuria. Evidence of weight loss, masses, lymph node enlargement, weakness or skin changes should lead to the evaluation for 24 The Bleeding and Clotting Disorders possible malignancy or collagen vascular diseases. Evidence of bleeding co- existing with thrombotic disease is seen in association with malignancies, essential thrombocytosis, lupus inhibitors, and certain drug reactions such as heparin—induced thrombocytopenia. CHAPTER 3 LABORATORY ASPECTS OF BLEEDING AND THROMBOTIC DISORDERS INTRODUCTION When a bleeding problem is suspected (see Chapter 2), it is necessary to decide which laboratory tests should be performed. This is especially impor- tant when the patient is going to have a surgical procedure. The source of the patient’s bleeding usually suggests the most likely cause, e.g. petechial bleeding from mucous membranes is probably due to vascular defects, platelet abnormalities, or von Willebrand’s disease. Muscle and joint bleeding or ecchymoses are indicative of clotting factor deficiency or deficiencies. These clues, however, are not infallible, and the workup of the bleeding problem should be as methodical as possible. One such approach is shown in Figure 6. Here, the pathophysiologic bases are shown along with key (decisive) test results. Whether these tests should be performed one by one or together as a “hemostasis screen” will be dictated by local circumstances. The discussion below applies in either case. Basic tests are: A. A complete blood count - including hemoglobin (Hb) or packed cell volume (PCV), white blood count (WBC), platelet count, erythrocyte sedimentation rate (ESR), and blood smear. B. The partial thromboplastin clotting time — a measure of the “intrinsic pathway.” C. The prothrombin clotting time — a measure of the “extrinsic pathway.” D. The bleeding time — a measure of vascular integrity and platelet number and function. In some laboratories, two other tests are considered part of this screen: E. Fibrinogen - a measure of the final common pathway with emphasis on circulating fibrinogen. F. Thrombin clotting time — a measure of the final phase of coagulation with emphasis on the contribution of thrombin. These tests are discussed below and in later chapters. A. The complete blood count The tests that compose the complete blood count will give an important series of diagnostic clues. Anemia could reflect blood loss or an underlying disease. In either case its presence and nature are important to establish. Leukopenia could suggest marrow damage, megaloblastic anemia, neoplasia, or overwhelming infection. Leukocytosis could suggest infection or hema- tologic malignancy. Thrombocytopenia is obviously a key finding requiring further evaluation. Thrombocytosis could reflect a myeloproliferative prob- Figure 6 - Flow chart for the interpretation of the three routine tests in bleeding disorders PLATELETS: ESTIMATE IN BLOOD SMEAR ABNORMAL PLATELET COUNT THROMBOCYTOPENIA THROMBOCYTOSIS POSSIBLE CAUSES MYELOPROLIF- ERATIVE DISEASE POSSIBLE CAUSES IMMUNE THROMBOCVTOPENIA onus nucnous (APLASTIC EFFECT on muons REACTION) Tmussusmu REACTION ALLERGIC nucnou HYPERSPLENISM DISSEMINATED INTRAVASCULAR GOAGULATION (BIC) VITAMIN DEFICIENCY (B12-F0LA‘I’E) MALIGNANCIES APLASTIC ANEMIA OTHER causes or PLATELET CONSUMPTION on roon PLATELET PRODUCTION 'REPEAT THE TEST ON I:I MIXTURE WITH NORMAL PLASMA. NORMAL I 1 NORMAL APTT NORMAL PROLONGED *- PROLONGED NORMAL PROLONG ED IBLEEDING TIME I ‘INHIBITOR SCREEN FACTOR VII I I NORMAL ABNORMAL POSSIBLE CAUSES * ' FACTOR VII INHIBITOR FACTOR XIII “ IN TRUE AFIBRINOGENEMIA THE BLOOD WILL NOT CLOT. POSSIBLE CAUSES POSSIBLE CAUSES DEFICIENCY THROMBOCYTOPENIA ABNORMAL PLATELET FUNCTIONS VON WILLEBRAND‘S DISEASE ' INHIBITOR SCREEN BLEEDING TIME FACTORS VIII, IX, ' INHIBITOR SCREEN FIBRINOGEN FACTORS II. V, X XI, XII W FACTOR VIII. IX. XI, XII. PRE~ KALLIKREIN OR HIGH MOLECULAR WEIGHT KININOGEN DEFICIENCY INHIBITORS VON WILLEBRAND'S DISEASE NOTE: IF INHIBITOR SCREEN IS POSITIVE, THE MOST COMMON INHIBITORS WILL BE LUI’US-LIKE INHIBITORS POSSIBLE CAUSES FACTOR X, V. II, OR I" DEFICIENCY INHIBITORS DIC LIVER DISEASE VIT. K DEFICIENCY Slaplosm b‘umom pue 5 Ipaala mu 9?. 27 lem. An increased ESR could suggest anemia, underlying inflammatory disease, or a protein disorder. In particular, examination of the blood smear can yield much important information. First, it is often possible to estimate the number of platelets. Usually there are four to eight platelets per 100 red cells. If this number is reduced, the cause of bleeding is probably thrombocytopenia, and the estimation should be confirmed with a platelet count. Second, the examina- tion of the blood smear can frequently give additional information on the cause of the bleeding disorder. Thus, blast cells indicate a diagnosis of leukemia; red cell fragments indicate disseminated intravascular coagulation (DIC); large platelets indicate increased production, as in idiopathic throm- bocytopenia purpura (ITP), or a myeloproliferative problem. The peripheral blood smear is sometimes helpful in identifying a problem of platelet function. For instance, when peripheral smears are made directly from the fingertip (i.e., without anticoagulant), platelets frequently clump together in groups of two to five. If abnormalities of platelet aggregation are present, whether caused by drugs that inhibit platelet function or diseases such as thrombocytopathia (acquired, e.g., severe uremia, or congenital, e.g., Glanzmann’s disease), the clumps may not occur in the blood smear. In such cases, bleeding time should be measured to confirm the presence of abnormal platelet function. Note, however, that platelets observed in a blood sample anticoagulated with ethylenediamine tetracetic acid (EDTA) do not normally clump, and examination of the blood smear may not be helpful in this situation. B. and C. The partial thromboplastin and prothrombin clotting times The partial thromboplastin and prothrombin clotting times will be consid- ered together. If both are normal, the patient’s bleeding is almost certainly due to vascular or platelet defects. This diagnosis would be confirmed by platelet count or by a bleeding time. 1. Abnormal activated partial thromboplastin time (APTT); normal prothrombin time (PT) If the APTT is prolonged beyond a reference normal value (p. 49) and the PT is normal, and if the prolongation of the APTT is corrected by repeating the APTT with a 1:1 mixture of patient’s plasma and normal plasma as substrate, then the patient has a deficiency of one or more of the following factors: VIII, IX, XI, XII, prekallikrein, or high molecular weight kininogen. Whether a factor deficiency is hereditary or acquired can usually be determined from the history and initial laboratory tests; acquired defects tend to be multiple, whereas hereditary defects are usually isolated. Factor VIII deficiency (hemophilia A or classical hemophilia) and factor IX deficiency (hemophilia B) are the two most common inherited disorders of clotting. Another form of factor VIII deficiency is von Willebrand’s disease. In this condition, a prolonged bleeding time and a low von Willebrand antigen (vWF2AG) are found. 28 The Bleeding and Clotting Disorders Factor XII deficiency is asymptomatic; it can be diagnosed very simply. Add fresh normal plasma to a glass tube and let it stand for a few minutes; then discard the plasma and rinse the tube 10 minutes with saline. Pour the patient’s plasma into this tube and repeat the APTT. If it is now normal, the diagnosis of factor XII deficiency is established. The factor XII (or glass contact factor) is adsorbed from the normal plasma to the glass and corrects the patient’s defect. Conversely, a circulating anticoagulant may be suspected because the prothrombin time or the partial thromboplastin time has not been corrected when the patient’s plasma is mixed 1:1 with normal plasma. The specificity of antibody can then be tested by a specific factor inhibitor assay. Some patients with systemic lupus erythematosus and other related immu- nologic disorders have been reported to have a circulating anticoagulant with immunoglobulin-like properties. This is not directed against a specific coagulation factor, but appears to interfere with phospholipid binding, which is needed for factor activation. These patients may have many of the serologic abnormalities observed in lupus. After the presence of a circulating antico- agulant has been demonstrated by the failure of the partial thromboplastin time to correct when the patient’s plasma is mixed 1:1 with normal plasma, the inhibitor can be characterized by a tissue thromboplastin inhibition test, both in PT and AP'IT test systems. The lupus anticoagulant can cause a prolonged PT and/or AP'IT. Bleeding in these patients occurs mainly when they have associated thrombocytopenia or an isolated factor II deficiency. 2. Normal APTT; abnormal PT If the APTI‘ is normal and the PT is prolonged beyond a reference normal value, and if that prolongation is corrected by repeating the prothrombin time with a 1:1 mixture of patient’s plasma and normal plasma as substrate, then the patient has factor VII deficiency. If, on the other hand, the l : 1 mixture fails to correct the prolonged prothrombin time, the patient has an inhibitor. 3. Abnormal AP'IT; abnormal PT If both PT and APTT are prolonged beyond reference normal values, and if these prolongations are corrected by repeating the tests with a 1:1 mixture of patient’s plasma and normal plasma as substrate, then the patient has a deficiency of one or more of the following factors: X, V, II, or I. Fibrinogen can be assayed easily be a modification of the thrombin time; the other factors require specific assays to determine which fact0r(s) is deficient. Abnormalities of the common pathway tend to be more commonly associated with multiple deficiencies, e. g. liver disease, vitamin K deficiency, and DIC, than with single factor deficiencies. D. Bleeding time (pp. 69 - 74) E. Fibrinogen concentration Fibrinogen concentration is an estimate of the production and function of fibrinogen, and of its utilization in the final common pathway of coagulation. 29 Decreased fibrinogen concentration may be acquired, for example, occuning in association with DIC and fibrinolysis. Less commonly it is due to hereditary hypofibrinogenemia or afibrinogenemia. Measurement of fibrinogen con— centration is one of the key tests for monitoring DIC or surgical bleeding. The action of fibrinogen can be decreased without affecting its concentra— tion when functionally abnormal fibrinogen is present. These abnormal fibrinogens can even act as inhibitors of fibrinogen function. F. Thrombin clotting time This test measures the time required for plasma to clot when thrombin is added. It generally reflects the final phase of clotting. Prolongation of the thrombin time is characteristic of hypofibrinogenemia, heparin use, and dysfibrinogenemias. It also occurs when circulating anticoagulant is present. APPROACH TO THE PATIENT WITH THROMBOTIC DISORDERS When a clotting disorder is suspected, the direction of the laboratory investigation is frequently suggested by data obtained during the history and physical examination. These data, however, are more likely to assist in focusing on a general medical area (e.g., occult tumor versus collagen vascular disorder) than on specific abnormalities of the hemostatic system (e.g., protein C versus antithrombin III deficiency). Unlike the laboratory evaluation of a bleeding disorder in that a “hemosta— sis screen” is used, the laboratory evaluation of a clotting disorder uses no standard “thrombosis screen” (at least none which helps to decide on further evaluation). This is because the tests useful for these disorders are, in general, specific for particular factors and thus lack.the broad coverage of diseases given by tests such as the prothrombin time and the activated partial thromboplastin time. In addition, the laboratory techniques used for these tests require specialized procedures and equipment, which are not always available. Without the use of screening tests, the laboratory work is best directed toward diseases suggested by the clinical evaluation. This means that most of the tests will be outside the scope of this book in that they will be directed at diagnosis of underlying medical disease. However, the tests below often provide useful information. A. The complete blood count As with the bleeding disorders, the complete blood count can give important descriptive clues to underlying disease (p. 25). In addition, polycy- themia should be ruled out. Red cell morphology can be particularly helpful in cases of DIC, metastatic tumor, hyperlipidosis, or protein disorders. Platelet numbers and morphology should receive special attention, since thrombocy- tosis can be associated with both clotting and dysfunctional bleeding. 30 The Bleeding and Clotting Disorders B. Observation of the blood clot (p. 64) Plasma above the clot should be observed to see if a high lipid content is present. The retraction of the clot can be severely disturbed in paraproteinemia (myeloma or Waldenstrom’s disease); a small clot with irregular shape is often found when it is being digested by enhanced fibrinolysis as part of DIC. No clot may be seen in severe DIC, whereas a small clot may be seen in dysfibrinogenemia. C. APTT and PT These tests may be helpful. Some patients with thrombotic disorders may have a shortened APTT or PT, but the significance of this is unknown and it does not diagnose a pre—thrombotic state. Persons with lupus anticoagulant (see Chapter 1) often have prolongation of the APTT and occasionally the PT. When present, the prolongation should be tested to determine if it is due to an inhibitor (see Measurement of the intrinsic system, p. 78). The lupus anticoagulant can be characterized by the tissue thromboplastin inhibition test (TI‘I), with abnormal AP'IT and normal PT (pp. 27-28). D. Specific tests for abnormalities These tests will be indicated in only a minority of the thrombotic events. 1. Fibrinogen levels by immunologic and functional assays. When fibrinogen levels are reduced by a clotting assay (p. 81) and are normal by an immunologic method, an abnormal fibrinogen should be sus- pected. Inhibitors may produce a similar pattern so that these tests are not specific. 2. Protein C assays. These determinations are beyond the scope of this book and should be measured in a suitable reference laboratory. Protein C may be decreased acutely after a large thrombosis has occurred, and a test performed during this period will often be spuriously low. Protein C should be measured after allowing this process to clear; at least 6 weeks following a thrombotic episode is usually adequate. Since protein C is a vitamin K-dependent factor, oral anticoagulants will decrease its activity. Thus when determinations are made on patients receiving oral anticoagulants, comparison is gener- ally made to another vitamin K-dependent factor, such as factor II or X, to determine if the value is lower that expected. 3. Protein S determination. Like protein C, protein S is a vitamin K— dependent factor and its measurement is not routinely available (see 2 above). Protein S exists in two states, one bound to another protein (C4bBP) and the other a free form. Only the free form is functional and therefore measuring the level of the free form is more useful in diagnosing protein S deficiency. 4. Antithrombin III deficiency. Several different methods have been developed to measure antithrombin III in blood. They do not all measure exactly the same form of antithrombin III and all may not diagnose some patients with the disease. 31 5. Measurements of plasminogen, plasmin, and tissue plasminogen activator. Tests of the plasminogen-plasmin system can be performed in specialized laboratories using specialized procedures. At present the prevalence of congenital abnormalities appears to be low. DISSEMINATED INTRAVASCULAR COAGULATION (DIC) DIC is a thrombotic syndrome that usually presents with bleeding because of its wide effect on the hemostatic system. Typically, the patient with DIC is bleeding from multiple sites. Common laboratory abnormalities include thrombocytopenia, prolongation of the thrombin time, and increased concen- tration of fibrin degradation products (FDPs). Other abnormalities that are frequently detected include hypofibrinogenemia and prolongation of the prothrombin time and partial thromboplastin time. The peripheral blood smear may show red cell fragmentation as a result of damage inflicted on the circulating cells by intravascular fibrin strands. HEMOSTATIC PROBLEMS ASSOCIATED WITH SURGERY Surgery patients, like other hospitalized or immobilized patients, are at increased risk for thromboembolism. In many cases prophylactic low—dose heparin regimens help prevent these problems without interfering with surgical care. Bleeding during or after surgery is sometimes a very difficult problem to evaluate. It may simply be caused by a problem in surgical technique, in which case reoperation may be necessary. Alternatively, it may be due to any one of a number of hemostatic problems, of which DIC, thrombocytopenia, and hypofibrinogenemia are the most common. Figure 7 is a useful guide. EVALUATION OF BLEEDING IN SURGERY PATIENTS GENERALIZED MICROVASCULAR BLEEDING? NO YES LARGER VESSEL PERFORM SCREENING TESTS BLEEDING SURGICALLY PLATELET COUNT CORRECTABLE AND BLOOD SMEAR EXAMINATION APTT (ACTIVATED PARTIAL THROMBOPLASTIN TEST) PT (PROTHROMBIN TIME) I REFER TO FIGURE 6. Figure 7 - Approach to surgical bleeding 32 The Bleeding and Clotting Disorders HEMATOLOGIC FINDINGS IN AIDS PATIENTS There are many hematologic abnormalities associated with human immu— nodeficiency virus—1 (HIV- 1) infection culminating in acquired immunodefi- ciency syndrome (AIDS). In the latent period, which ranges from months to years, there are no specific features, but thereafter lymphoid function deterio- rates with resulting opportunistic infections and neoplasms. In early stages of the active disease the blood shows lymphopenia, i reactive lymphocytes, and non—specific red cell changes. Neutropenia can sometimes follow. Thrombo- cytopenia occurs from production of abnormal antibodies. The bone marrow is often initially hypercellular with increased plasma cells and lymphocytes. Bizarre megakaryocytes are sometimes seen, as are dyspoietic normoblasts. Increased storage of iron, characteristic of chronic inflammation, can occur. Evidence for secondary malignancies (Kaposi’s sarcoma, B-cell lymphoma) and a wide range of secondary opportunistic infections (viral, bacterial, fungal, protozoal) can often be found. Note: All blood smears and bone marrow aspirates are stained with Wright’s Stain unless otherwise specified. All bone marrow biopsies are stained with H & E, unless otherwise specified. Code for magnification: (L.P.) = Low power magnification (dry) (H.P.) = High power magnification (dry) (L.O.) = Low oil magnification (H.O.)= High oil magnification 33 Hemostasis , aim» Left - Petechiae are seen on the arm of a patient with Immune Thrombocytopenia Purpura (ITP). The le- sions are multiple, small, and discrete. Right - This is a massive series of ecchymoses in a patient with factor VIII deficiency who had been given a subcuta- neous injection in his left buttock. ,. ”W , Left, Vascular purpura: This patient had systemic amyloidosis with periorbital purpura, which is very characteristic of this disease. Right - This patient had drug—induced Henoch Shoenlein Purpura which is characterized by a peripheral vasculitic reaction, here manifested by petechiae and ecchymoses. / Left » A normal peripheral blood smear is seen with a central neutrophii. One platelet isjust to the left of the neutrophil. Two other platelets can be seen in the field. Right - This is a bone marrow biopsy of a normal patient. The cellularity and the distribution of cells is normal. The larger cells seen in the field are megakaryocytes. Left - This is a muscle hematoma in a patient with factor VIII deficiency. Right — This patient had mas- sive sepsis with severe disseminated intravascular clotting associated with ecchymoses and ecchymotic vesicles. Left - A malnourished patient with peripheral ecchymoses. This patientwas diagnosed as having scurvy. Right - Multiple small telangiectases are seen on the inner and outer mucous areas of the lip in a patient with Hereditary Hemorrhagic Telangiectasia. Left, Immune thrombocytopenic purpura (ITP): A large platelet and a small platelet, as well as some hypochromic red cells, are seen in this peripheral blood smear. Right, Infectious mononucleosis: Thrombocytopenia is often seen in association with infectious mononucleosis. Reactive lymphocytes are seen in this field but no platelets are present. 