Articles on Medical Diseases and Conditions

Category: Blood Coagulation

  • Investigation of Bleeding Disorders

    There is no standardized protocol for workup of bleeding problems. Also, one should differentiate between workup of a patient who is actively bleeding and the use of screening tests in persons who are not bleeding. Any algorithm presented could be criticized by someone who prefers a different approach or different test procedures. Nevertheless, I will present one possible workup for persons who are actively bleeding. This includes bleeding time (to test for platelet and capillary fragility abnormalities), inspection of a peripheral blood smear for platelets and RBC morphology, APTT (to test for intrinsic pathway abnormalities), PT (to test for extrinsic pathway abnormalities), and FDP test (for DIC). If bleeding time is abnormal, a platelet count should be done. If the PT and PTT results are both abnormal, the fibrinogen level should be determined. If the APTT is the only abnormal finding, a simple correction experiment is done using the APTT and patient plasma diluted 1:1 with fresh normal plasma. If the APTT is corrected, the defect is presumed to be a factor deficiency within the intrinsic pathway. If the abnormal APTT is not corrected, or if there is time-dependent noncorrection, a circulating anticoagulant or an inhibitor should be suspected. If the PT is the only abnormal finding, liver disease or unsuspected coumarin intake should be suspected. If both the APTT and PT results are abnormal, unsuspected heparin effect (e.g., heparin flushes) are possible, as well as DIC, coumarin therapy, severe liver disease, and circulating antithrombins or fibrinolysins. The results from the FDP test should help support or rule out DIC.

    If at all possible, the results of abnormal screening tests should be verified by a redrawn blood specimen, since a frequent cause of incorrect results is a nonoptimal specimen.

    A frequent obstacle to correct diagnosis is transfusion of blood or blood products before a bleeding disorder is suspected, or empirical attempts at therapy before diagnostic tests are ordered or a consultation is obtained. If there is any question of abnormal bleeding, a citrate anticoagulant coagulation test tube, an EDTA-anticoagulated hematology test tube, and a nonanticoagulated serum tube should be obtained in case coagulation tests are needed later. The citrate tube should be kept in the refrigerator.

  • Bleeding Problems in Surgery

    Bleeding constitutes a major concern to surgeons. Problems may arise during operation or postoperatively, and bleeding may be concealed or grossly obvious. The major causes are the following:

    1. Physical defect in hemostasis—improper vessel ligation, overlooking a small transected vessel or other failure to achieve adequate hemostasis, or postoperative tissue devitalization and release of a vessel ligature.
    2. Unrecognized preoperative bleeding problem— a coagulation defect is present but was not recognized before surgery. This may be congenital (e.g., hemophilias), secondary to a disease that the patient has (e.g., cirrhosis), or due to medications (e.g., aspirin or anticoagulant therapy).
    3. Transfusion reactions or complications from massive transfusion.
    4. Intraoperative DIC.
    5. Unexplained bleeding difficulty.

    Unusual bleeding has some correlation with the type (magnitude) of the operative procedure, the length of the operation, and the particular disease involved. The more that any of these parameters is increased, the more likely that excessive bleeding may occur. In most cases, the defect can be traced by means of laboratory tests or, retrospectively, by reestablishing physical hemostasis. In some cases laboratory workup is hindered by transfusions administered before a coagulation problem was considered. Sometimes the source of bleeding is never uncovered, even after thorough investigation. This fact cannot be used as an excuse for inadequate workup, because proper therapy depends on finding the etiology. This is why some knowledge of blood coagulation mechanisms is necessary.

  • Platelet Defects

    According to one report, platelet counts on capillary (fingerstick) blood average ±3% lower than on venous blood samples and about 25% of the capillary samples were 25% or more below venous results. Platelet-associated abnormality is most commonly produced by decreased number (thrombocytopenia) or defective function (thrombocytopathia). A bleeding tendency may also appear with a greatly increased platelet count (thrombocytosis), usually not until the count exceeds 1 million/ mm3. Clinically, purpura is the hallmark of platelet abnormality. Most other types of coagulation disorders do not cause purpura.