34 The Bleeding and Clotting Disorders Left - Bone marrow aspirate showing megakaryocytosis in lTP. The megakaryocytes present show varying stages of maturation from blast to mature megakaryocyte. Right - Bone mar- row biopsy from a patient with AIDS. Megakaryocytosis and other general hypercellularity can be appreciated. Often such patients will have thrombocytopenia. Left - Myelodysplasia: These very abnormal plate- lets are seen in the peripheral blood of a patient with myelodysplastic syndrome. Right - Agnogenic my- eloid metaplasia and splenectomy: Giant platelets, thrombocytosis, and abnormal megakaryocyte nucleii comprise the peripheral blood smear picture seen here. 9.. Left - These three blasts are from a patient who had megakaryoblastic crisis of Chronic Myelogenous Leukemia (CML). The large megakaryoblasts are characteristic. Right - This is a blood smear from a patientwith Acute Non-Iymphoblastic Leukemia - M- 7 type. The patient also had Down’s syndrome. Left, Microangiopathic thrombocytopenia: This pa- 00 34:" Wt? o. VMM tient had marked thrombocytopenia in association with hemolysis; both of which can be suspected by the red cell fragments and polychromatic cell in the field. Right - Thrombocytosis: This patient with iron deficiency anemia has a microcytic hypochromic blood smear and the commonly encountered throm- bocytosis. Left, Myelodysplastlc syndrome: This bone marrow aspirate shows a strikingly abnormal megakaryo- cyte. Nuclear dyspoiesis is present as is a grossly abnormal cell morphology. Right, Essential throm- bocythemia: The megakaryocytes in this bone marrow aspirate are strikingly abnormal in terms of their nuclear ploidy and morphology. Left - This giant platelet is from a patient with Ber- nard—Soulier Syndrome. Right - This giant platelet is seen in a patient with May-Hegglin Anomaly who alsoshowsabnormalneutrophilswith spindle-shaped dohle bodies in their cytoplasm. CHAPTER 4 QUALITY ASSURANCE GENERAL CONSIDERATIONS In the broad sense, quality assurance includes correct specimen collection and handling, good methods, reliable reagents and reference materials, proper maintenance of equipment, adequate records, an efficient system for report- ing results, and a set of procedures designed to check the reliability of the reported results. Quality assurance is not difficult to achieve, and it is essential for good laboratory practice. In laboratory work both accuracy and precision are important and must be kept within acceptable limits. For this purpose, a quality assurance program is essential. Accuracy and precision are maintained by internal quality control, which usually consists of tests on control materials and analysis of data from tests on patient specimens. Internal quality control determines whether results are sufficiently reliable to be released to the clinician. Accuracy is further controlled by external quality assessment, which is an independent retrospective analysis to ensure comparability of results between laboratories and between methods. Accuracy and precision are not the same thing; a test may be reproducible, that is, precise, but highly inaccurate. The difference between accuracy and precision is shown by the following illustration of individual shots in relation to the center of a target (Figure 8). ACCURATE ACCURATEf PRECISE— NOT ACCURATE & PRECISE NOT PRECISE NOT ACCURATE —NOT PRECISE Figure 8 - An explanation of the contrast between accuracy and precision Thus, accuracy is a measure of the closeness of an estimated value to the true value, and precision is the closeness with which repeat measurements on one sample agree. The average result calculated from all the measurements is called the mean. The spread of a group of values around the mean is expressed through the standard deviation (SD) (p. 37). 36 The Bleeding and Clotting Disorders Accuracy can be checked only by the use of reference materials. Partici— pation in an external interlaboratory quality control program (external quality assessment) is a good way to confirm accuracy. Precision can be checked by a) repeated testing of specially prepared control specimens or b) duplicate or replicate testing of routine patient specimens. It is also important to determine when a test procedure begins to drift, that is, when there is a gradual change in test performance due, for example, to the deterioration of reagents or a change in apparatus. This is detectable by means of a control chart (p. 39). REFERENCE AND CALIBRATION PREPARATIONS A reference standard is a substance (e.g., hemoglobin standard) that has been characterized by chemical or physical means and has been assigned a value. There are both international and national reference standards. Intema— tional reference standards are manufactured by or on behalf of an international organization (for example, the World Health Organization or the Intema- tional Council for Standardization in Haematology [ICSH]). They provide a measure against which national reference materials and calibrators (p. 36) can be controlled. They are produced in limited amounts only and must not be used as working materials in the routine laboratory. They are provided on request to national authorities and sometimes to individual workers from a country that has no national body responsible for producing appropriate standards. A reference material is a substance (e.g., reference plasma) or device (e.g., reference light absorbing filter) that conforms to a national or international reference standard. This material is used to ensure accuracy of a test procedure in routine practice. A calibrator is a substance or device that also conforms to a reference standard or specifications. It is used to calibrate an apparatus or to adjust a measurement to obtain accurate results. A control is a substance (e. g., plasma control) used to check the precision (that is, reproducibility) of a test in routine practice; it must be similar in performance to the patients’ specimens alongside which it is analyzed. A control is not intended to check accuracy. However, just as a calibrator may sometimes be used as a control material, a control may be used as a calibrator, provided it has the necessary characteristics described in these definitions. Controls are usually more expensive than calibrators and available only in limited amounts. THE USE OF CONTROL SPECIMENS Instead of testing every patient’s specimen in duplicate, a check for precision can be performed on control specimens. To check precision, one does not have to know the actual value of the control specimen. If, however, the value has been reliably determined, for example, by a reference center, the 37 control material can also be used to check accuracy (p. 40). If available, controls with high, low, and normal values are preferred. The control specimens may be anticoagulated whole blood, preserved pooled red cells, plasma, or serum, depending on the test. The preparation of each of these materials is described under the individual test. At least one control specimen should be used for every batch of specimens to be tested, even if the batch contains only one specimen. If the batch is large, there should be one control for every 20 patient specimens. Since controls introduced into the test systems are intended to simulate random sampling, they must be treated exactly like the patient specimens. Results of the analysis of the control specimens are plotted on a chart, as described below. Standard deviation To understand the significance of the standard deviation (SD) in the context of quality control, one must appreciate the way in which test results are likely to lie above and below the mean. The distribution of these results can be defined as a Gaussian curve, which has the following appearance (Figure 9). The measure of the spread is expressed by the SD. Simply speaking, SD is just a yardstick that we use. The area under the center of the curve, i 1 SD, equals 68% of the whole area; i 2 SD equals 95%; i 3 SD equals 99.7%. If the limits are set at i 2 SD and 100 measurements are done on the same specimen, then 95% of the 100 will fall within i 2 SD. This also means that by chance alone 5 out of 100 determinations will be outside the 95% range. Most laboratories set their quality control limits at i 2 SD. Y )_< i—i'lSD'—| i——1'ZSD i—-i3SD—————| Normal Distribution Curve (Gaussian Curve) Figure 9 - Gaussian distribution curve 38 The Bleeding and Clotting Disorders Calculation of the standard deviation 2 The SD is calculated from the formula SD = w n _. in which d equals the difference of individual results from the mean and n equals the number of measurements. Modern hand-held calculators can provide the SD directly from the test results. The SD can, however, also be obtained, slightly more laboriously, by direct calculation. The method for this calculation is shown in the following example with prothrombin time: a. Carry out 20 consecutive measurements of prothrombin time on a specimen. Each measurement is referred to as “x” and the number of measurements as “n.” b. Tabulate the measurements in a column; calculate mean of measure- _ 2 ments (2?) and calculate (x — i) and (x — x) as follows: Test i (x—i) (x—i)2 1 12.5 +0.4 0.16 2 11.8 -0.3 0.09 3 12.2 +0.1 0.01 4 l 1.7 -0.4 0.16 5 12.0 -0.1 0.01 6 12.6 +0.5 0.25 7 12.6 +0.5 0.25 8 12.7 +0.6 0.36 9 12.3 +0.2 0.04 10 12.0 —0.1 0.01 11 12.0 —0.1 0.01 12 11.7 -04 0.16 13 11.4 —0.7 0.49 14 12.2 +0.1 0.01 15 12.7 +0.6 0.36 16 12.2 +0.1 0.01 17 11.7 -04 0.16 18 12.2 +0.1 0.01 19 11.5 -0.6 0.36 20 12.0 -0.1 0.01 39 _ 2 2 _ 0. Calculate ASL. In the example this will be: , 0.15 = 0.39; that is, 0.4 (to nearest 1 decimal place). Thus, SD = 0.4 sec. Coefficient of variation (CV) The CV relates the SD to the actual measurement so that measurements at different levels can be compared. CV (as a %) is calculated by E X 100 % mean Thus, using the example given, CV = 0"1/121 X 100 = 3.3% _ 2 Sum ofx = 242.0, n = 20, X = 12.1, Sum of (x—i) = 2.92 2 E x—i 2-92 SD: {EL = ——19 = , 0.153 =0,39_ Preparation of a control chart Before using a control specimen, one must first make a series of at least 10 measurements of the constituent in question and calculate the mean and SD of the results, since these measurements are used to construct the chart (p. 38 for calculation of SD). These measurements should be made with great care under conditions as ideal and as constant as possible so that optimal results can be obtained. On the control charts the mean is drawn to represent the baseline; lines representing i 2 SD are drawn above and below it (see Figure 10). A separate control chart should be made for each type of test. +‘ . 280— 0.: 0". -.‘.. ‘0 28D- —111111111111111 0»— Figure 10 - Quality control chart 40 The Bleeding and Clotting Disorders Subsequently, the control specimen is used along with routine specimens and plotted on the chart. The following guidelines will help the laboratory workers decide whether this test is out of control, that is, whether something 1s gomg wrong: - a value lies entirely outside the i 2 SD control limits — several consecutive values show a rising tendency — several consecutive values show a falling tendency — several consecutive values lie on one side of the mean - two or more results per 20 values lie on the + 2 or - 2 SD lines Examples of the way in which deteriorating performance can be detected are shown as the following points along the x—axis in Figure 10. Point A: Value outside + 2 SD; this was because of a pipetting error. Point B: Several values have occurred consecutively on one side of the mean and there is a rising tendency; this was because of deterioration of the diluent. Point C: In good control after correction of fault. The validity of the control chart depends on the control specimen retaining its stability. Accuracy The control tests described above do not check for accuracy. Accuracy can be checked in control tests only if the true value of the control material is known. It can also be checked by measuring reference material that has a stated value determined previously in a reference institute (central laboratory or international reference laboratory). When this stated value is provided, it usually also includes the SD, which is the limit of precision obtained in the reference laboratory. The essential test for accuracy is that reference preparations and reference materials be used as described above. External quality assessment is another procedure that will help ensure that accuracy is maintained. EXTERNAL QUALITY ASSESSMENT (EQA) Extemal quality assessment is an important and even essential supplement to an internal quality control system. Even when all possible precautions are taken to achieve accuracy and precision in the laboratory, errors will arise that are only demonstrable through this procedure. The principle of external quality assessment is based on the procedure of sending samples of the same material from one center to a large number of laboratories for testing. All the laboratories send test results back to the center, where they are analyzed. This analysis includes the mean, the SD, and an indication of acceptable performance in comparison with those of other 41 participants and often with the performance in the reference center itself. The World Health Organization (WHO) offers the opportunity for a few main laboratories in various countries to participate in such a scheme at an intema— tional level, and these laboratories are then encouraged to establish national or regional schemes for other laboratories in their country. Information on the International EQA Scheme (IEQAS) can be obtained from WHO (p. 40). Acceptable results (good performance) We shall not discuss acceptable performance in an external quality assessment program, since this is the concem of the control laboratory, which will establish the appropriate criteria for reliability. Acceptable performance with respect to precision is based on the distribution of results in repeated testing on the same material. The SD of repeat tests should, in general, be no greater than 10% of the reference normal range. Control of morphology The morphologic appraisal of blood and bone marrow smears is highly subjective. It also depends upon the smear being well made and well processed, with good quality stains. The reliability of this procedure should be checked on a regular basis by supervisors and by the exchange of slides with other laboratories. Junior personnel should, whenever possible, show supervisors all important abnormalities, such as early or reactive leukocytes, thrombocytopenia, or major red cell changes. Visits by supervising senior personnel to smaller laboratories should include the examination of blood slides. Regularly scheduled departmental discussions of preparations of special interest are an important means of stimulating interest in and further- ing knowledge of correct morphologic evaluation of blood films and bone marrow smears. The careful appraisal of blood films is a useful check for gross errors in red cell indices and leukocyte and platelet counts and provides useful clinical information. CALIBRATION, CARE, AND MAINTENANCE OF LABORATORY INSTRUMENTS Centrifuge l. The speed of the centrifuge (revolutions per minute [rpm]) should be checked with a tachometer every six months. 2. Timers should be checked against a clock or stopwatch monthly. 3. Rubber strips in the lid of the centrifuge should be checked monthly and replaced when worn. 4. Brushes should be inspected every 6 months and replaced when there is poor contact with the rotor. 42 The Bleeding and Clotting Disorders 5. The heads of the centrifuge should be cleaned with soap and water only (but see also p. 53). 6. When a centrifuge is loaded, care must be taken to balance tubes placed in the head by weighing them in their buckets, adding water to the bucket (not into the tubes) as required. Microscope The microscope should be thoroughly cleaned by a trained person every 12 months. The oculars and objectives, including the oil immersion lens, should be cleaned daily with lens paper or clean cotton cloth. Xylol may be used very sparingly, but other solvents, especially alcohol, should never be used, especially on the objectives. When the microscope is not in use, it should be covered to prevent excessive dust from accumulating. In the tropics it should be stored, if possible, in a cabinet heated by an electric light bulb, to prevent fungi from growing in the lenses. Pipettes Although pipettes obtained from reputable manufacturers are generally reliable, it is good practice to check any new “1—mar ”capillary pipette before it is first used. The procedure described below may also be adapted to disposable pipettes. Clean unscratched glass is necessary. Acid cleaning is generally considered the best method for cleaning glassware for coagulation studies. The glassware must be thoroughly rinsed to remove all traces of acid, because this could change the pH. Disposable glassware and plastic ware are very useful and eliminate the time-consuming process of acid washing. Siliconized glassware must be kept separate from nonsiliconized glassware. Detergents are not used in cleaning glassware for coagulation studies because the last traces are difficult to remove and these traces may affect the results of the tests. Equipment Mercury Tuberculin syringe Thick-walled rubber or plastic tubing 50—ml beaker Weighing bottles Analytical balance 99:5935’.‘ 43 Cleaning procedure 1. Soak pipettes in 3N (3 mol/L) HCl overnight. 2. Rinse at least 10 times with tap water and 10 times with distilled or deionized water. 3. Dry the glassware in the air. Calibration procedure 1. Place mercury in the beaker. Weigh a weighing bottle and record the weight. 2. Connect the tip of the syringe to the pipette with a short length of thick- walled rubber or plastic tubing. 3. Immerse the tip of the pipette in the mercury. Slowly withdraw the plunger, thus filling the pipette with mercury. When the graduation mark is reached, empty the mercury into the weighing bottle, and again weigh it and read the weight. Establish the weight of the mercury by subtracting the weight of the empty weighing bottle from its final weight with mercury. The determination should be done twice. 4. Establish the volume of the mercury (in microliters) by dividing the weight (in mg) by the factor 13.54. 5. If the volume differs by more than 1% of the stated volume, the pipette either should not be used or the appropriate correction factor should be applied. The actual volume of the pipette (in ul), for example, 21 ul, divided by the claimed volume of the pipette, for example, 20 ul, gives the correction factor for the pipette. In the example, the factor would be 21 20 = 1.05. Use of 1-mark capillary pipettes Only good quality pipettes that perform within an accuracy of 1% should be used. If a pipette becomes chipped, it must be discarded. The pipettes should be Cleaned in acid (p. 42). Draw up the sample to just above the mark, and wipe the outside clean with a piece of tissue paper; at the same time, remove the excess blood from the pipette by gently touching the tip. Place the tip of the pipette at the bottom of the test tube containing reagent, and expel the contents by gentle pressure on the attached teat. Then partly withdraw the pipette from the solution and rinse it three times with reagent from the upper layer in the test tube. Blood should not be drawn up far in excess of the mark and then allowed to drain out to the mark, because a certain amount of the excess may adhere to the wall and then be rinsed out with the reagent, thus giving falsely high values. 44 The Bleeding and Clotting Disorders SPECIAL ASPECTS OF QUALITY CONTROL FOR COAGULATION STUDIES If test results are to be valid, quality control within the laboratory must be adequate. In any quality control program, four variables must be considered: specimens, reagents, equipment, and techniques. Steps to ensure that these variables are properly controlled are discussed below. Specimens The practical aspects of blood collection are described in Chapter 6. The specimen must be collected in such a manner that the integrity of the coagulation factors is preserved. The coagulation system consists of enzymes and proenzymes, which are easily activated or denatured. In blood collecting, the first step is to identify the patient, and to write his or her name and any identifying number on the container that will receive the blood. Then, reassure the patient and endeavor to relieve any apprehension before beginning. As far as possible, blood should be collected each time under the same conditions, e.g., when the patient is resting, in the morning, and before meals (p. 57). In blood that is drawn slowly or obtained with difficulty, the clotting mechanism may be activated, and test results will show excessively high coagulation factor activity and a falsely low number of platelets, many of which may function abnormally. Therefore, an experienced person should collect the blood. Use the two-syringe technique to collect blood for most coagulation tests. Draw the first milliliters into one syringe and discard it or use it for chemistry or other blood tests. The prothrombin time (PT) and activated partial thromboplastin time (APTT) may be run from the first tube, if necessary. Use the blood collected in the second syringe for most coagulation tests. Siliconized or plastic syringes should be used, if available, in drawing the blood. For accurate results, the tube must contain within 10% of the stated volume. Compare each tube with a reference tube containing the minimum acceptable volume. Discard any tubes that do not contain the minimum volume, and collect another sample. If the blood foams while it is being collected in evacuated tubes, fibrinogen and factors V and VIII may be denatured. The needle should not be smaller than 21 gauge. Remove the needle and empty the syringe by placing the tip of the nozzle against the side of the tube and allowing the blood to run down the side to prevent frothing or excessive turbulence. Immediately mix the blood by gentle inversion. These procedures are necessary because coagulation proteins may be denatured by vigorous mixing. Store blood for the APTT and specific factor assays on melting ice or refrigerated at 4°C-10°C until tested, but for not longer than 2 hours. 45 If platelet—rich plasma is desired, centrifuge the blood at 200—400;; at room temperature (20°C—25°C) for 10 minutes. Platelet-poor plasma is used for most coagulation tests, since platelets contain platelet factor 3, platelet factor 4, and several of the blood coagulation factors. For platelet-poor plasma, the blood must be centrifuged a second time at 1000 X g or greater for 20 minutes. It is best to store blood and plasma in plastic tubes and make all transfers with plastic pipettes. Glass surfaces may activate the clotting sequence, thereby invalidating the results of many tests. Unstoppered tubes left standing at room temperatures will lose C02, thus causing a change in pH. If all tests cannot be completed within 4 hours, freeze small aliquots (0.5 to 1 ml) of the plasma and store them (preferably at -70°C, or for shorter periods, at -20°C in a regular freezer). Quick—thaw the plasma at 37°C immediately before it is tested. If the plasma is stored in small aliquots, one tube can be thawed for each test, and very little plasma is wasted. Various drugs and physiologic conditions (e. g., pregnancy, excitement, or vigorous exercise) affect some of the coagulation tests. Aspirin and other anti- inflammatory drugs inhibit platelet aggregation. Platelet adhesion, platelet aggregation, and the activity of factors I (fibrinogen), 11, VII, VIII, IX, X, and XII are all increased to a statistically significant degree by oral contraceptives. Factor VIII activity can be increased very rapidly by strenuous exercise or adrenalin infusion. Reagents Sodium citrate should be used as the anticoagulant. It is superior to sodium oxalate in several ways; one of the most important is that factors V and VIII are more stable in citrate than in oxalate. In one study, factor V activity decreased as early as 15 minutes after the specimen was drawn. In addition, APTT tests performed on plasma samples collected in citrate were much more sensitive to the presence of heparin than those collected in oxalate. This is important when the AP’TT test is used to monitor patients who are on heparin therapy. Many thromboplastins and activated partial thromboplastins are commer- cially available. Thromboplastins have shown wide variability in their sensitivity to factor VII. Laboratory variability in the 1—stage prothrombin time test results has been attributed to differences among thromboplastins. Variability in other tests has been attributed to differences among activated partial thromboplastin reagents. To overcome variability in thromboplastins and partial thromboplastin, use standardized reagents where possible. Inter— national (WHO) reference preparations of thromboplastins are available for calibrating manufactured preparations (p. 97). The choice of which thromboplastin or which activated partial thrombo- plastin to use depends on the reagent’s reproducibility and sensitivity and on 46 The Bleeding and Clotting Disorders the procedure used. Certain thromboplastins and activated partial thromboplas- tins are not compatible with certain instruments. Turbid or paiticulate throm— boplastin and activated partial thromboplastins may not work in instruments that detect endpoints via an optical system. The manufacturer’s directions as to the use of the thromboplastin and activated partial thromboplastin must be strictly followed. In many instances, the reagent must be mixed immediately before use in order to resuspend particles in the reagent. Store the reagent at the temperature the manufacturer recommends and, during tests, do not keep it at 37°C for longer than the manufacturer recommends. The concentration of calcium chloride used in the APTT test is critical and is related to the concentration of the sodium citrate used as anticoagulant. Some of the coagulant procedures require the use of buffers to ensure that the reactions take place in a physiologic pH range. These buffers must be carefully prepared and the pH checked before they are stored at 4°C—10°C. Reagent—grade deionized water should be used, if available, in testing and in prepaiing reagents. Water with a high pH will prolong the results if the system is not properly buffered. Anticoagulants prepared with water that contains ammonia will cause rapid deterioration of factor V and thus prolong the prothrombin time. Store the thromboplastin, activated paitial thromboplastin, buffers, and anticoagulants at 4°C- 10°C, but keep the calcium chloride and water at room temperature. Equipment Clean, unscratched tubes are used for storage and testing. As for pipettes, acid cleaning (3 mol/L HCl) is generally considered the best method for cleaning glassware for coagulation studies. The glassware must be thor— oughly rinsed to remove all traces of acid because this could change the pH. Disposable glassware and plastic ware are very useful and eliminate the time— consuming process of acid washing. Siliconized glassware must be kept separate from nonsiliconized glassware. Manual endpoint detection requires a temperature—controlled water bath and stopwatch or foot timer. The temperature of the water bath must be 37°C i 1°C. A lighted water bath facilitates reading the endpoint. If the tube is removed from the water bath for reading, the temperature of the plasma in the tube drops rapidly. For this reason, the use of a hook to detect the clot is recommended instead of the tilt-tube method. Automatic endpoint detection devices are widely used. Most instruments are in fact semiautomatic, since some nonautomatic pipetting is usually necessary. However, instruments that will pipette both the plasma and the reagents are available. The reproducibility of endpoint detection provided by the automated and semiautomated devices greatly improves the comparabil- 47 ity of results obtained in different laboratories. Regardless of whether a water bath or an automated instrument is used to detect endpoints, the temperature must be checked and recorded each day of testing. In a laboratory with resources to purchase an automated instrument, the choice of such an instrument depends on its precision, dependability, and ease of use and repair. Instrument evaluations can be found in the scientific literature. Additional information can be obtained from reviewing results of proficiency testing and external quality assessment programs. The time required for clots to form varies, depending on whether optical, mechanical, or electrical endpoint detection devices are used. Generally, the endpoints obtained with optical devices are shorter than endpoints Obtained with electrical devices. Therefore, alternating between types of endpoint detection devices is not advisable. The main coagulation instrument and the back-up instrument should have the same type of endpoint detection device. If different systems are used, each should have its own normal range estab- lished. Even under these conditions, results obtained with different instru— ments may vary. Some instruments with an optical system cannot accurately test lipemic, hemolyzed, or jaundiced plasmas. Synthetic substrates have been developed for the determination of endpoints in clotting assays. How— ever, the reactions in these assays are such that the endpoints may not have the same clinical significance as the measurement of clot formation. Techniques Many variations in test results are caused by faulty technique. Minor variations in technique, reagents, temperature, and pH cause substantial variation in test results. The incubation time and temperature are critical parameters of control in the 1—stage prothrombin time test. The plasma must not be maintained at 37°C for longer than 10 minutes, and the thromboplastin for no longer than the manufacturer recommends. Reactions involved in blood coagulation are enzymatic; therefore, opti- mum conditions in terms of pH and ionic strength must be established whenever an assay procedure is developed. Most routine assays are per- formed in buffer systems covering the physiologic pH range of the blood. The pH of the reaction mixture must be 7.2 — 7.4. The successive steps of the test procedure must be exactly defined as to the amounts of reactants used, the order of adding them, and the incubation times for the different reactants. Incubation periods for the activated partial throm- boplastin reagent and plasma mixture must be exactly the same for each testing. The mixing procedure determines the amount of contact activation in the APTT. Inconsistent technique in mixing the activated partial thrombo- plastin and plasma during the incubation procedure will cause variable results. The plasma samples are checked for hemolysis, jaundice, lipemia and clots. Erythrocytes contain phospholipids, which, when released, contain a 48 The Bleeding and Clotting Disorders coagulant activity similar to that of platelet factor 3. This phospholipid could shorten the APTT of hemolyzed plasma samples. Any clots in a sample, no matter how small, would invalidate all results obtained. Dilution errors cause variable results. The proportion of blood to antico— agulant must be precise. Patients with a high packed—cell volume may have erroneously prolonged prothrombin times because of the higher concentra- tion of citrate in the plasma. After the samples are centrifuged, the packed cell volume (PCV) of red cells is noted, as samples from patients with high PCVs may give misleading results. In such cases, it may be advisable to obtain another sample containing a smaller amount of citrate. We recommend a chart (Figure 1 1) for determining the volume of citrate to use when collecting blood from patients with high PCVs, i.e., above 0.65. Despite all precautions, biologic reagents and plasma change during a work period. It is wise, therefore, to minimize this change by designing the assay appropriately. Steps should also be taken to eliminate observer bias, especially in manual procedures. One of the ways to eliminate observer bias and systematic bias caused by time trends is to take one reading of each of several samples and another reading in reverse order. For example, four samples are tested in the order 1, 2, 3, 4, 4, 3, 2, 1. The extent of the time trend is apparent from the trend of differences between duplicates. 1.6— 1.5- 1.44 1.3- 5 1.2- E 1.1- E 2 : 910- : ’5 9- E “5 ' : (c .8- i 2 : E '7‘ ' Ttol = I O 2 5‘ 5 Volume .5- i 10ml .4- g 7 ’3‘ ' 5m: .2. 5 m .1- 5 2'0 2'5 3'0 3'5 4'0 4'5 50 5'5 6'0 6'5 7'0 7'5 8'0 Hemotocrit (°/o) Figure 11 - Chart for determining volume of citrate when collecting blood from patients with high PCVs 49 CONTROLS Normal and abnormal controls are included in each assay. Lyophilized controls in both the normal and abnormal range are commercially available. Commercial firms maintain that these controls usually work best when they are prepared by the manufacturer who made the thromboplastin and activated partial thromboplastin reagents. “Homemade” controls may be fresh, frozen, or lyophilized. Fresh plasma must be tested immediately; frozen plasma controls can be used for 3-4 months, and the lyophilized plasma controls for at least a year. If the plasma is to be used for a control chart, it should be prepared from at least 10 normal persons, both male and female. Plasma from pregnant women and women who are taking oral contraceptives should not be used, since the activities of many of the clotting factors are elevated in these conditions. REFERENCE VALUES (NORMAL VALUES) The phrase “normal values” indicates good health, which is regarded as a normal condition. This standard is applicable worldwide, and a person is said to be in good health when such a person meets all the standard criteria of normal health, the only allowed variables being those of age, sex, and geographic location (altitude). The values of statistical normality based on relative frequencies define the upper and lower limits of variation; the central 95% values within this range are accepted as the normal range for a given homogeneous population of normal people. The laboratory must have suitable scales of normal values for comparison. The term “reference values” refers to the health characteristics of a given defined group or population. It may be a normal population as described above, but it could also refer to a community of people who have an endemic disease or environmental condition, including smoking, in which it is impor- tant to be able to identify the patients who may have a superimposed illness. It is, of course, essential to define the reference population. Blood type (Group ABO) must also be taken into account. In establishing reference values in any age group and for each sex, a sample size of 40 persons per category is usually adequate. These donors should represent the age range of the population and should be healthy persons taking no medication. The simplest method for calculating reference values, using a selected, apparently normal population, is as follows: 50 The Bleeding and Clotting Disorders 1. .U‘PP’N Calculate mean and standard deviation (SD) using the formula: Sum of all results 11 2 SD = Emil where . n _ x = individual measurement i (mean) = )7 = mean 11 = number of values From these date, calculate the range, )7: — 3SD to x + 3SD. Eliminate from the original data any values outside these limits. Recalculate i and SD from the remaining values. The accepted normal range is then i i ZSD, which includes 95% of all normal subjects. SUMMARY In summary, the following quality control measures are taken: 1. S” 93”.“? Carefully collect blood with the two-syringe technique using plastic or siliconized syringes with large— gauge needles. Store plasma in tightly—capped plastic tubes and test it within 4 hours. Freeze and store plasma that cannot be tested within 4 hours. Use a concentration of 0.1 1 mol/L sodium citrate to anticoagulate the blood. Strictly follow the manufacturer’s directions for storage and use of products. Maintain the reaction temperature at 37°C i 1°C. Check pH and molarity of reagents used in tests (p. 105). Ensure that 3 Procedures manual is available to all personnel. Do not keep the plasma at 37°C for longer than 10 minutes and the thromboplastin or activated partial thromboplastin for longer than the manufacturer recommends. 10. Eliminate observer bias and time trends by appropriately designing the experiment. 11.Measure all samples in duplicate unless precision is checked by a control chart. 12. Include controls each day on which tests are performed. 13. Establish reference values for each analysis. CHAPTER 5 LABORATORY SAFETY All laboratory workers must be aware of potential hazards and must know how to ensure adequate protection. Hazards fall into the following categories: 1. Biohazardous specimens 2. Toxic chemicals 3. Physical accidents 4. Electrical and fire hazards MICROBIOLOGIC SAFETY All specimens from patients should be regarded as potentially infectious for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and other blood-bome pathogens. They must always be handled carefully to minimize exposure of skin and mucous membranes to blood and other body fluids of patients. This practice is an integral part of universal precautions. Collection of blood and other body fluids Universal precautions include thorough handwashing and in particular washing hands and other skin surfaces immediately if they are contaminated with blood or other body fluids. In case of accidental skin puncture wash the affected part gently in running tap water without scrubbing. Disposable plastic or thin rubber gloves should always be wom when performing phlebotomy on an agitated or uncooperative patient, when collecting capillary blood, and in all cases if the health—care worker has cuts, scratches, or other abrasions or skin breaks on his/her hands. A fresh pair of gloves must be used for each patient. Institutions that consider that routine gloving for all phlebotomies is not necessary should periodically reevaluate their policy. Gloves should always be available to health-care workers who wish to wear them for phlebotomy. Care must be taken to prevent injuries when handling syringes and disposing of the needles. Do not recap used needles by hand; do not detach the needle from the syringe or break, bend, or otherwise manipulate used needles by hand. Used disposable syringes and needles (and other sharp items such as glass slides) must be placed in a puncture-resistant container for disposal. All patient specimens must be sent to the laboratory in individual closed plastic bags, and request forms should be kept separate from the specimens, to prevent them from becoming contaminated should there be any leakage from the specimens. 52 The Bleeding and Clotting Disorders Cleaning syringe and needle NOTE: It is important to make clear from the outset that this applies only to syringes and/or needles that are reusable. If the syringe and/or needle are reusable, wash them thoroughly in tap water to remove all traces of blood after they are used. Soak needles overnight in a biodegradable detergent (e. g., Decon 90), then rinse thoroughly under running tap water, soak in two changes of distilled (or demineralized) water, and dry in an oven at 60°C. Place syringes (with pistons and barrels separate) in a large pan containing detergent (e.g., Decon 90), then rinse thoroughly under running tap water, soak in two changes of distilled (or demineralized) water, and dry in an oven at 60°C. Place syringes (with pistons and barrels separate) in a large glass or metal tube, and plug the container with nonabsor- bent cotton wool. Place each needle in a small test tube with a pad of nonabsorbent cotton wool at the bottom of the tube to protect the tip of the needle, and plug the open end. Sterilize in a hot air oven at 180°C for 30 minutes, or autoclave at 121°C at 1.4 kg/cm2 (20 lb/sq.in) for 20 minutes. (It is preferable to use disposable needles whenever possible; if non-disposable needles are used, inspect them between each use to ensure that the point has not become hooked; if it has, discard the needle). Disposable syringes and needles must not be reused. Laboratory tests Whenever any material of human origin is handled in the laboratory, mouth pipetting is forbidden. Eating, drinking, smoking, and applying make—up must not be allowed in the laboratory working area. Handwashing, as described above, and other techniques to avoid contamination by specimens must always be observed. The following established precautions are also recommended for situa— tions in which there is an increased risk of contamination with blood and other body fluids. 1. Use a dedicated part of the laboratory. 2. Wherever possible use disposable plastic containers instead of glass— ware. Do not use broken or chipped glassware and take great care not to prick yourself with sharp-pointed instruments such as scissors or needles. After sharp instruments such as scissors have been in contact with patients or patient material, they should be sterilized before reuse. 3. Wear close—fitting disposable plastic or thin rubber gloves. Wear glasses or goggles and a disposable plastic apron over the normal laboratory protective clothing when carrying out procedures in which aerosol spray may occur. 4. Keep the bench area clear of all equipment other than that required for the immediate procedure. 53 5. Centrifuge specimens only in sealed centrifuge buckets to minimize droplets or aerosol spray. 6. For the “housekeeping” and decontamination procedures described below wear general purpose utility gloves (e.g., rubber household gloves). These gloves may be washed and decontaminated for reuse but should be discarded if they begin to peel or crack, or if they have punctures, tears or other signs of deterioration. 7. Immediately after completion of the work, disinfect the working area with freshly prepared solution of sodium hypochlorite (household bleach) at a dilution of 1%; soak pipettes in 25% solution for 30 minutes or longer; and use a 10% solution for cleaning up blood spillage or breakage in a centrifuge. Rinse bleach off since it is corrosive. 