    Thrombocytopenia

    Decrease in number is the most common platelet abnormality. In general, such conditions may be classified according to etiology:

    1. Immunologic thrombocytopenia
    Drug-induced thrombocytopenia
    Idiopathic thrombocytopenia
    Posttransfusion thrombocytopenia
    Other thrombocytopenias with an immunologic component
    2. Hypersplenism
    3. Bone marrow deficiency
    4. Other causes

    Immunologic thrombocytopenia

    Drug-induced thrombocytopenia. This syndrome occurs due to idiosyncratic hypersensitivity to certain drugs. It may develop during initial, continued, or intermittent use of the drug. Once hypersensitivity begins, platelet depression follows swiftly. The bone marrow most often shows a normal or increased number of megakaryocytes, which often display degenerative changes. The most frequently associated drugs are heparin, quinidine, quinine, cimetidine, and various sulfonamide derivatives; but other drugs (potassium chloride and furosemide, among others) have been incriminated in rare instances. Of course, this effect is uncommon even with the relatively frequent offenders. Platelet antibodies have been demonstrated in many cases. Intravenous heparin causes thrombocytopenia below 100,000/mm3 (µL) in about 10%-15% of patients (range, 1%-30%). About 2% develop prolonged decrease below 100,000/mm3. It has been estimated that 33%-66% of patients who receive heparin intravenously develop some degree of platelet decrease from baseline levels. Thrombocytopenia has even been reported with heparin flushes. Decrease in platelets occurs about 5-7 days (range, 2-15 days) after start of therapy. The degree of thrombocytopenia is most often mild or moderate but in some cases may be less than 50,000/mm3. Diagnosis of immune thrombocytopenia can be assisted by platelet-associated IgG measurement in the presence of the offending drug. However, at present none of the methods is easy; none detects all cases; false positive results are sometimes reported in nonimmune thrombocytopenias, and small degrees of hemolysis may interfere.

    Idiopathic thrombocytopenic purpura. ITP may exist in either an acute or chronic form. The acute form is usually seen in children, has a sudden onset, lasts a few days to a few weeks, and does not recur. The majority of cases follow infection, most often viral, but some do not have a known precipitating cause. The chronic form is more common in adults; however, onset is not frequent after age 40. There are usually remissions and exacerbations over variable lengths of time. No precipitating disease or drug is usually found. Platelet antibodies have been demonstrated in 80%-90% of patients with chronic ITP. The methods used are generally based on measurement of platelet-associated IgG. Some patients eventually are found to have systemic lupus erythematosus or other diseases.

    Clinically, there is purpura or other hemorrhagic manifestations. The spleen is usually not palpable, and an enlarged spleen is evidence against the diagnosis of ITP. Bone marrow aspiration shows a normal or increased number of megakaryocytes, although not always.

    Posttransfusion purpura. Platelets contain certain blood group and tissue antigens on their surface; the most important of these are ABO, HLA, and platelet-specific antigen (PLA). Posttransfusion purpura usually (not always) occurs in patients whose platelets are PLA1 negative, who have been sensitized to the PLA1 antigen by previous transfusions or by pregnancy, and who are then administered blood products containing PLA1-positive platelets. An alloantibody is formed in response to sensitization. Once the antibody is formed, it exhibits rather unusual behavior for an alloantibody since it attacks both PLA1-positive or PLA1egative platelets, whether belonging to the patient or a donor. The syndrome is uncommon in spite of the fact that about 2% of Europeans are PLA1 negative. The great majority of reported patients have been female. Onset of thrombocytopenia most often occurs about 7 days after transfusion (range 2-14 days). Thrombocytopenia is often severe. The episodes last about 3 weeks, with a range of 4-120 days. They may or may not recur with future transfusion. If massive transfusions with stored bank blood are given during a short time period, thrombocytopenia frequently develops, usually ascribed to dilutional factors and to the low functional platelet content of stored bank blood. This takes at least five units of blood and usually more than 10, given within 1-2 days’ time, and may or may not be accompanied by a bleeding tendency. However, stored blood deficiency of factors V and VIII (the unstable clotting factors) may contribute to any bleeding problem.