8. If it is suspected that a tube has broken during centrifugation, the centrifuge motor must be switched off and the lid left Closed for 30 minutes. If breakage is discovered only on opening the centrifuge, the lid must be replaced immediately and left closed for 30 minutes. After this time, while wearing protective gloves (see 6), pick up glass debris with forceps and place all broken tubes, glass fragments, buckets, and the rotor overnight in 2% activated glutaraldehyde, formalin, or a similar germicide, then reswab with the germicide (see Disinfection below), wash with water, and dry. 9. Automated blood cell counters, coagulometers, and other automated equipment should be disinfected after use by flushing them several times with 10% sodium hypochlorite solution, glutaraldehyde (freshly diluted to 2% in 3 g/L sodium bicarbonate), or a similar germicide, followed by thorough rinsing in water. Hypochlorite should not be used in instruments with metal surfaces because it causes corrosion. 10. Place all disposable material in robust, labeled plastic bags which should be transported without spillage to the autoclave indicated on the label. 11.All persons should wash their hands after completing laboratory activities and should remove protective clothing before leaving the laboratory. 12. Disposable equipment (e. g., gloves) must not be reused, as they may retain contaminated material and may deteriorate when cleaned. Disinfection The most commonly encountered dangerous pathogens in hematology and blood transfusion laboratories are hepatitis viruses and HIV. Phenolic disin— fectants are ineffective against these viruses and have been omitted from consideration. As a general rule, hypochlorites are cheap and are the first choice for disinfecting all but metal surfaces, whereas glutaraldehyde is more expensive but is useful for metal (or metal containing) surfaces or instru- 54 The Bleeding and Clotting Disorders ments. Both hypochlorite and glutaraldehyde are inactivated by organic material such as blood and therefore have a very limited “bench-life.” Disposable gloves should be worn when using any disinfectant. Hypochlorites (Chlorous, Domestos, DBX) These substances attack metal to a varying degree and must not be used on metals or moving parts of machinery. They are suitable for blood and viruses but not for mberculous material. Commercial products usually contain 100,000 ppm available chlorine. They should be used as follows: 1. General use: 1% dilution 2. Contaminated glassware jars: 2.5% dilution 3. Blood spillage: 10% dilution Fresh working hypochlorite solution must be made daily. Aldehydes These consist of formalin and glutaraldehyde (Cidex). Formalin is a 30 w/ v solution of formaldehyde and for general use is diluted 1 in 10 v/v. Formaldehyde gas, prepared by boiling together equal volumes of formalin and water, is useful for disinfecting fume cupboards but is too irritant for general use. Formaldehyde is used as a fixative in some flow cytometric applications. Formaldehyde mixed with oxidant results in the formation of a carcinogenic substance. For this reason formalin and bleach must not be mixed. Glutaraldehyde does not readily penetrate organic matter and is most effective on surfaces that have already been cleaned. It is less irritating than formalin and is most useful for disinfecting centrifuges and other metal equipment. It is most efficient at pH 7—8 but deteriorates after becoming alkaline. For use as a disinfectant, 2% glutaraldehyde is diluted in 0.3% bicarbonate buffer. In recent years, however, the safety of glutaraldehyde has been questioned because of increasing evidence of its irritant properties, particularly to the skin and eyes. In addition, it is hepatotoxic, carcinogenic, and teratogenic. An airborne exposure limit of 0.2ppm has been enforced, but when skin contact occurs, overexposure may occur even within the statutory airborne limit. Proxygen-containing compounds are now being used as an effective alternative with proven antibacterial, antifungal, and antiviral actions. It is often helpful to add a compatible detergent to the disinfectant to be used as this aids cleaning and therefore efficient disinfection, since disinfectants are most active on clean surfaces. There are, however, constraints on this procedure. For example, hypochlorites are compatible with anionic deter- gents (e. g., Teepol, HB6, or Triton GRS) and with non—ionic detergents (e. g., Nonidet P40 or Triton X-100) but not with cationic detergents such as cetrimide. 55 Waste disposal Blood and other potentially infected body fluids can safely be poured down a drain provided that the drain is connected to a sanitary sewer. Laboratory waste, such as blood specimen containers, used syringes, swabs, tissues, and dressings, should be incinerated or, alternatively, auto- claved and then disposed of in a rubbish dump. Boxes containing needles and other sharp objects should be incinerated without opening. Soiled laundry should be placed in leak—proof labeled bags for transport to the laundry where the items should either be washed in hot water (> 70°C) with detergent for 25-30 minutes before being rinsed, 0r subjected to a routine low- temperature washing machine cycle with suitable disinfectant. EXPOSURE TO TOXIC CHEMICALS A number of reagents used in hematologic tests contain chemicals that are extremely toxic (e.g., potassium cyanide) or potentially carcinogenic (e.g., benzidine). However, in the amounts required in test reagents the risk from these substances is negligible when used with reasonable care under normal working conditions. Reagents should be prepared from hazardous substances only by experienced workers; the following precautions should be observed: 1. Store the chemical in a secure place with restricted access. 2. Weigh the desired amount of chemical in a fume cupboard if available. 3. Wear plastic or rubber gloves and protective clothing. INJURIES Injuries may be caused by sharp instruments, needles, or broken or chipped glassware. These injuries can be prevented by being aware of the potential hazards and paying attention to good laboratory practice. An untidy cluttered bench increases the risk of injuries. ELECTRICAL HAZARDS AND FIRES These hazards arise from carelessness or improper use of electrical equipment, incorrect or poorly maintained fittings and connections, and the use of long trailing leads. In order to prevent electrical hazards, all equipment that uses electrical power should be certified by the manufacturers as conforming to safety requirements and should be installed by a trained electrician. Every power plug should be independently fused at appropriate amperage and, if possible, only one piece of equipment should be connected into each power point. 56 The Bleeding and Clotting Disorders Most fires result from accidents with flammable substances such as alcohol and solvents. All manipulations with such substances must be carried out away from naked flame. Bulk stocks should be kept in a storage area separated from the laboratory and clearly marked as FIRE RISK. MANAGEMENT OF INJURIES AND EXPOSURES Every laboratory should have a staff member trained in general laboratory safety. All injuries and exposures to biological, chemical, and electrical hazards should be reported to and recorded by him or her. There must be a defined plan of action for treating the exposed or injured person. CHAPTER 6 GENERAL PROCEDURES BLOOD SAMPLING Collecting capillary blood for blood films Capillary blood may be collected most conveniently from the fingertip. From infants it may be collected conveniently from the heel or large toe. For details of the containers and slides to be used, see the appropriate heading below. Procedure 1. Clean the skin thoroughly over the site to be punctured and the surrounding area with a cotton pad soaked in a suitable disinfectant, e.g., 70% alcohol. 2. Wipe the area dry with a sterile, dry gauze pad. 3. Puncture the skin, using a single stroke with a sterile disposable lancetz. Use a quick stroke, sufficiently deep to ensure a free flow of blood. Blood flow may be stimulated by gentle pressure exerted along the side of the finger a slight distance from the puncture site. 4. The blood must be free flowing. Too much squeezing will dilute it with tissue fluid and can make it unsuitable for examination. 5. Always discard the first drop of blood by wiping it away with a dry gauze pad, leaving the puncture site clean and dry. 6. Obtain the desired specimen from subsequent drops of blood, which are usually aspirated directly into the appropriate pipettes or placed on glass slides. Collection of venous blood for coagulation studies Venous blood can be collected most conveniently from the veins in the bend of the elbow, preferably just above one of the venous branches at this site. A tourniquet can facilitate collection. For young children, other sites may be used; if they are, it is better for an experienced worker to collect the sample. Use a sharp needle, not smaller than 21 gauge, and a 5 ml or 10 ml syringe. Both needle and syringe must be clean, dry, and sterile. With practice, blood may be obtained very satisfactorily by using a needle only or a needle with an attached piece of tubing and letting the blood run directly into the tube or 2 The best instrument for capillary puncture is a sterile disposable lancet, which must not be used for more than one patient. If a cutting needle is used, sterility must be ensured by adequate flaming to prevent transfer of disease, particularly hepatitis. Flaming, however, tends to blunt the needle and to leave some soot on the point. 58 The Bleeding and Clotting Disorders tubes. For this method, the needle must be 19 gauge. For coagulation studies the first 2 to 3 ml should be discarded, but this part of the sample can be used for a blood count, platelet count, or erythrocyte sedimentation rate (ESR). Procedure 1. Disinfect the skin over the site to be punctured by wiping with a swab soaked in a suitable disinfectant, e.g., 70% alcohol. 2. Prepare the syringe and needle, using a careful aseptic technique. 3. Apply a toumiquet to the arm (above the site to be punctured) tightly enough to restrict the flow of venous blood. 4. Disinfect the skin again over the site to be punctured; wipe the skin dry with a sterile, dry cotton swab or gauze square. 5. Ensure that the syringe plunger is as far into the syringe barrel as it will go, i.e., that the syringe does not contain air. Insert the syringe needle into an enlarged vein with one steady motion. 6. Slowly draw back the syringe plunger and collect 2 t0 3 ml of blood into the syringe. 7. Remove the syringe from the needle and attach another syringe containing the anticoagulant sodium citrate. If the concentration of sodium citrate is 0.11 mol/L, then enough sodium citrate should be placed in the syringe to achieve a final concentration of 1:10 with blood, e.g., 0.5 ml for a 5 ml blood sample or 1.0 ml for 10 ml blood sample. 8. Slowly draw back the syringe plunger and collect the required volume of blood into the syringe. 9. Release the tourniquet, place a dry gauze square over the puncture site, and withdraw the needle from the vein in one motion. 10. Maintain gentle pressure on the puncture site for a moment with the sterile cotton pad, then ask the patient to continue to hold this pad in place with some pressure while you process the blood in the syringe. 11.When the blood is collected with a needle only, the same procedures and amount of anticoagulant apply. Graduated tubes should be used so that the appropriate volume of blood to citrate is used. BLOOD FILMS Principle A drop of blood is spread on a slide, stained, and examined under the microscope. In this way, red blood cells, leukocytes, and platelets may be studied. The purpose is to establish the morphologic features of each cell type and to assess the relative frequency of different leukocytes. The slide is stained with one of the Romanowsky stains. These stains vary in their staining 59 properties, and it is essential to select one and to become familiar with it. Wright’s stain is fairly reliable and simple to use, but others are equally satisfactory. For example, ICSH has recommended a standardized stain con- sisting of pure Azure B and Eosin Y, the two components essential for producing the Romanowsky effect. This gives consistently reliable results because the purity of the dyes ensures that the staining will not vary. It is particularly satisfactory for demonstrating malaria or other blood parasites. Wright’s stain is also fairly reliable for staining blood films and is simple to use. Others (e. g., Leishman’s, May-Griinwald and Giemsa) are equally satisfactory, although different batches of stain may give variable staining. The ICSH stain and Wright’s stain are described below. Equipment 7. QMPP’N?‘ Microscope 25- x 75-mm glass slides Rectangular cover glasses Neutral mounting material Immersion oil Slide rack Pasteur pipettes Wright’s stain Reagents 1. Wright’s stain 1 g Absolute methyl alcohol, acetone free 600 ml Add the alcohol slowly, a few milliliters at a time, and mix well with the aid of 10 to 20 glass beads. Keep well stoppered to prevent evaporation. Store in dark for 2 to 3 weeks, mixing at frequent intervals. Filter before using. Leishman’s stain is an adequate substitute and may be preferred in areas where malaria or other blood parasites are prevalent. Buffer: phosphate buffer, 67 mmol/L, pH 6.8 (Reagent 15, p. 106) Fixative: absolute methyl alcohol. The bottle must be kept well stoppered to prevent hydration. This is particularly important in tropical, humid atmospheres. One way to prevent this is to use small bottles of alcohol (pint—size or less)so that the alcohol is used soon after the bottle is opened. Specimen EDTA venous blood sample in EDTA (at 1.5 mg/ml blood) or capillary blood. 60 The Bleeding and Clotting Disorders Procedure 1. Make a pusher slide by breaking off one corner of a 25- X 75 -mm glass slide, choosing one with a smooth edge. A piece of stiff plastic of similar size may be used instead. 2. Put a small drop of blood about 2 to 3 mm from the end of a clean, dry slide. Place the pusher slide at an angle of 30° to 45° to the slide, and then move it back to make contact with the drop. The drop should spread out quickly along the line of contact of the pusher with the slide. The forward movement of the pusher spreads the blood on the slide. The film should be about 300 mm long (Figure 12). The pusher should not be lifted off the slide until the last trace of blood has been spread out. The thickness of the film can be regulated by changing the angle at which the pusher slide is held, by varying the size of the drop of blood, and by varying the pressure and the speed of spreading. 3. Allow the film to dry in the air. Write the patient’s name and the date on the edge of the film in pencil. 4. Fix the film by placing the slide in absolute methyl alcohol for 2 to 3 minutes. The smear will change from red to light brown. This fixation is desirable, even though the stain is in absolute methyl alcohol. 5. Completely cover the slide with stain. After 3 minutes, add an equal volume of buffer. Blow gently to ensure uniform mixing. A green metallic sheen will appear. 6. After an additional 5 minutes, rinse thoroughly with tap water. Run the stream very slowly at first and then more briskly in order to free the film of all traces of excess stain. Wipe the back of the slide to remove all traces of stain. Air dry with the slide in an erect position on a slide rack. When completely dry, cover the film with a rectangular cover glass, fixing it in position by spreading a drop of mounting material between slide and cover glass. For a temporary mount, immersion oil may be used. A cover glass is essential because it permits the film to be examined at a low power. The practice of examining a slide solely with the oil immersion lens must be avoided. If the slide is used as a temporary mount, the cover glass can be reused. 7. Study red cell morphology on all slides. The area suitable for the grading of red cells should be an area where most of the red cells are just touching but not overlapping. The film should be scanned to obtain an overall estimate of the total leukocytes and to identify any unusual or abnormal cells. The numbers of platelets should also be estimated, and the presence of abnormally large platelets should be noted. 61 Pusher Drop of blood Drop of blood spread evenly along edge of pusher Figure 12 - Illustration of method for preparing blood film ICSH Stain Reagents l. Azure B: Azure B 3 g Dimethyl sulfoxide (DMSO) 300 ml Preheat the DMSO to 37°C before adding the Azure B, then stand for 30 minutes at 37°C, shaking vigorously for 30 seconds at 5 minutes intervals. 2. Eosin Y: Eosin Y 1 g DMSO 250 m1 Prepare in the same way as Azure B. 3. Stock stain: Slowly mix the Azure B-DMSO solution with the Eosin—DMSO solution over a 30—minute period, using a magnetic stirrer if available. Keep in a dark cupboard at room temperature in a tightly stoppered brown bottle. Under these conditions the stock will remain stable for 3 months. 62 The Bleeding and Clotting Disorders 4. Staining solution: Stock solution 1 volume HEPES buffer (p. ) 14 volumes Prepare fresh each day; it is stable for about 8 hours. HEPES Buffer; pH 6.6: HEPES 2.38 g Add distilled water to make 1 liter Adjust pH to 6.6 with 1 mol/L NaOH Then add 50 m1 of DMSO Phosphate buffer pH 5.8; 67 mmol/L: KH2P04 (9.1 g/L) 92.2 ml NaZHPO4 (9.5 g/L) 7.8 ml 7. Fixative: Stock solution 1 volume Methanol 14 volumes Specimen EDTA venous blood samples or capillary blood. Procedure l.-3. As above for preparation of film 4. 5. 6. Fix the film by placing the slide in fixative solution for 3 minutes Completely cover the slide with staining solution. After 10 minutes replace the staining solution with phosphate buffer for 1 minute. Rinse thoroughly in tap water, and continue the procedure as in 6 and 7 above. Sources of error . Glassware should be absolutely free of oil and grease. Wash slides with soap and water, rinse, and if possible, store in 95% ethyl alcohol. If needed, dry the slides with a clean towel. . Blood films must be completely dry before staining; otherwise the wet areas will wash off the slide. Slides should be fixed as soon as possible after preparation, since leukocytes tend to become distorted and disintegrate. If the smear is excessively blue, it may be too thick or it may have been insufficiently washed or stained too long, or the stain, buffer, or water used may have been excessively alkaline, or the stain itself may be chemically contaminated. If the stain is too red, the stain, buffer, or water may have been excessively acid or the stain contaminated. 63 7. Stain precipitate on film indicates the need for filtration; the stain must be filtered each day before use. 8. Many hematologists believe that the best cell morphology is achieved by using blood that contains no anticoagulant. However, if anticoagulated blood is used, the film should be developed immedi— ately, if possible, or less than 1 hour after the blood has been collected. Longer delay may lead to some decrease in the quality of the morphol- ogy. The different appearances of platelets when anticoagulated and coagulant-free blood is used are referred to below. 9. The pusher may not be clean, or the edge of the pusher may not be smooth. 10. The slide may not be clean and free from dust, oil or grease, and finger marks. The slides should be stored in a jar of alcohol; they should be removed and wiped with a lint-free cloth just before use. 1 l. The film may have ridges, waves, or holes instead of being smooth and even. 12. Inadequate fixation may affect subsequent staining and morphologic appearances. This is especially likely to occur if the stock of methyl alcohol is not “absolute” and if it has absorbed water, e.g., from a humid atmosphere. Estimation of platelet count and platelet morphology in a blood film A thin blood film is needed, since the number of platelets has to be counted in relation to red cells. Count the number of red cells in a high-power field (X10 eyepiece and X40 objective), and count the number of platelets. Platelets normally clump in groups of 2 to 5; therefore, counts can be more easily performed in films prepared from EDTA—treated blood. The normal value is 4—8 platelets per 100 red cells. A reasonable estimate of the platelet number can be obtained by counting the platelets present in relation to 500 red cells. Twenty platelets counted per 500 red cells is about 150 X 109/1 but could be 75 —300 X 109/L, depending on the technical limitation of the procedures. This estimate, of course, is only true for patients without anemia; the range of the estimate is even greater in patients with anemia. If the platelets are not clumped in a blood film made directly from a puncture wound, their function may be clearly abnormal. Failure of platelets to clump, in connection with a very much prolonged bleeding time, indicates Glanzmann’s thrombasthenia, provided that the intake of platelet function— inhibiting drugs and the existence of other acquired platelet abnormalities, such as uremia, are excluded. The diameter of platelets is normally about 2— 3 tun or 1/3 of the red cell diameter. When platelets are longer than this, one has to consider a high platelet production level, as in idiopathic thrombocy— topenic purpura (ITP). Platelets that are nearly as large as red cells occur in 64 The Bleeding and Clotting Disorders the myeloproliferative disorders and in two rare disorders ,i.e., Bemard- Soulier’s platelet abnormality and May—Heggelin anomaly. OBSERVATION OF THE CLOT Principle Examination of a clot found in a tube gives information on (a) the concentration of fibrinogen, (b) the number and function of platelets, and (c) the activity of the fibrinolytic system. Equipment Water bath; glass round-bottom tubes; wire hook. Specimen Clotted blood from anticoagulant-free blood in a glass tube. Procedure 1 . Place the tube containing the blood in the water bath at 37°C for 3 hours. 2. If there is no visible clot surrounded by serum, try to find a clot by manipulating the wire hook, and putting the clot on filter paper. If this attempt is not successful, pour the contents of the tube carefully into another empty tube to see if a small clot is present. Result 1. About 30% of the total volume in the tube should be clot (Figure 13). 2. A very large clot with a weak structure is found in cases of thrombo— cytopenia or with very severely disturbed platelet function such as thrombasthenia. Also, in cases of paraproteinemia (myeloma or Waldenstrém’s disease) retraction of the clot can be severely dis- turbed. 3. A small clot with a regular shape is found if there is a low fibrinogen concentration. 4. A small, irregular clot is found when the clot is being digested by enhanced fibrinolysis as part of disseminated intravascular coagula- tion (DIC). 5. No clot at all is found in complete afibrinogenemia, as seen in congenital afibrinogenemia or in severe DIC. Sources of error 1. Dirty tubes. 2. Contamination of the blood with an anticoagulant (citrate, EDTA). 65 V Figure 13 - Examples of clots found in normal persons and in patients with some coagulation abnormalities: (1) normal; (2) thrombocytopenia; (3) low fibrino- gen; (4) enhanced fibrinolysis PLATELET COUNTS Principle Blood is mixed with a diluent that causes hemolysis of red cells. A hemocytometer is filled with the diluted fluid, and the platelets are counted under the microscope, preferably by using phase-contrast, if available. Equipment 1. 99%.“ Flat-bottom, thin counting chamber (phase-contrast hemocytometer with Neubauer ruling). Preferably, a phase-contrast microscope equipped with long-working- distance phase condenser, if avaiable; otherwise an ordinary light microscope. ZO-LLl pipette 2—m1 graduated pipette 12-X 75-mm tube Mechanical mixer 66 The Bleeding and Clotting Disorders Reagent Diluting fluid: 1% ammonium oxalate in distilled water. Store in the refrigerator, and always filter just before using. Specimen EDTA-treated venous blood or capillary blood. Procedure 1. S” If the blood sample is from a finger prick, the puncture must be clean and the blood free flowing. Wipe away the first drop of blood. If the blood sample is from venous blood, it must be collected into a dry plastic (or siliconized glass) syringe with a short needle not smaller than 21 gauge. The needle must be removed before the blood is delivered into a plastic container with EDTA. The blood and antico- agulant must be mixed gently, to avoid frothing, without any delay. Pipette 0.38 ml of diluting fluid into a test tube. Fill the 20411 pipette to the mark, and wipe off the outside of the pipette. Expel the contents of the pipette into the diluting fluid, and wash out the pipette by drawing up the blood and expelling it into the tube a few times. Mix for at least 10 minutes by hand or, preferably, by mechani— cal mixer. Fill the hemocytometer as described below. Cover the chamber with a petri dish for 10-20 minutes to allow the platelets to settle. Leave a piece of wet cotton or filter paper in the dish to prevent evaporation. Using a phase—contrast microscope, count the platelets in the large squares of 1 mm (= 0.1 ul). Count the platelets in as many squares as necessary to reach a count of at least 100. The platelets appear round or oval, and their intemal granular structure and purple sheen allow them to be distinguished from debris. Ghosts of the red cells that have been lysed by the ammonium oxalate are seen in the background. If phase contrast is not available, an ordinary light microscope can be used, provided the condenser is racked down to provide a low intensity of light. Calculate the number of platelets per liter of blood according to the formula below. The hemocytometer The hemocytometer counting chamber, with Neubauer or improved Neubauer ruling, is so constructed that the distance between the underside of the cover glass and the surface of the chamber is 0.1 mm. The surface of the chamber contains two specially ruled areas with dimensions as shown in Figure 14. The central 1 mm2 has double or triple boundary lines. In the central 67 Figure 14 - Hemocytometer counting chamber (a) Neubauer and (b) improved Neubauer areas are 25 squares in the improved Neubauer and 16 squares in the Neubauer ruling. Each square has an area of 0.04 mm2 (0.2 X 0.2 mm). These squares are in turn divided into smaller squares, each 0.0025 mm2 (0.05 X 0.05 mm). The outer quadrants of the ruled area are each 1 mm2 and are divided into 16 squares. 68 The Bleeding and Clotting Disorders Calculations The formula for calculating the cell count is: Count (cells/L) = N X D/AX 10 X 106 Where N = total number of cells counted D A 10 = dilution = total area counted (in mmz) = factor to calculate volume in pl from area (in mmz) and depth of chamber (0.1 mm) 106: factor to convert count/ul to count/ul Sources of error in cell counting l. 2. When capillary blood is used, a free-flowing drop must be obtained. When anticoagulated blood is used, the specimen must be carefully mixed by inverting the tube of blood at least 20 times before a sample is taken. Do not shake the tube, because shaking introduces foam, which makes accurate pipetting impossible. Tilt the well—mixed tube to an angle of 45° or slightly more, and pipette from the lip of the tube, following the same procedures as for capillary blood. The blood sampling pipettes must be clean and dry. The pipette must be filled quickly, and the blood must be drawn accurately by using a pipette suction device attached to the pipette, filling up to the desired line. If the line is overshot slightly, the excess blood may be expelled by touching the tip of the pipette on a piece of filter paper or soft tissue. If the line is overshot more than slightly, a fresh pipette must be used. No air bubbles should be present in the blood column. The outside of the pipette must be wiped free of blood (being careful not to pull blood from the tip) before it is introduced into the diluting fluid. After the contents of the pipette have been discharged into the diluent, the diluting fluid must then be drawn into the pipette with steady suction several times, to ensure that all the blood is discharged into the fluid. The tube containing the diluted blood must be shaken gently for at least 2 minutes by hand or, preferably, in a mechanical shaker. After the tube has been shaken the chamber is immediately filled by means of a Pasteur pipette or capillary tube. The chamber is filled by capillary action, with the flow of fluid from the pipette or capillary regulated so that it fills quickly and smoothly. It must be filled completely, but fluid must not spill over into the moats. Allow the cells to settle in the counting area for 10 to 20 minutes, then proceed with the counting. 69 10. The hemocytometer Chamber and cover glass must be clean and dry 11. before they are used. Grave errors are introduced by fingerprints or an oily film. A sufficient number of cells must be counted to reduce error due to chance distribution of cells. In practice, at least 100 cells should be counted. As a further check on correct distribution of cells in the chamber, the number of cells counted in each area (i.e., in the large squares) should not differ by more than 10%. Controls 1. 2. Two dilutions must be made, and the mean of the two counts taken; the two counts should agree within 10%. The platelet count on the chamber should be correlated with the platelets seen on the smear. Sources of error in platelet counting 1. 4. 5. Blood obtained by a venipuncture is preferable to capillary blood, because platelets adhere to the wound and successive dilutions from a finger prick are not always reproducible. The general errors of pipetting and hemocytometry are described above. In addition, special attention must be paid to ensuring that the counting chamber is scrupulously clean, since dirt and debris may be counted as platelets. Wash the chamber with soapy water, then rinse with distilled water, allow to drain dry, and wipe with lint-free tissue. Be sure that the cover slip is clean before using it. The presence of platelet clumps precludes reliable counts. If the sample contains clumps, a fresh sample must be collected. The ammonium oxalate diluent should be kept refrigerated and must be discarded if there is evidence of bacterial contamination. The specimen must be counted within 3 hours of collection. Electronic counting Platelet counts can be performed with greater precision by means of an electronic cell counter. However, this requires an aperture tube with a smaller orifice (50 um) than is used for red cells and leukocytes. Also, calibration is more complex because pulses due to small platelets must be distinguished from those generated by electrical interference and other “noise”. In the simpler instruments the count can only be carried out on a blood sample after separation of the platelets from the red cells; preparing this suspension may also be a cause of error. 70 The Bleeding and Clotting Disorders BLEEDING TIME Principle The bleeding time is a measure of vascular and platelet integrity. Platelets adhere to exposed collagen (subendothelial) and subsequently to each other to form an aggregate that plugs the wound. For the aggregation of platelets a plasma factor, the von Willebrand factor, is also necessary (p. 3). The bleeding time is measured by determining the time required for bleeding to stop from small subcutaneous vessels that have been severed by a standardized incision. One of the major problems encountered in perform- ing the bleeding time is reproducibility. For that reason, three generations of tests have been developed, each with increasing standardization of a wound of uniform depth and length. The oldest method is the Duke method, which is performed by puncturing the ear lobe with a lancet. The drawback of this method is that it is impossible to standardize the depth of the incision. In addition, if the patient has a significant bleeding disorder, bleeding into the soft subcutaneous tissue in the ear lobe could lead to a large hematoma. The Duke method is not recommended and will not be described here. The Ivy method is more standardized than the Duke method. This method uses a lancet (known as a stylet) to produce an incision in the volar side of the forearm. The blade has a shoulder that limits the depth of the cut. In addition, because the volar side of the forearm is large enough to accommodate several incisions, two or three wounds can be made and the results averaged. Furthermore, this method provides improved standardization of the pressure in the vascular system, because a sphygmomanometer cuff around the upper arm (40 mm Hg) maintains venous pressure within narrow limits. More recently, the template method has been introduced; it allows a standard cut to be made under the same conditions as the Ivy test. The cut is made with a surgical blade in a holder that allows the blade to cut 1 mm deeper than the template is thick. The bleeding time is longer than with the Ivy method. The template method is very reproducible, but the relatively large incision may produce scars, which can result in keloid formation. Furthermore, in follow-up examinations the patient might complain about having several additional incisions made on the arm. Some commercial versions are avail- able that reduce these problems. 71 The Ivy method Equipment 1. Sphygmomanometer 2. A stopwatch (preferably three Stopwatches) 3. Circular filter paper 4. Alcohol for disinfection 5. Cotton-wool pads or gauze 6. Disposable lancets (2 mm pointed blades with shoulders) 7. Sterile bandages Procedure 1. Apply the manometer cuff around the upper arm; gently cleanse the forearm with an alcohol pad and allow to dry. 2. Inflate the cuff to 40 mm Hg, and wait 30 seconds to allow capillary filling to equilibrate. 3. Make three cuts on the lower arm, preferably on the anterior side where there is no hair; avoid superficial veins. 4. Start one stopwatch for each puncture wound when bleeding begins; in general, bleeding starts within 15—30 seconds. 5. If bleeding does not start within 30 seconds, spread the wounds slightly between two fingers (this does not change the test result). 6. Gently blot the blood with a circular filter paper at 15—second intervals; try to avoid direct contact of the filter paper with the wound. 7. The endpoint is reached when blood no longer stains the filter paper. Record the time at this point for each puncture wound. 8. Average the bleeding times of the three wounds. 9. Clean the puncture sites and apply a sterile bandage. Results In general, 95% of all values are less than 4 minutes. If the bleeding time is between 3 and 4 minutes the possibility of a mild abnormality should be considered. Sources of error 1. 2. 3. No bleeding occurs because of too gentle an incision. Severe bleeding indicates that a superficial vein has probably been cut. If the filter paper touches the wound, a platelet aggregate might be removed, resulting in prolonged bleeding. 72 The Bleeding and Clotting Disorders Figure 15 - Illustration of method for performing template bleeding time The template method Equipment QMPP’N?‘ 7. 8. Sphygmomanometer Stopwatch (preferably two or three Stopwatches) Circular filter paper Alcohol for disinfection Cotton—wool pads or gauze Template, blade handle, and gauge (see Figure 15). Similar templates are available commercially Surgical blade (No. 11) Sterile bandages Procedure 1. Mount the surgical blade on the handle. Standardize the depth of the blade by placing the handle on the gauge. Adjust the blade so that the 9. 73 tip just touches the foot of the gauge. Be sure to keep the blade sterile while handling it. Tighten the screw holding the blade. . Apply the sphygmomanometer cuff on the upper arm; gently cleanse the forearm with an alcohol pad and allow to dry. Inflate the cuff to 40 mm Hg and wait 30 seconds to allow the capillary filling to equilibrate. Place the template on the forearm about 5 cm from the antecubital fossa. Apply firm pressure to the template while introducing the blade at a right angle on the upper portion of the template slot. This guides the blade to make the appropriate incision, which is usually 1 mm deep and 9 mm long; however, different incision lengths have been proposed (see the Results section p. 73). Make the incision smoothly and rapidly. Start the stopwatch immediately. Make a second (or third) incision parallel to the first. Under normal conditions the first full drop of blood appears in between 15 and 20 seconds. Gently blot the blood with a circular filter paper at 30-second intervals. The endpoint is reached when blood no longer stains the filter paper. The last drop is usually a mixture of red blood cells and serum. Record the time at this point. Average the bleeding times of the two (or three) incisions. 10. Clean the wounds, pull the sides together, and apply a bandage or adhesive strip, which must remain in place for 2 or 3 days to minimize scarring. 11.After a test, the template and gauge must be washed thoroughly with surgical soap, then rinsed well with water, and autoclaved or sterilized by a gas such as ethylene chloride. 12. New or sterilized blades should be used each time. Results Normal: 2—7 minutes with 9-mm length incision. Reference time in health and disease must be established if another incision length is used. In general, faint linear scars may persist. In some persons keloids may form at the incision. The patient must be advised of both possibilities before the test is done. Sources of error 1. Too much pressure on the template will permit too deep an incision, resulting in an erroneously prolonged time; too little pressure results in the reverse. 74 The Bleeding and Clotting Disorders 2. Severe bleeding indicates that a superficial vein has probably been cut. 3. If the filter paper touches the wound, a platelet aggregate might be removed, resulting in prolonged bleeding. Interpretation The test is highly variable and must be interpreted with caution in the light of the clinical features. Prolonged bleeding times are demonstrable in patients with: 1. Thrombocytopenia: In general a platelet count of 50 x 109 /L is sufficient for a normal bleeding time. Below 10 X 109 /L the bleeding time test is nearly always infinite; the bleeding time should not be done in a thrombocytopenic patient, particularly if it is known or suspected that the platelet count is less than 10 X 109 /L. 2. Acquired platelet-function abnormalities: Drugs, such as aspirin and indomethacin, block the release of vasoactive substances (e.g. ADP), from the platelets. The clinical effect lasts for about 4 days. In uremic patients, prolongation of the bleeding time usually becomes apparent at creatinine concentrations of 5 mg/dl (450 mol/L). The toxic sub— stance is unknown but is usually dialyzable. In patients with hematologic malignancies, especially when the megakaryocytes are abnormal in the myeloproliferative disorders, platelet abnormalities may be present. A prolonged bleeding time can, therefore, be seen in thrombocythemic patients. In reactive thrombocytosis, such as that seen after splenectomy, a normal bleeding time will always be found. In DIC there is a mild to moderate thrombocytopenia. Because the circulating platelets have been aggregated and subsequently released from microthrombi by the action of the fibrinolytic system, they have become degranulated and are functionally abnormal. 3. Von Willebrand’s disease: An autosomally dominant disorder, char- acterized by a deficiency of the von Willebrand factor (measured as a protein by the immunoelectrophoretic assay of VWF3Ag, and/or as an activity by the ristocetin—cofactor assay in a platelet-aggregation test), and a deficiency of factor VIII activity. The von Willebrand factor is essential for the aggregation of platelets. Von Willebrand’s disease exists in mild, moderate, and severe (homozygote) forms. The severe form has an infinite bleeding time. 4. Thrombasthenia (Glanzmann’s disease): An autosomally recessive disorder, manifest only in homozygotes. Platelet morphology is nor— mal, but clumping in blood films is absent. The bleeding time is extremely prolonged. 5. Congenital thrombocytopathia: Bernard-Soulier’s platelet abnor- mality is an autosomally inherited disorder of platelet function with mild thrombocytopenia. Platelet morphological examination shows 75 Figure 16. - Illustration of hook and waterbath used for clotting tests giant forms. The bleeding time is extremely prolonged. Storage-pool disease is an autosomally dominant disorder, with a mild to moderate prolongation of the bleeding time. 6. Afibrinogenemia: In congenital afibrinogenemia, a mild prolongation of the bleeding time occurs. 76 The Bleeding and Clotting Disorders MEASUREMENT OF THE EXTRINSIC SYSTEM Prothrombin time (one-stage) Principle The prothrombin time is the time required for plasma to clot after tissue thromboplastin and an optimal amount of calcium chloride have been added. Reference method for the l—stage prothrombin time test is that of the International Council for Standardization in Hematology/International Council on Thrombosis and Haemostasis (ICSH/ICTH). The method described on page 76 is a modification of the ICSH/ICTH reference method for the one-stage prothrombin time test. Equipment 1. 2. 3. 4. A waterbath, thermostat set at 37°C, containing a tube rack. Preferably, the waterbath should be made of glass or perspex. The tube can then remain in the waterbath when the test is performed (Figure 16). Wire hook. A handle containing a metal hook, made from a paper clip, that can be moved up and down in a test tube. Test tubes (round-bottom glass tubes) that fit the tube rack. Stopwatch. Reagents 1. 2. 3. Platelet-poor citrated plasma A thromboplastin reagent, commercial or homemade. A calcium chloride solution, 0.025 mol/L (in some commercial reagents this has already been added to the thromboplastin). Procedure No. 1 for commercial thromboplastin-calcium reagent 1. Add blood to 0.11 mol/L sodium citrate in a ratio of nine parts blood to one part citrate. Centrifuge blood at 3000 rpm (l600X g) for 15 minutes to obtain platelet-poor plasma. Incubate plasma at 37°C for 5 minutes. To a test tube containing 0.2 ml prewanned thromboplastin—calcium, add 0.1 ml prewarrned plasma. Start the stopwatch. Record the time required for clot formation by pulling the wire hook up and down every second. The end point is identified by the formation of a fibrin strand attached to the wire hook. If duplicate tests are done, the difference in duplicates of all samples must be less than 5% of the prothrombin time. Clean hooks between each sample by wiping with 1% phosphoric acid, rinsing with distilled water, and allowing to dry. 77 Procedure No. 2 for homemade or commercial noncalcium thromboplastin 1. Prepare the plasma as for Procedure No.1 above 2. Incubate plasma at 37°C for 5 minutes in a test tube. Add 0.1 ml of prewarmed thromboplastin and incubate for 30 seconds. 