    Other thrombocytopenias with an immunologic component

    Neonatal thrombocytopenia may be due to antiplatelet antibodies in maternal blood that are produced when fetal platelets contain an antigen (most commonly PLA1) that is absent on maternal platelets. Analogous to Rh immune disease, the mother produces IgG antiplatelet alloantibody that crosses the placenta to the fetal circulation. The mother and fetus are usually ABO group compatible. Infants often respond poorly to random platelet transfusion but much better to washed maternal platelets. The mother is usually not thrombocytopenic and the maternal antibody does not react with maternal platelets. Neonatal thrombocytopenia may also be produced by maternal antibodies associated with maternal ITP. About 50% of the infants of these mothers have severe thrombocytopenia. The mother’s platelet count does not reliably predict the infant’s count. In one study, about 25% of women with autoimmune thrombocytopenia and platelet counts more than 100,000/mm3 (100 Ч 109/L) delivered infants with platelet counts less than 50,000/mm3. Neonatal thrombocytopenia may also be due to other causes, such as intrauterine viral infection or neonatal sepsis.

    Narcotic addicts and clinically healthy homosexual males have a high incidence of thrombocytopenia. Most, but not all, display reactive screening tests for HIV-I infection. Some have demonstrable antiplatelet antibodies and some do not.

    Hypersplenism

    Hypersplenism was discussed in Chapter 5. The syndrome may be primary or secondary; if secondary, it is most commonly due to portal hypertension caused by cirrhosis. There may be any combination of anemia, leukopenia, or thrombocytopenia, but isolated thrombocytopenia is a fairly frequent manifestation. The spleen is usually palpable, but not always. Bone marrow megakaryocytes are normal or increased. The thrombocytopenia seen in lupus erythematosus is usually associated with antiplatelet antibodies, but there may be an element of splenic involvement even though the spleen is often not palpable.

    Bone marrow deficiency

    This condition and its various etiologies were discussed in Chapter 4, the principal causes being metastatic tumor to bone, aplastic anemia, and myelofibrosis. This group forms a large and important subgroup of the thrombocytopenias and is the reason why bone marrow examination is frequently indicated in a patient with thrombocytopenia.

    Thrombocytopenia is a very frequent feature of acute leukemia and monocytic leukemia, even when the peripheral blood WBC pattern is aleukemic. It may also occur in the terminal stages of chronic leukemia.

    Other causes of thrombocytopenia. A miscellaneous group remains that includes various unrelated disorders, some of which will be discussed.

    Microangiopathic hemolytic anemia. This is a group of conditions that share the hematologic picture of hemolytic anemia, thrombocytopenia, a considerable number of red cell schistocytes in the peripheral blood, and a tissue histologic picture of fibrin thrombi in small blood vessels. This group includes DIC, thrombotic thrombocytopenic purpura (Moschowitz’s disease), the hemolytic-uremic syndrome, the prosthetic valve hemolytic syndrome, cancer chemotherapy (rarely, cancers without chemotherapy, mostly adenocarcinomas such as prostate or stomach), Zieve’s syndrome, sepsis, and the HELLP preeclamptic syndrome. Thrombotic thrombocytopenic purpura (TTP) will be presented here as a representative example of the group. The other conditions are discussed separately elsewhere. TTP is a very uncommon disorder that occurs most frequently in young adults, although it may occur at any age. There is a characteristic triad of severe microangiopathic hemolytic anemia (96%-98% of cases), thrombocytopenia (83%-96%), and neurologic symptoms (84%-92%) that typically are multiple and shifting. About 75% of patients have the complete triad. Some also include renal disease (76%-88%) and fever (59%-98%). Hemoglobin is less than 10 gm/100 ml in about 90% of cases. The peripheral blood usually contains many schistocytes. Nucleated RBCs are present in about 20%. The direct Coombs’ test is positive in 6% (0%-7%). The white blood cell count is increased in about 55%. Serum bilirubin levels are elevated to some degree in about 80% with the unconjugated fraction predominating. Serum LDH levels are increased, and haptoglobin levels are decreased. In textbook cases, PT, APTT, fibrinogen, and FDP are all normal. However, in several series about 18% had elevated PT; 7% had elevated APTT; 7% decreased fibrinogen; and about 25% elevated FDP.