3. Add 0.1 ml of prewarmed CaCl2 to the incubation mixture, and mix well by shaking. Start the stopwatch. 4. Record the time required for clot formation (identified by a fibrin strand from the hook to the liquid) while pulling the wire hook up and down every second. 5. If duplicate tests are done, the difference in duplicates of all samples must be less than 5% of the prothrombin time. Results For each new batch of thromboplastin (whether commercial or homemade) normal values have to be established by assaying plasma from about 20 normal men or women who are not on oral contraceptives. These tests need not necessarily be done all on the same day; performing the tests on different days adds a day-to—day variability. The mean and SD are calculated from the results (p. 37). Interpretation The PT test is prolonged in patients on oral anticoagulant therapy and also in patients with a deficiency of one or more of the following factors: I (fibrinogen), II (prothrombin), V, VII or X. For example, these factor deficiencies are found in patients with a circulating anticoagulant, vitamin K deficiency, intestinal malabsorption, liver disease, or obstructive jaundice. Controls A specimen of freshly drawn normal plasma (preferably from a group of previously tested patients) is tested in duplicate each time prothrombin time tests are performed. The mean of the duplicates and difference between duplicates are recorded on quality control charts. If the results of the normal control are outside two standard deviations of the computed mean (p. 37), do the following: 1. Check to make sure the temperature is at 370 i 1°C. Be sure the pipettes are properly calibrated. Check the molarity of the CaCl2 solution. Take blood from another normal person. .U‘PPJP Reconstitute a new vial or thaw a new tube of thromboplastin, and repeat the test. 6. Make sure the pH of the reaction mixture is about 7.4. 78 The Bleeding and Clotting Disorders 7. The results of patient samples should not be reported unless the control mean is within the computed :2 SD limits. Sources of error 1. Because cold activates factor VII in some plasmas, the blood must be kept at room temperature until tested. 2. Platelet—poor plasma should be removed from the cells within 1 hour of collection. The tube should then be capped until just before testing to prevent loss of carbon dioxide and a change in pH. 3. The test must be performed within 4 hours, or the plasma must be quick—frozen and stored frozen in plastic tubes at -70°C or below. 4. Although all materials used in the test must be warmed to 37°C before use, the plasma must not remain at 37°C for longer than 10 minutes, and the thromboplastin must not remain at 37°C longer than specified in the manufacturer’s directions. 5. Mix thromboplastin well before each use. .0“ The 9:1 ratio of blood to sodium citrate should be precise. 7. If the PCV of the blood sample varies from normal, the concentration of anticoagulant in the plasma will be changed. This is especially true when the PCV value is high, as in polycythemia. The volume of citrate to add to maintain a constant concentration of citrate may be calculated from the graph (see Figure 11). 8. Use distilled or deionized water to reconstitute reagents. 9. Follow the directions in the manufacturer’s package insert sheet. The preparation of brain thromboplastin Under running water, wash small pieces of brain (animal, i.e., ox or rabbit). Clean blood vessels and all other tissue. Remove the cerebellum and as much white matter as possible. Use about 150 ml prewarmed (37° - 39°C) saline per 100 g brain tissue. Blend the brain and saline (reagent 2, p. 105) in portions: - with an electric household mixer (or blender), 10 minutes per portion, or — by hand in a mortar, until an even dispersion without particulate matter is obtained. Mix all the portions together in a bowl or glass pot that is placed in a waterbath. After 2 hours of incubation, add buffered saline (reagent 2, p. 105) amounting to 1/3 of the volume of the dispersed brain. Subsequently, centrifuge in portions for 30 minutes at 1250 g (about 3000 rpm). Collect the superna— tants. Adjust the pH to about 7.3 with HCl or NaOH as indicated. Perform a prothrombin time test on a freshly drawn normal plasma. The PT (prothrom— bin time) should be between 10 and 14 seconds. If a short time (< 10 seconds) is found, add a sufficient volume of buffered saline to the thromboplastin until 79 the PT is > 10 seconds. Then divide the thromboplastin into small aliquots and freeze. The thromboplastin must be kept frozen (-20°C) in stoppered glass tubes. MEASUREMENT OF THE INTRINSIC SYSTEM Activated Partial Thromboplastin Time (APTT) Principle This test measures the intrinsic procoagulant activity of plasma. The partial thromboplastin is a substitute for platelet factor 3. Contact activation is standardized by adding an activator (kaolin, celite, or ellagic acid) to the reagent. This test does not measure activities of factors VII and XIII. Equipment 1. A waterbath with thermostat and tube rack (p. 75) 2. Round-bottom glass test tubes 3. Stopwatch 4. A wire hook Reagents 1. Citrated platelet poor plasma (spun at 3000 rpm for 15 minutes) 2. 3.8% inosithin or Alcolec 3. Veronal buffer (about pH 7.3) or commercial APTT reagent 4. Celite suspension or kaolin suspension (reagent 5, p. 106) 5. 0.025 mol/L CaCl2 6. Freshly drawn normal control plasma Procedure 1. Prepare the APTT reagent the day of testing by adding 3.4 ml of Veronal buffer to 3.5 ml of celite suspension and 0.1 ml of 3.8% inosithin or Alcolec. Mix well. 2. In a test tube at 37°C, add 0.1 m1 plasma to 0.1 m1 well—mixed celite- inosithin or celite—alcolec. Start stopwatch and swirl to mix. 3. Incubate at 37°C for exactly 4 minutes. Swirl again. 4. Add 0.1 ml prewarmed 0.025 mol/L CaC12, swirl again and start stopwatch. 5. Record the time required for clot formation while pulling the wire hook up and down each second. 6. Each patient and control plasma must be tested in duplicate. 80 The Bleeding and Clotting Disorders Results Normal (freshly drawn) plasma specimens from 20 persons should be tested to establish normal reference values (especially when a homemade APTT reagent has been used). The APTT of the patient should be within 10 seconds of the normal control (freshly drawn plasma) that is tested simulta— neously. If the patient’s APTT is prolonged, the inhibitor screening test should be carried out (p. 80). Sources of error 1. The tests must be performed at 37°C. 2. All plasma specimens are preferably kept on melting ice until tested. They should be tested within 2 hours of collection. 3. Exactly the same technique must be used for each test to obtain accurate, reproducible results. The incubation time, pH, temperature, and amount of contact activation influence the results. The test tubes must be swirled the same each time. Inhibitors Principle A plasma with a prolonged clotting time due to a coagulation factor deficiency is corrected by adding 10% - 20% normal plasma. A plasma with a prolonged clotting time due to a circulating anticoagulant (inhibitor) is not corrected by adding 50% normal plasma. The test system selected is the one that showed the abnormality (prothrombin time or APTT). Equipment A waterbath with thermostat set at 37°C A wire hook Test tubes, round-bottom glass 95“.“? Stopwatch or watch with a second hand 5. Pipettes Reagents Citrated patient plasma. Procedure for detecting inhibitors This procedure can be performed using the APTT or the prothrombin time test. The reagents used will depend upon the test to be performed (pp.74, 78). 81 1. Perform the APTT or the prothrombin time test in duplicate on each of the following mixtures: Plasma Tube: 1 2 3 4 5a Normal (ml) 0.0 0.1 0.25 0.4 0.5 Patient (ml) 0.5 0.4 0.25 0.1 0.0 2. Keep the plasma mixtures on melting ice and mix just before testing. 3. After testing, incubate the tubes for exactly 1 hour at 37°C, and repeat the test. 4. The presence of an inhibitor is suggested when the addition of 50% normal plasma does not correct the prolonged APTT or PT of the patient’s plasma. If adding 10% of patient plasma significantly pro- longed the APTT or PT of the normal plasma, the patient’s plasma probably contains an inhibitor. 5. Some inhibitors appear only after incubation for an hour at 37°C. Factor VIII inhibitors are usually higher in titer after incubation, whereas those associated with lupus erythematosus are not. 6. If correction does occur, the patient has a clotting factor deficiency with no inhibitor (p. 28). Sources of error As with the API l or PT test, good technique is essential; timing, incubation temperature, and contact activation all greatly influence the results. The same technique must be used each time. FIBRINOGEN (CLAUSS METHOD) Principle The enzyme thrombin converts the soluble plasma protein fibrinogen into its insoluble polymer, fibrin. At high thrombin concentrations (approximately 100 National Institutes of Health [NIH] units/m1) and low fibrinogen concen- trations (0.05-0.8 g/L), the reaction is determined by the fibrinogen concen— tration. When plotted on double logarithmic paper, the thrombin clotting time is linear when compared with the fibrinogen concentration (Figure 17). Equipment A waterbath, thermostat set at 37°C, containing a test tube rack (p. 49). A wire hook Test tubes, round-bottom glass tubes Stopwatch or watch with a second hand P9P!" 82 The Bleeding and Clotting Disorders THROMBIN CLOTTING TIME IN SECONDS J: a: 0 O I IllllI 0) O I N O I .1 (J # mmumwo IIIITTI I '0 I 1 I lll|llll 1 I IIlJllJ 0.2 0.3 0.4 0.6 0.81.0 2,0 3.0 4.0 6.08.0101) FIBRINOGEN IN GRAMS/LITER Figure 17 - Example of a fibrinogen calibration curve Reagents l. 2. Thrombin, bovine; 50 NIH units/ml Purified fibrinogen of known concentration. If purified fibrinogen to produce the standard curve is unobtainable, it is possible to use the following table (Table 5) for approximate levels of fibrinogen. The results, however, will be approximate, and the laboratory must estab— lish a normal range by analyzing samples from 20 normal persons (p...). . Barbital buffer, pH 7.5 (reagent 1, p. 105) Platelet-poor plasma 5. Normal control plasma Procedure 1. Make serial dilutions in barbital buffer of purified fibrinogen to give 2. concentrations of approximately 0.80-4.0 g/L. Dilute each dilution 1:10 in barbital buffer. Dilute plasma samples (from patients and controls) 1:10 with barbital buffer. 83 Table 5 - Approximate fibrinogen levels by the Clauss method Thrombin clotting time Fibrinogen (glL) (seconds) 6 4.00 7 3.45 8 3.00 9 2.70 10 2.45 11 2.25 12 2.08 13 1.90 14 1.80 15 1.68 16 1.58 17 1.49 18 1.40 19 1.33 20 1.27 21 1.22 22 1.18 23 1.11 24 1.08 25 1.00 26 0.98 27 0.96 28 0.92 29 0.89 30 0.86 3. Thaw 1 vial of thrombin (100 NIH units/ml) (reagent 10, p. 106). Add 7. an equal volume of buffered saline to yield 50 NIH units/m1. Mix and keep at room temperature. Incubate 0.2 ml of each dilution at 37°C for 4 minutes. Add 0.2 ml thrombin, mix by shaking, and Start watch. Record time required for clot formation While pulling the hook up and down each second. Test each dilution in duplicate. Calculations 1. Plot the mean of the duplicates of each dilution of the purified fibrinogen on double logarithm paper. Draw a line of best fit. Read the concentrations of the control and patient dilutions from the graph. 84 The Bleeding and Clotting Disorders 3. If the fibrinogen concentration is greater than 4.0 g/L, make a 1:20 dilution of the original plasma and retest. Multiply the concentration taken from the curve by two. If the fibrinogen concentration is less than 0.8 g/L, make a 1:2 or 1:5 dilution of the original plasma and retest. The value taken from the curve is divided by five for the 1:2 dilution and by two for the 1:5 dilution (see Figure 17). Results Normal values are usually between 1.6 and 3.2 g/L. Controls A normal plasma control is tested simultaneously. Interpretation 1. 2. 3. Decreased fibrinogen levels are consistent with: a. Hereditary hypo- or afibrinogenemia, some dysfibrinogenemias. b. Disseminated intravascular coagulation. 0. Liver disease. (1. Fibrinolysis. Normal fibrinogen levels with prolonged TT can be associated with hereditary and acquired dysfibrinogenemias. Increased fibrinogen levels occur with the use of cryoprecipitate. Sources of error 1. 2. 3. Dilution must be carefully made. Samples above 4.0 g/L or below 0.8 g/L must be retested at appropriate dilutions. Results may be affected by the presence of heparin or fibrinolytic degradation products in the patient’s plasma. Significant levels of either of these may cause the test to indicate a falsely low fibrinogen level. The thrombin solution will probably only be stable at -20°C for 1 month. Thrombin time Principle The time required for plasma to clot when thrombin is added is a function of the final phase of coagulation. It is affected by the concentration of fibrinogen and the presence of fibrin-split products and antithrombin agents (e.g., heparin). 85 Equipment 1. 2. 3. 4. A waterbath with thermostat set at 37°C containing a test tube rack (p.75) A wire hook Test tubes, round—bottom glass tubes Stopwatch or watch with a second hand Reagent 1. 2. 3. 4. Thrombin (approximately 5 NIH units/m1) (reagent 11, p. 106) — keep on melting ice Veronal buffer, pH 7.3 (reagent 13, p. 106) Patient plasma keep on melting ice Normal control plasma Procedure 1. 2. 3. Add 0.1 ml veronal buffer to 0.1 m1 of plasma. Incubate at 37°C for 4 minutes. Add 0.1 ml thrombin and mix by shaking. Simultaneously start stopwatch. Record time required for clot formation while pulling the hook up and down each second. If the patient’s thrombin time is more than 2 seconds longer than that of the control, perform these mixing tests: a. One volume (0.2 m1) control plasma plus one volume (0.2 ml) of patient’s plasma. b. One volume (0.1 ml) control plasma plus nine volumes (0.9 ml) of patient’s plasma. Interpretation Prolongation of the thrombin time is consistent with: 1. .U‘PP’!" Hypofibrinogenemia, usually 0.9 g/L or less. The presence of heparin or heparin-like anticoagulants. Decreased ability of fibrinogen to clot in fibrinolytic states. The presence of an abnormal fibrinogen. If the thrombin time of the mixture of patient and control plasmas is closer to that of the patient’s plasma alone than to that of control plasma alone, and if the patient has not received heparin in the previous 6 hours, abnormal fibrino gen or fibrin breakdown products are probably present in the patient’s plasma. 86 The Bleeding and Clotting Disorders Sources of error 1. The main source of error is the rapid inactivation of the thrombin solution due to the adsorption of thrombin to surfaces. This adsorption is particularly pronounced with glass. 2. The thrombin solution must be kept on melting ice after thawing. 3. The frozen thrombin solution will not be stable at -20°C for longer than 1 month. ERYTHROCYTE SEDIMENTATION RATE (ESR) Principle The erythrocyte sedimentation rate (ESR) measures the sedimentation rate of red cells in plasma. The numerical value in millimeters is obtained by measuring the distance between the lowest point of the surface meniscus to the upper limit of the red cell sediment in a column of anticoagulated and diluted blood that has stood in the selected tube for 60 minutes. The method described, which is based on Westergren’s, is a standardized routine method as recommended by ICSH. Equipment 1. Westergren tubes meeting standards recommended by ICSH 2. Sedimentation tube holder Reagent Trisodium citrate dihydrate anticoagulant-diluent solution: Na3C6HSO7' 2H20 32.08 g Distilled H20 to 1 liter Filter through a sterile membrane, with a maximum bore diameter of 0.22 pm, into a sterile container. Do not add preservatives. Keep refrigerated. The solution should be satisfactory for several months, but discard if it becomes turbid. Specimen A venous blood sample is obtained, and four volumes of blood are added to one volume of anticoagulant diluent. Alternatively, blood anticoagulated with solid EDTA (1.5 mg/ml) is a suitable specimen, but it must be diluted, four volumes of blood to one volume of diluent reagent, just before the test is set up. 87 Procedure 1. 2. 3. Fill a clean, dry standard Westergren tube with the blood and adjust the level to the “0” mark. Place the tube in a strictly vertical position on the bench, not exposed to direct sunlight, and free from vibrations and draughts. After exactly one hour, read the distance from the bottom of the surface meniscus to the top of the column of sedimenting red cells (where the full density is apparent) in mm and record the reading as the ESR value; for example, ESR = 26 mm in 1 hour. Results In healthy persons the upper limits are 10 mm/l hour in men, 12 mm/l hr in women, and 10 mm/l hr in children. The normal values increase with age, e.g., for persons over 60 years old, to 14 mm/l hr in men and 20 mm/l hr in women. Controls None available at present except good technique. Sources of error 1. .05"? S If a specimen is kept at room temperature, the test must be set up within 2 hours after collection; if kept at 4°C, within 6 hours. Blood in EDTA can be used up to 24 hours if kept at 4°C. The ESR depends on the room temperature at which the test is performed. The normal range usually quoted relate to 18°C—25°C. If the temperature is higher, a different normal range must be established. Contamination with skin—cleaning materials during the venipuncture should be avoided. Any clots in the blood will invalidate the test. Strict attention should be paid to dilution and mixing. The glass tubes should be cleaned in an acetone-water system (cleaned in water, then alcohol, and finally acetone). Alternatively, they may be rinsed in water and left to drain and dry overnight. Use of dichromate and detergent mixtures is not recommended. The tube must be kept absolutely vertical. If the tube does not conform to the ICSH specifications, e. g., the tube has different bore or is made of plastic instead of glass, a test must be carried out to Check that results are reproducible and comparable to the standard method. If not, a calibration graph must be prepared relating the results with the modified method to those obtained with the standard method over a range of ESR measurements. If the relation- ship is constant (e.g., linear) throughout the range a correction factor can be applied; if not, it is essential to establish normal reference limits with the modified method. 88 The Bleeding and Clotting Disorders CHAPTER 7 SPECIALIZED PROCEDURES ONE-STAGE FACTOR VIII ASSAY Principle The factor VIII assay described here is based on the activated partial thromboplastin time test (APTT). The ability of the patient’s plasma to correct the APTT of plasma congenitally lacking factor VIII is compared with that of normal plasma. Equipment PP’PE‘ Waterbath with thermostat set at 37°C A wire hook Test tubes, round-bottom glass Stopwatch or watch with a second hand Pipettes Reagents 1. 9:59P 6. Lyophilized, frozen, or fresh congenital severe factor VIII-deficient plasma (substrate). If another factor assay is to be performed, then the plasma substrate should be deficient in that factor. Normal reference plasma Normal control plasma Veronal buffer, pH7.3 (reagent 13, p. 106) 0.025 mol/L CaCl2 AP'IT reagent, commercial or homemade, (see reagent 4, p. 105) Procedure 1. 2. Obtain blood by using the two—syringe technique and prepare platelet- poor plasma. Keep plasma on melting ice or at 4°C during preparation. Prepare AP’IT reagent (reagent 4, p. 105). Mix well by inversion each time before use. Reconstitute if lyophilized, or thaw frozen factor VIII-deficient plasma (substrate). If several vials of substrate are used, pool them and place on melting ice. 90 The Bleeding and Clotting Disorders 4. Reconstitute or thaw normal reference plasma and make the following dilutions: 1:10 1:20 1:40 1:100 100% 50% 25% 10% Plasma (ml) 0.1 0.1 0.1 0.1 Verona] Buffer (ml) 0.9 1.9 3.9 9.9 Place the dilutions on melting ice. 5. In a test tube at 37°C, pipette 0.1 ml of APTT working reagent, 0.1 m1 of substrate plasma, and 0.1 ml of diluted plasma. Start stopwatch and swirl tube to mix. 6. Incubate 4 minutes at 37°C. Just before the end of the 4—minute incubation period, swirl test tube again. At exactly 4 minutes, add 0.1 ml CaCl2 and start watch. 7. Record time required for clot to form by using the wire hook described on page 75 or by tilting. 8. Test each plasma dilution in duplicate. 9. Prepare dilution of normal control plasma at 1:10 and 1:40 as above. Repeat steps 5 —8. 10. Dilute patient plasma 1:10 and 1:40 and repeat steps 5-8. 11.Frozen patient plasma must be quick—thawed at 37°C and diluted immediately before testing. 12. When severely deficient plasma is tested, the reference curve must be diluted further, that is, 1:200 (= 5%) or 111,000 (2 1%). Do not dilute patient plasma less than 1:10 (as 1:5 or 1:2); the results are not linear because of the high concentrations of the rest of the clotting factors. Calculations 1. On double logarithmic graph paper, plot % activity against the time in seconds required for each reference plasma dilution to clot. Draw lines of best fit for the two components of the curve. The line should be linear between 10% - 100% activity (Figure 18). Read the patient’s results from the curve: the 1:10 dilution is read directly from the curve, but the 1 :40 dilution is read from the curve and then multiplied by four. The mean of the two results is taken. The patient’s curve must be parallel to the reference curve for valid results. Controls A normal plasma sample or, if available, normal pooled plasma (p. 49) is tested simultaneously. If available, a moderately deficient plasma is also tested simultaneously as a check on the sensitivity of the assay. 91 TIME (SECONDS) 8 O w # O O I I N O I 10 I I I 1 I l l I I I I I l I l L l I l l I l I I I I I 1 2 3 4 5 6 7 8 10 20 3O 40 60 80100 200 300 600 1000 FACTOR vm 40° 80° Figure 18 - Example of a factor VIII reference curve. (Two linear graphs have been drawn from the data points.) Interpretation 1. Normal = 50% — 150% activity 2. Mild factor VIII deficiency = 5% — 25% activity 3. Moderately severe = 1% - 5% activity 4. Severe = less than 1% activity 5. This test alone is insufficient to diagnose carriers of factor VIII deficiency. Occasionally their factor VIII activity may be below the safe hemostatic level of the coagulation factor. Sources of error 1. Patient blood should be drawn with plastic or siliconized syringes by the two—syringe technique and kept on melting ice. The blood must be centrifuged within 30-60 minutes in a refrigerated centrifuge and aliquots of the plasma quick—frozen and stored until tested. The plasma must be tested within 4 days after the blood is collected. 2. All equipment should be plastic or siliconized. 3. Exercise and stress increase factor VIII activity; therefore, have the patient rest for 10-15 minutes before drawing blood. 4. The technique must be identical each time: the incubation time must be exactly 4 minutes, and the mixing of plasma with celite-inosithin or Alcolec must be identical each time (that is, swirl six times). 92 The Bleeding and Clotting Disorders 5. A buffer time test is performed with each new substrate by substituting 0.1 m1 of veronal buffer for the plasma dilutions. The time should be longer than the 1% normal reference plasma dilution for best sensitiv— ity. The substrate (factor VIII—deficient plasma) must have less than 1% activity and no inhibitors to factor VIII. Plasma must not be reconstituted, quick-thawed, or diluted until immediately before testing. FACTOR ASSAYS IN THE EXTRINSIC PATHWAY 1 Factors in the extrinsic pathway are measured by the prothrombin time. The procedure is similar to that used for measuring factor VIII, but the assay is performed by using the prothrombin time test, and a plasma substrate deficient in the factor to be measured (see factor VIII assay). EUGLOBULIN LYSIS TEST Principle Euglobulins are proteins that precipitate when plasma is diluted in water. Plasmin, plasminogen, and fibrinogen are all included in the precipitate. When euglobulins are redissolved and the fibrin clotted with thrombin, the activated thrombin will activate plasminogen to plasmin. The amount of time required for the plasmin to lyse fibrin is the euglobulin lysis time. Equipment >1 QMPPP?‘ Centrifuge Test tubes Parafilm or clean rubber or plastic stoppers Glass rods Waterbath with thermostat set at 37°C Stopwatch Pipettes Reagents JePJPT‘ Citrated plasma (p. 57) Acetic acid 1% Veronal buffer, pH 7.3 Bovine thrombin, 100 units/ml in veronal buffer 93 Procedure 1. Add 0.5 m1 of cold (temperature of melting ice) citrated plasma, 7.5 ml of water, and 0.13 ml of acetic acid to a test tube. The pH of the mixture of the plasma and acetic acid should be about 5.2-5.3. Cover the tube and invert five times, and centrifuge at 1200X g for 4 minutes. Remove the supernatant, invert tube, and allow to drain for 1 minute. Stir the sediment with a glass rod. Leave the rod in the tube. . Add 0.5 ml of veronal buffer to the sediment, stir with the rod, and warm to 37°C until the sediment dissolves. Add 0.45 ml of veronal buffer with 0.05 ml of thrombin, and mix immediately and thoroughly. 7. Inspect the tube every 5 minutes for 30 minutes, then every 10 minutes for 2 hours, or until lysis is complete. Interpretation < 2 minutes = severe fibrinolysis, explains severe bleeding 2-10 minutes = moderate fibrinolysis, explains postoperative or posttraumatic bleeding 10-30 minutes = mild fibrinolysis, but not explanation for bleeding 30 minutes-2 hours = physiologic enhanced fibrinolysis 2-4 hours = normal > 4 hours = possibly defective fibrinolysis Sources of error 1. The euglobulin lysis time can be moderately shortened by leaving the tourniquet in place for a prolonged period before venipuncture. This is because plasminogen activator is released from the endothelial cells. The lower the pH of the plasma—acid mixture, the longer the euglobulin lysis time. If the sample is not adequately drained, inhibitors of lysis may drain back onto the sediment and cause prolongation of the lysis time. Similarly if an increased amount of fibrinogen is left in the tube (only 50% to 60% is normally in the euglobulin portion), the sediment to be dissolved will increase, which will result in a longer lysis time. 94 The Bleeding and Clotting Disorders FACTOR XIII, QUALITATIVE Principle Factor XIII (fibrin-stabilizing factor) converts soluble fibrin into insoluble fibrin in the presence of calcium ions. In the absence of factor XIII, the bonds between the fibrin molecules are broken by 5 mol/L urea or 1% monochloroacetic acid. Equipment 12- X 75-mm glass tubes 37°C incubator Stopwatch F‘E’JNT‘ Pipettes 5. Parafilm or clean rubber or plastic stoppers Reagents 1. Patient’s plasma 2. 5 mol/L urea or 1% monochloroacetic acid 3. 0.025 mol/L CaCl2 4. Normal control plasma Procedure 1. Place 0.2 ml of plasma in a glass test tube. 2. Add 0.2 ml of CaClz. Cover tube and mix by inversion. 3. Allow to clot in a 37°C incubator for 30 minutes without disturbing the tube. 4. After incubation, loosen the clot from the wall of the tube by gentle tapping. 5. Add 3 ml of urea or monochloroacetic acid. Cover the tube and allow to stand at room temperature for 24 hours. 6. Check for dissolution of the clot at 30 minutes (with monochloroacetic acid, check at 10 and 30 minutes) and at 1, 2, 4, and 24 hours. Result Normal — no clot dissolution within 24 hours. Controls A lyophilized pool of normal plasma should be tested simultaneously. 95 Sources of error The test system is sensitive to various levels of plasma fibrinogen. Therefore, this test is not an absolute measure of factor XIII. The effect of fibrinogen level is most obvious below 0.8 g/L and above 8.0 g/L. VITAMIN K1 TEST Principle When a prolonged prothrombin time (PT) has been found in a patient, repeating the PT after the administration of vitamin K may help to differen- tiate between liver parenchymal disease and vitamin K deficiency, and give some clues as to the cause of vitamin K deficiency. Reagents Sterile vitamin K1 for injection. Procedure 1. Administer 10 mg vitamin K1, intravenously (slowly) or subcutane- ously. 2. Repeat PT 24 hours after intravenous administration or, in case of subcutaneous administration, after 48 hours. Results When the PT remains prolonged, the patient’s liver is diseased and not able to synthesize the coagulation factors in normal quantities. When the PT is corrected (i.e., becomes normal or decreases considerably), the resorption of the fat-soluble vitamins K1 and K2 from the gut is severely decreased. In a jaundiced patient, this decrease is most likely due to the absence of bile salts in the gut resulting from a complete block of the Choledochus duct, which impairs fat resorption. If a prolonged PT in a nonicteric patient is corrected by parenterally administered vitamin K1, the vitamin K deficiency can be due either to sprue (celiac disease), or to intoxication with warfarin or coumarin drugs (vitamin K antagonists). These drugs are not only used as oral anticoagulants but also as rodent killers, and, therefore, accidental intake might occur, especially in children. Because such an intoxication might happen with a large dose, 10 mg of vitamin K1 may be just sufficient to give a slight reduction of the PT. Because vitamin K1 resorption is normal in this condition , the test may be extended by the oral administration of 100 mg of vitamin K1- 96 The Bleeding and Clotting Disorders Sources of error 1. See sources of error of the PT (p. 77). 2. The vitamin K1 preparation must be stored in a refrigerator and should be used before its expiration date. 3. The absorption of Vitamin K1 into the blood from intramuscular administration is unpredictable. Results following intravenous admin— istration can be interpreted only in the case of a positive result, that is, correction of the PT. CHAPTER 8 CONTROL OF ANTICOAGULANT THERAPY ORAL ANTICOAGULANTS Oral anticoagulant control is based on the l-stage prothrombin time (PT) test with thromboplastin. The PT should be expressed as the ratio of the PT of the patient’s plasma to that of normal control plasma. To control anticoagulant therapy reliably, the laboratory worker/physician must be able to compare results between laboratories and also from the same laboratory with different batches of thromboplastin. To achieve this, WHO has recommended that all thromboplastins be calibrated by comparison with an International Sensitivity Index (ISI). WHO established a primary interna- tional reference standard of human brain and subsequently reference stan- dards of human, ox, and rabbit thromboplastins. These preparations were calibrated relative to the original primary standard, which was given an ISI of 1.0. Because of the risk of biohazard, human brain should not be used as a source of thromboplastin. Other thromboplastin reagents can be standardized by the procedure described below, in which the reaction of the reagent is compared with that of a reference standard or even a commercial reagent with an established ISI. The ISI should thus be determined on all new batches of thromboplastin, both homemade and commercial products, if the manufac- turer has not already provided this measurement. For this purpose the reference standard and the new batch should be from the same species, that is, rabbit for rabbit, ox for ox. In addition, for use in oral anticoagulant control, the prothrombin ratio should be reported in a standardized way as an International Normalized Ratio (INR). The use of this ratio will be described below. A. Standardization of a thromboplastin working preparation For standardization of commercial reagents or of a national reference, standard measurements of prothrombin time are obtained on plasma from 20 normal persons/donors and 60 anticoagulated patients. The tests can be performed over a period of 10 days. For a thromboplastin that will be used in one laboratory, a reasonably reliable level of control may be possible where the tests are reduced to two normal specimens and six abnormal specimens on 2-3 days (i.e., a total of 4-6 normal specimens and 12-18 abnormal speci- mens). 98 The Bleeding and Clotting Disorders Method 1. On each day prepare freshly drawn plasma from two normal donors and from 6 patients who are on oral anticoagulant therapy and, preferably, who have been on this therapy for at least six weeks. Perform a PT test on each plasma in duplicate with both the working preparation and the reference preparation. The testing should be in the following order: Order of test 1. Normal 1 with reference preparation 2. Normal 1 with working preparation 3. Patient 1 with reference preparation 4. Patient 1 with working preparation 5. Patient 2 with reference preparation 6. Patient 2 with working preparation 15. Normal 2 with reference preparation 16. Normal 2 with working preparation If any duplicate result differs by more than 10%, repeat the tests on that plasma. Plot results of PT (in seconds) on double-logarithmic graph paper, with the PT of the working preparation (WP) on the horizontal axis and the PT of the reference preparation (RP) on the vertical axis (Figure 19). Draw a best-fit straight line for all the plots. This is the calibration line. Estimate the slope,3 i.e., ratio of RP/WP either by means of a calculator or by a computer that has an x - y function or as follows: 3 This method is adequate for clinical purposes. A more accurate statistical method should be used by a control laboratory. In this method the slope is calculated from the following formula: Slope ofRP/WP=m+ , m2+1, wherem= xww XI [(y—rf-Dx—if 22(y—yxx-rc) where : = logarithm of each individual PT with the reference preparation = mean of logarithms of all the individual PTs with the reference preparation = logarithm of each individual PT with the working preparation = mean of log of all the individual PT with the working preparation 99 50- 30- ————— Caiibration line (the International Sensitivity Index is defined in terms of the slope of this linei 20 Prothrombin time of Reference Preparation 10 l I l l l 1 0 20 30 40 50 60 Prothrombin time of working preparation Figure 19 - Double-logarithmic plot of prothrombin times for determination of the International Sensitivity Index. (In this example, the ISI of the working preparation is 1.05.) From the graph read several values of x and the equivalent values of y in the range of 20-60 sec. From the mean values calculate the slope by the formula: b = (y-a) + x where y = log of mean value for reference preparation x = log of mean value for working reagent a = log of intercept on y b= slope Then calculate the ISI of the working preparation from the formula: ISIofWP=ISIofRPXb 100 The Bleeding and Clotting Disorders B. Measurement of prothrombin ratio (PR) on patient’s plasma and calculation of International Normalized Ratio 1. Determine normal PT; mean of 20 tests on a pool as described on page 49. 2. Measure the PT on the patient’s plasma as described on page 74. 3. Calculate the prothrombin ratio: PR = PT of patient’s plasma established value PT 4. Convert PR to the INR by the formula: INR = (PR 151 of Ihromboplastin) This calculation can be performed either with a calculator that has an “xy function” or by means of log tables: INR = antilog [(log PR) x 181]. With some commercial reagents, a graph or table is provided showing the INR for various measurements of RT or PR. This graph will be applicable only to that batch of reagents provided that the mean normal PT obtained (see above) is the same as that indicated by the manufacturer. IN TE RPRE TA TION In patients receiving oral anticoagulants such as warfarin the INR should be kept within the recommended therapeutic range by adjusting the dosage with regular checks on the PT, usually at 2—3 week intervals. The following limits are recommended: Clinical state INR - Prophylaxis of deep-vein thrombosis before surgery 2.0 — 2.5 — Prophylaxis for hip surgery and for operations for fractured femur 2.0 - 3.0 - Treatment of deep-vein thrombosis or pulmonary embolism 2.0 — 3.0 - Transient episodes of ischemia 2.0 - 3.0 — Recurrent deep—vein thrombosis or pulmonary embolism 3.0 — 4.5 - Myocardial infarction 3.0 - 4.5 - Arterial grafts 3.0 - 4.5 — Cardiac prosthetic valves and grafts 3.0 - 4.5 If the INR is prolonged (> 5.0), spontaneous bleeding is likely to occur. If the patient reports any hemorrhage, the INR must be checked immediately. The INR must also be monitored carefully in patients receiving other drugs, especially those known to increase or to decrease the effects of anticoagulants (Table 6). Oral anticoagulants may harm the fetus. They should be stopped and replaced by heparin at the first sign of pregnancy. 101 Table 6 - Clinical factors and drugs that interfere with oral anticoagulant effect INCREASED EFFECT CL|N|CAL CONDITIONS Hepatic disorders Malnutrition Steatorrhea Diarrhea Visceral carcinoma Fever and infection Cardiac failure Renal impairment Thyrotoxicosis X-ray therapy Alcoholism DECREASED EFFECT Antacids Barbiturates Cholestyramine Corticosteroids Diuretics Estrogens Oral contraceptives Phenytoin Rifampicin DRUGS Acetylsalicyclic acid Allopurinol Ampicillin Anabolic steroids Aspirin Chloramphenicol Clofibrate Cytotoxic drugs Neomycin Phenylbutazone Sulfonamides Thyroxine Tricyclic antidepressants HEPARIN ANTICOAGULATION Unlike oral anticoagulant therapy, heparin anticoagulation is not well standardized. The use and limitations of laboratory tests are described below, but the patient’s clinical response to heparin therapy must always be checked because it is an excellent guide to the adequacy of the heparin dose. Laboratory control of heparin anticoagulation is based upon measurement of the prolongation of the clotting process by the inhibition of several reactions within the clotting system. Heparin produces this inhibition by forming a complex with antithrombin III in the patient’s blood. This complex reshapes the antithrombin III molecule so that it is able to combine with the active 102 The Bleeding and Clotting Disorders (enzymatic) portion of activated factors II, IX, X, XI, and XII that are formed during the clotting process. Factor VII is not affected by the heparin— antithrombin III complex and thus only two factors in the extrinsic pathway are inhibited, whereas five factors in the intrinsic system are affected by heparin. Several options are available for monitoring the effect of heparin anticoagulation. The tests of the intrinsic system are more sensitive because of the large number of factors in that system that are affected. Thus, the whole blood clotting time, the recalcifrcation time, the activated partial thrombo- plastin time, and the kaolin and plasma clotting time have all been utilized. However, the whole blood clotting time and the recalcification time do not have well—controlled contact activation of factor XII; they depend on regular tilting of the tube during the test and are subject to much intertechnician variations. As it is almost impossible to control the intertechnician effect, these two tests are not recommended. The activated partial thromboplastin time (APTT) is a commonly used test to control heparin therapy. After a baseline APTT is determined on the patient, and intravenous heparin admin- istered, the ratio of the APTT measured on plasma taken subsequently from the heparinized patient is divided by this baseline value. The ratio is usually kept at 1.5 to 2.5 on patient blood receiving a continuous infusion of heparin, and it is adjusted by controlling the rate of infusion. Heparin is not a well—standardized material and varies in its source (usually pig gut or beef lung) and in the content of low and high molecular weight varieties of heparin molecules. The composition is important, in that the low molecular weight varieties of heparin will produce anticoagulant effects in the patient without producing much change in many of the monitoring tests. The use of low dose heparin and bolus dose regimens of heparin to achieve full heparinization further complicate the monitoring of heparin therapy because of the half—life of circulating heparin and the relationship of the time of the blood collection to the last dose of heparin. Good clinical and laboratory practice dictate that the treatment method and the monitoring test used be standardized to suit local circumstances. Oral anticoagulation is often begun during the lst week of anticoagulation on patients who have been receiving heparin therapy for thrombotic disease (see oral anticoagulation). The oral anticoagulant will usually produce a greater effect on the extrinsic clotting system during the first 24—48 hours, than on the intrinsic system because of the shorter half-life of factor VII. Also, the degree of reliability of the tests used to monitor heparin will decrease during this 24-48 hours because the effect of the oral anticoagulant on the vitamin K- dependent factors occurs. Heparin therapy is usually tapered as the effect of the oral anticoagulants increase. Besides checking for the occurrence of bleeding, laboratory personnel should be watchful for thrombocytopenia associated with heparin therapy, 103 and patients on heparin therapy should have routine platelet counts (p. 65). This is especially true for patients who have had prior heparin therapy. Sometimes the patient’s APTT or other laboratory test appears to be unresponsive to heparin in spite of very large doses of heparin. This occurs especially when patients have 'a very high activity of circulating factor VIII and in this instance the test does not give a good reliable measure of the degree of anticoagulation. 104 The Bleeding and Clotting Disorders APPENDIX Reagent Test Storage Monochloroacetic acid, 1% Factor XIII Make fresh Barbital buffer, pH 7.5 Fibrinogen (Clauss) 4° C Brain thromboplastin Prothrombin time 4° C Buffered saline Various tests 4° C 0.025 mol/L Calcium Chloride APTT, PT, Factors II, 4° C V, VII, VIII, IX, X, XI, Celite-lnosithin or -Alco|ec XII Make fresh working reagent APTT, Factor assays 2% Celite Room temp. APTT, Factors VIII, IX, 3.8% Inosithin or Alcolec XI, Xll -20° C (American Lecithin Company) APTT, Factors VIII, IX, 1.0% Kaolin, pH 7.2 Xl, XII 4° C Saline, 9 g/L Kaolin clotting time 4° C 0.11 moi/L Sodium citrate Various tests 4° C Thrombin, freeze-dried Anticoagulant 4° C Thrombin, 5 NIH units/ml Various tests ~20° C (1:200 dilution) Thrombin time 5 mol/L Urea Make fresh Veronal buffer, pH 7.35 Factor Xlll 4° C Various tests Preparation 1. Barbital buffer, pH 7.5. Dissolve 2.05 g of sodium diethylbarbiturate, 2.75 g of diethylbarbituric acid, and 7.30 g of sodium chloride in 750 ml of deionized water; adjust pH to 7.5. Dilute to one liter with deionized water. Buffered saline — 7 parts saline; 3 parts veronal buffer, pH 7.3. 0.025 mol/L calcium chloride. Dissolve 0.555 g of anhydrous calcium chloride in deionized water and dilute to 200 ml in a volumetric flask. AP’IT reagent. Prepare fresh daily by adding 3.4 ml of veronal buffer, pH 7.3, to 3.5 ml of well—mixed 2% celite suspension or 1% kaolin suspension and 0.1 ml of 3.8% inosithin or Alcolec. Mix well by inversion each time before use. 2% celite suspension. Place 5 g of celite in a 250-ml volumetric flask, and dilute to the mark with 9 g/L saline. Store at room temperature. Mix well before use. 106 The Bleeding and Clotting Disorders 6. Monochloroacetic acid 1% (0.105 mol/L). Dissolve 1 g of crystalline monochloroacetic acid in 100 ml of distilled water. Keep in tightly stoppered bottle at 4°C. 7. 3.8% inosithin or Alcolec. In a blender or tissue grinder, homogenize 3.8 g of inosithin or Alcolec (American Lecithin Company) in 100 ml of 9 g/L saline. Dispense in small aliquots and freeze at —20°C. May be thawed and refrozen repeatedly. 8. 9 g/L saline. Dissolve 18 g of sodium chloride in deionized water and dilute to 2 liters in a volumetric flask. 9. 0.11 mol/L sodium citrate (32g/L) 4 Na3C6HSO7' 2HZO. Dissolve 32 g of dihydrate sodium citrate in deionized water and dilute to one liter in a volumetric flask. 10. Thrombin, 100 units/ml. Reconstitute each vial of thrombin by adding saline to make a concentration of 100 units/ml. Dispense in l-ml aliquots and freeze at -20°C. 1 1. Thrombin, 5 units/ml. Thaw a vial of thrombin (100 u/ml). Dilute with saline until the thrombin time is between 25 and 35 seconds. This is usually about 5 units/ml. 12.5 mol/L urea. Place 30 g of urea in a 100-ml Erlenmeyer flask. Add deionized water up to approximately 90 m1. Heat and mix on heated magnetic stirrer until all urea is dissolved. While solution is still hot, add a small scoop of resin beads (Analytical Grade Mixed Bed Resin, AG 501-X8 [D], 20—50 mesh, fully regenerated, Bio—Rad Laborato- ries). Mix well and filter through glass wool into a 100—ml volumetric flask. Flush urea through by rinsing with deionized water. When cool, dilute to 100 ml with deionized water. Mix well. Make fresh on day of use. 13. Veronal buffer, pH 7.3. Dissolve 5.88 g of sodium diethylbarbiturate and 7.34 g of sodium chloride in 600 ml of deionized water. Add 215 ml of 0.1 N HCl. Check pH and adjust to 7.3, if necessary; then dilute to one liter with deionized water. 14.EDTA. Sodium or potassium salts, 1.5 +25 mg/ml of blood. 15. Phosphate buffer, pH 6.8. NaZHPO4 (18 mmol), 2.56 g; KHZPO4 (49 mmol), 6.66 g; distilled water to one liter. 16. Fixative — absolute methyl alcohol. The bottle must be kept well stoppered to prevent hydration. This precaution is particularly impor- tant in tropical, humid atmospheres. 4 32 g/L is actually 0.109 mol/L 107 108 The Bleeding and Clotting Disorders 109 GLOSSARY OF TERMS Activated Partial Thromboplastins: Mixtures of contact activators (kaolin, ellagic acid, or Celite) with organically extracted brain and lung phospholipids used to initiate intrinsic clot formation. Adenosine Diphosphate (ADP): A purine nucleotide that induces platelet aggregation. Afibrinogenemia: The absence of fibrinogen in the blood. This disorder may be inherited or may occur in disseminated intravascular coagulation. Aggregometry: Quantitative measurement of platelet cohesion in a photometer, which measures changes in light transmission through a plasma suspension of platelets. The suspension is prepared from whole blood, then activated with a variety of agonists. The pattern of light transmission change is recorded and analyzed. Alpha Granule: The storage organelle in platelets that contains various hemostatic proteins. Anticoagulant: A substance that inhibits the formation of clots. Those used in blood specimen collection include EDTA, citrate, and oxalate, all of which bind calcium to prevent coagulation. Heparin, an anticoagulant that occurs in vivo, and is used in collection, acts against thrombin to prevent coagulation. 2-Antiplasmin: A naturally occurring inhibitor of fibrinolysis. Antiplatelet Antibody: An immunoglobulin specific for platelet antigens, such as P1A1. The antibody may be responsible for an autoimmune or alloimmune disorder. Antithrombin III (ATIII): An alpha globulin that is synthesized by the liver and is a naturally occurring inhibitor of coagulation. Arachidonic Acid: A 20—carbon fatty acid with four unsaturations, abundant in the phospholipid bilayer that forms most membranes. It is the substrate for prostaglandin and thromboxanes. Bernard-Soulier Syndrome: A rare, autosomal-recessive bleeding disor- der, in which platelets demonstrate abnormal adhesion and thrombocyto- penia. There is a normal aggregation response to ADP, epinephrine, and collagen, but no response to ristocetin even when von Willebrand factor is added. Bleeding Diathesis: A predisposition or tendency to bleed. Bleeding Time: The time required for a standard wound to stop bleeding under controlled conditions of intracapillary pressure and skin thickness. The bleeding time test is the initial screen for assessment of platelet function in the clinical laboratory. 110 The Bleeding and Clotting Disorders Capillary Fragility: The unexpected escape of red cells through capillary walls as a result of capillary disorders such as vasculitis. Fragility results in the appearance of petechiae, ecchymoses, and purpura in the skin. Clot: A gel—like mass formed in whole blood, composed of fibrin, platelets, and erythrocytes, or in plasma, composed of fibrin and platelets. Coagulation: The process by which several glycoproteins interact with platelets to form an insoluble blood clot to stop blood loss or flow. Complete thromboplastin: Acetone—dried extracts from animal brain or lung tissues. Coumarin (Warfarin): An antagonist to the production of the vitamin K— dependent factors, used routinely as a therapeutic anticoagulant. Cryoprecipitate: A preparation used to restore coagulation in patients with hemophilia or von Willebrand disease. The cryoprecipitate is prepared by freezing and slowly thawing fresh plasma. Cryoprecipitate is rich in Factor VIII, von Willebrand factor, and fibrinogen. DDAVP: 1-Desamino-8-D—arginine—vasopressin, an analogue of antidi- uretic hormone used as a vasoconstrictor. DDAVP stimulates release of Factor VIII and von Willebrand factor from endothelium. Disseminated Intravascular Coagulation (DIC): A disturbance in the hemostatic balance, that produces the release of activated procoagulants into the circulation. Both platelets and coagulation factors are consumed, fibrin is deposited in small vessels in many organs, and the fibrinolytic system is activated. Dysfibrinogenemia: A qualitative abnormality in the fibrinogen molecule due to a possible amino acid substitution. The condition is usually an autosomal-dominant trait. More than 40 abnormal fibrinogens have been reported, with only a few of the actual defects known. Defects can result in abnormal aggregation of fibrin monomer, abnormal release of fibrinopep— tides, or abnormal crosslinking. The thrombin, prothrombin, and Reptilase times in these patients are prolonged. Ecchymosis: Hemorrhage into the skin greater than 3 cm in diameter. Essential Thrombocythemia: A myeloproliferative disorder character- ized by proliferation of megakaryocytes, extreme thrombocytosis, and neutrophilic leukocytosis. Platelet counts regularly go above 1000 X 103/L, and a frequent sign is gastrointestinal hemorrhage. Fibrin: The insoluble protein that is formed when thrombin acts on fibrinogen. Fibrin Clot: A thrombus composed of platelets and fibrin. Fibrin Degradation Products: The breakdown products of fibrinogen and fibrin that result from activation of fibrinolysis. There are four main fibrin degradation products (X, Y, D, E). All can act as strong anticoagu- lants and are seen in patients with disseminated intravascular coagulation, renal transplant patients, and patients with primary fibrinolysis. 111 Fibrinogen: A large glycoprotein that circulates in the plasma that is the precursor of fibrin. Fibrinogenolysis: Pathologic process, resulting from excess free plasmin destroying fibrinogen and other coagulation factors. Fibrinogenolysis is a component of disseminated intravascular coagulation. Fibrinolysis: The process of breaking down or lysing fibrin through enzymatic dissolution. Glanzmann’s Thrombasthenia: A rare, autosomal—recessive congenital bleeding disorder, in which platelets function abnormally although they are normal in number. There is usually no aggregation response to ADP, epinephrine, or collagen. Hematoma: A swelling filled with clotted blood that has escaped from a blood vessel into the tissues or a cavity. Hemophilia: A group of hereditary bleeding disorders of coagulation in which one of the coagulation proteins is missing or defective. Hemorrhage: Uncontrolled bleeding into the skin or surrounding environ- ment. Hemostasis: A physiologic system based on blood vessel integrity, platelet function, and a set of plasma proteins, which functions to preserve blood vessel integrity and prevent hemorrhage. Hemostatic Plug: The thrombus formed to arrest the flow of blood. Heparin: A complex heteropolysaccharide with a structure similar to that of heparan sulfate, but with more sulfate groups. In vitro, heparin is used as an anticoagulant to prevent the tube of blood from clotting so that is can be tested. Hypofibrinogenemia: An abnormally low concentration of fibrinogen in the blood. This is usually inherited as an autosomal-recessive trait and may occur in disseminated intravascular coagulation. Immune Thrombocytopenia Purpura (ITP): A chronic or acute ac- quired thrombocytopenia caused by the presence of circulating antiplatelet autoantibodies or isoantibodies or deposition of immune complexes. Inosithin: A soybean phospholipid that is useful as a partial thromboplas- tin. Lupus Inhibitor: A circulating anticoagulant seen in 5% to 10% of patients with lupus erythematosus that causes a prolonged APTT. The lupus inhibitor is often associated with a thrombotic tendency. May-Hegglin Anomaly: A disorder of autosomal-dominant inheritance characterized by the presence of Dohle bodies in leukocytes, increased platelet size, and occasional thrombocytopenia. Megakaryocyte: A bone marrow cell that is the precursor to the platelet. Megakaryocytes are uninucleate but multiploid and are the largest cells found in the bone marrow. Platelets are formed from megakaryocytes by cytoplasmic fragmentation. 112 The Bleeding and Clotting Disorders Partial Thromboplastin: A lipid reagent used as a substitute for platelets in clotting tests. Petechia: A hemorrhage into the skin of less than 3 mm in diameter. Plasminogen Activator: A heterogeneous group of serine proteases capable of converting plasminogen to plasmin; for example, tissue plas- minogen activator (endothelium, uterus, heart), circulating plasma plas— minogen activator, urokinase, and kidney tissue cell cultures. Platelet (Thrombocyte): An anucleate cell produced by cytoplasmic fragmentation of a bone marrow cell known as the megakaryocyte. The platelet is responsible for initiation of plasma coagulation in the case of vascular trauma. Platelet Factor 3: Fragments of plasma membrane phospholipid released from the platelet upon activation that serve as a point of assembly for certain of the plasma coagulation reaction complexes. Platelet-Poor Plasma (PPP): The plasma preparation used for most coagulation studies and for setting 100% transmission of light in platelet aggregometry. It is prepared by centrifugation of whole blood at 1600g to 2000g for 5 to 10 minutes. Platelet-Rich Plasma (PRP): The supernatant of whole blood required for platelet aggregometry, having a high concentration of platelets. It is prepared by centrifugation of whole blood at 75g to 100g for 5 to 10 minutes. Protein C: A vitamin K-dependent protein possessing anticoagulant properties characterized by inhibitory action against Factor V and VIII. It has the additional property of stimulation fibrinolysis. Protein C is con- verted to its enzymatic form (protein Ca) by a complex of thrombin and thrombomodulin, an endothelial cell thrombin receptor. Protein S: A vitamin K-dependent factor that markedly enhances the activity of protein C. Purpura: A hemorrhage into the skin between 3 mm to 10 mm in diameter. Also, a group of hemorrhagic diseases characterized by hemorrhage into the skin, mucous membranes, and serosal surfaces. Ristocetin: An antibiotic that induces agglutination of platelets in the presence of von Willebrand factor. It is used to produce platelet agglutina— tion. Platelets will not agglutinate to ristocetin in von Willebrand disease or Bemard—Soulier syndrome. Storage-Pool Disorder: A thrombocytopathy in which platelet secretion (release) is impaired, probably because of diminished levels of ADP or serotonin in dense body granules. Aggregation patterns are usually dimin— ished or may show primary aggregation followed by disaggregation, and no aggregation with collagen. Thrombasthenia: A rare, severe congenital bleeding disorder, transmitted as an autosomal-recessive trait, in which platelets function abnormally 113 although they are normal in number. There is usually no aggregation response to ADP, epinephrine, or collagen, a prolonged bleeding time, and normal clotting factors; also called Glanzmann’s thrombasthenia. Thrombocythemia: A fixed increase in circulating platelets associated with an increase in megakaryocyte number and volume. The term is usually used in reference to myeloproliferative disorders such as essential thrombo- cythemia in which the platelet count is frequently greater than 1,000,000/L. Thrombocytopenia: A decrease in the number of circulating platelets; a platelet count below 1.5 X lO3/L. Thrombocytosis: An increase in the number of circulating platelets; platelet count above 4 X 103/L. B-Thromboglobulin: A protein contained in the alpha granules of platelets that is released upon activation and that neutralizes heparin weakly. Thrombomodulin: A cofactor found on the endothelium that helps to inactivate thrombin. Thromboplastin: A substance having procoagulant properties that is found in most tissues. Thrombopoietin: The cytokine or cell growth factor that induces forma— tion of the megakaryocytic cell line. Thrombosis: Formation of a clot in a blood vessel, a lung, or the heart. The clot is composed of platelets, fibrin, and usually erythrocytes. Thrombotic Thrombocytopenic Purpura: A clinical condition marked by increased platelet consumption and thrombi in the microcirculation. Thromboxane: A prostaglandin product of platelet activation formed from arachidonic acid through the cyclooxygenase pathway. This sub— stance is one of the most potent aggregating agents and produces strong contraction of vascular muscles and platelet shape change. Von Willebrand Disease: An autosomal-dominant bleeding disorder in which the bleeding is from the mucous membranes and skin. There are many variants, with mild to severe expression depending on the amount of decrease of the von Willebrand factor. Von Willebrand Factor: That part of the Factor VHI molecule responsible for platelet adhesion and function. It has been referred to as Factor VIII/ vWF, ristocetin cofactor, and VIIIR2Ag in the past. 114 The Bleeding and Clotting Disorders 115 Index A Abruptio placentae 16 Acceptable results 41 Accuracy 35, 40 Acquired immunodeficiency syndrome (AIDS) 32, 34 Acquired platelet-function abnormalities 10-12 Activated partial thromboplastin time (APTT) 31, 44, 79-80, 102 Adhesion 3, 5, 12, 13, 45, 70 Afibrinogenemia 26,75, 109 Aggregation 13 Alcolec 79, 105 Amniotic fluid embolism 16 Anemia 9, 11, 27 Antithrombin III 8, 15, 30, 109 APPT reagent 30 APTT reagent 79, 105 Automated coagulometers 53 B Bemard-Soulier syndrome 12, 34, 64, 74, 109 Biological safety 51 Bleeding time 11, 13, 25, 28, 70, 109 Blood count 25-27, 29 Blood film 11, 58-75, 61, 63-75 Blood sampling 57 Blood smear 25, 26, 27, 31 Bone marrow 3, 4, 11 Buffered saline 78, 105 Burns 17 C Calcium chloride 46, 76, 79, 105 Calibration 36, 41—43, 99 Calibrator 36 Capillary blood collection 57, 68 Celite 109 Celite suspension 79, 105 Centrifuge 41 Chemical safety 55 Circulating anticoagulant 17, 28 116 The Bleeding and Clotting Disorders Clauss method 81 Clinical aspects 19-24 Clot 5, 8, 16, 17, 30, 64, 65, 110 Clot retraction 5, 12 Coagulation factor disorders 5, 12-17 Coagulometers. See Automated coagulometers Coefficient of variation (CV) 39 Common pathway 5, 8, 28 Contraceptives. See Oral contraceptives Control 36-37, 49, 69, 77 Control chart 39 D Disseminated intravascular coagulation (DIC) 10, 16—17, 26, 31, 110 E Electrical safety 55-56 Erythrocyte sedimentation rate (ESR) 25, 27, 58, 86 Euglobulin lysis test 92-93 External quality assessment 35, 40—41 Extrinsic coagulation pathway 5 Extrinsic system 5—6, 7, 76 F Factor IX 7, 13, 14,27, 45 Factor VIII 7, 12,14, 27, 33,45, 91 Factor VIII assay 89-92 Factor XIII assay 94-95 Factors 15, 16, 92 Fibrin 1, 7, 110 Fibrinogen 3, 5, 7, 9, 28, 30, 64, 65, 81, 111 Fibrinogen calibration curve 82 Fibrinolysis 8, 65, 111 Fire safety 55-56 Fixative 62, 106 G Gaussian curve 37 H Heat stroke 17 Hematocrit. See Packed cell volume (PCV) Hemocytometer 65, 66-67 117 Hemoglobin 25, 36 Hemolytic transfusion reactions 17 Hemophilia A 12, 14, 27 Hemophilia B 13, 14, 27 Hemostasis 1-17 normal 1-9 pathologic 8-12 Heparin 102 Heparin anticoagulation 10 1 — 103 Hereditary platelet disorders. See Platelet: disorders: hereditary History 19, 23 Hook 75, 76, 80 I Infection 25 Inhibitors 12-17, 80 Inosithin 79, 106, 111 Internal quality control 35 International Normalized Ratio (INR) 97, 100 International Sensitivity Index (181) 97, 99 Intrinsic coagulation system 6-8, 79-86 Ivy method 71 K Kaolin 79, 105, 109 and plasma clotting time 80 clotting time 79 L Laboratory safety 51-56 Leukocytosis 25 Liver disease 26, 28 Lupus erythematosus 17, 28 M Mean 35 Megakaryocytes 34, 111 Metastatic carcinoma 17 Microscope 42 Monochloroacetic acid 106 Myeloblastic leukemia 17 Myeloproliferative disorders 9, 26 118 The Bleeding and Clotting Disorders N Normal control of clotting 8 Normal plasma pool 94 Normal reference values. See Reference values Normal values 49 0 One stage prothrombin time 97 Oral anticoagulants 30, 97-100 Oral anticoagulation therapy 97-103 Oral contraceptives 77 P Packed cell volume (PCV) 25, 48 Partial thromboplastin time 6, 7, 25, 27 Phosphate 59, 62, 106 Phospholipids 7, 17, 28, 109 Physical examination 20, 23 Pipette 42 Plasma clotting time 102 Plasma pool, normal 94 Platelet 112 aggregation 45, 109 count 25, 31, 63-75 count estimate 27, 63-75 disorders 10—12 hereditary 12 dysfunction 10 "Factor 3" 3, 6, 48, 79, 112 function 64 plug 3-5 structure 4 Platelets 1, 7, 8, 9-12, 26, 44 Precision 35 Protein C 23, 30, 112 Protein S 23, 30, 112 Prothrombin ratio (PR) 100 Prothrombin time (PT) 5, 7, 27, 30, 44, 76, 80, 99 Q Quality assurance 35—50 Quality control 39, 44-49 R Recalcification time 102 Reference material 36 Reference preparation 36, 99 ' Reference range 49 Reference values 49 Release reaction 3 Retained dead fetus 16 Romanowsky stain 58, 59 S Saline 28, 78, 105 Screening tests 29, 31 Shock 17 Smoking 20, 23 Sodium citrate 45, 50, 58 Standard deviation (SD) 35, 37 Storage-pool disease 75, 112 Surgery 31 Synthetic substrates 47 T Template method 72-75 Thrombasthenia 63, 74, 112 Thrombin l, 5, 7 Thrombin time 5, 29, 84 Thrombocythemia 11 Thrombocytopenia 9, 25,26, 31, 33, 34, 65, 113 Thrombocytosis 11-12, 25, 26, 113 Thromboplastin 79, 113 Thromboplastin calibration 97-99 Thromboplastin working preparation (WP) 97 Thrombotic disease 20-21, 24 Tissue plasminogen activator (TPA) 8, 31 Toxemia of pregnancy 16 Tumor 9 U Universal precautions 51 Urea 106 119 120 The Bleeding and Clotting Disorders V Vascular component 1-5 Vascular defects 8-9, 25 Venomous snake bites 1, 17 Venous blood collection 57-58 Veronal buffer 79, 105 Vitamin K 95 Vitamin K deficiency 13, 26, 28 Vitamin K1 15 Vitamin K1 test 95-96 von Willebrand 13, 14, 25, 26, 27, 70, 74 W White blood count (WBC) 25 Wright’s stain 59 PUB LLLLLLLLLLLLLLL APR 201993 \\\\\\\\\\\\“N\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ 3333333333