    Thrombi composed of platelets with some fibrin occur in capillaries and small arterioles. Diagnosis is most definitively made through biopsy. Renal biopsy was the original recommended procedure; however, gingival or skin biopsy (if possible, of a petechia) is more common now. However, diagnostic yield from these sources is less than 40%. Differential diagnosis includes the other microangiopathic hemolytic anemias. Systemic lupus, autoimmune hemolytic anemia, and Evan’s syndrome also enter consideration of hemolytic anemia, but these anemias usually are not microangiopathic, and the results of the Coombs’ test are usually positive.

    Megaloblastic anemia. Thrombocytopenia occurs as a frequent manifestation of untreated well-established B12 and folic acid deficiency anemias, sometimes even when the anemia is mild. In chronic iron deficiency anemia, platelet counts are normal and may at times actually be somewhat increased.

    Infections. Thrombocytopenia has been reported in 18%-77% of patients with bacteremia. Neonatal thrombocytopenia always raises the question of sepsis. However, thrombocytopenia is not limited to actual septicemia. In one study of patients who had surgery because of intestinal perforation with peritonitis, all patients showed platelet count decrease that reached its lowest point 3-5 days after surgery at mean platelet levels about 55,000/mm3. This was followed by slow platelet count increase that reached the 100,000/mm3 level about postoperative day 10. This thrombocytopenia did not seem to produce an increased tendency to bleed and was not related to DIC, although DIC can develop in septic patients. Thrombocytopenia can occur in nonbacterial infections. It is especially associated with the congenital rubella syndrome and with HIV-I virus infection (2.6%-90% of HIV non-AIDS patients and 11% in patients with less than 250 CD4 lymphocytes). However, thrombocytopenia may occasionally and transiently be found in the early stages of other virus infections such as Epstein-Barr infectious mononucleosis. The hemolytic-uremic syndrome is a microangiopathic hemolytic anemia with thrombocytopenia and renal failure, usually following infection. It is most often seen in younger children, usually before age 5 years, and most often following onset of gastroenteritis. Today, possibly the most common cause is verotoxin-producing Escherichia coli 0157:H7, although viruses and a number of bacteria have also been incriminated.

    Rheumatoid-collagen diseases. Thrombocytopenia is reported in about 15% (range, 5%-26%) of patients with systemic lupus.

    Hypertension of pregnancy.—Thrombocytopenia has been associated with preeclampsia (pregnancy-induced hypertension) in 16% of cases in one study. Preeclampsia itself is reported in about 8% of pregnancies (range, 5%-15%), about 80%-85% of which are first pregnancies. About 3%-12% of preeclamptic patients (more often severe) develop microangiopathic peripheral smear changes with relatively normal coagulation studies (HELLP syndrome), a laboratory picture similar to microangiopathic hemolytic anemia. Thrombocytopenia, usually mild and transient, has been reported in about 10% (range, 0.3%-24%) of all pregnancies.

    Thyrotoxicosis. Some degree of thrombocytopenia has been reported in 14%-43% of patients with Graves’ disease. Severe thrombocytopenia with bleeding is uncommon.

    Uremia. As noted in the discussion of the bleeding time test, up to 50% of patients in uremia are reported to have some degree of thrombocytopenia in addition to various platelet function defects.

    Artifactual thrombocytopenia. Apparent thrombocytopenia is occasionally encountered when platelet counts are performed by particle-counting machines. Some of the causes are platelet satellitosis around neutrophils in EDTA-anticoagulated blood, many giant platelets, improperly prepared specimens (platelet aggregates), and platelet cold agglutinins. Peripheral blood smears may also falsely suggest some degree of thrombocytopenia due to platelet clumping or uneven distribution if the slide is not properly made.

    Platelet function defects

    Platelet function usually refers to the role of platelets in blood coagulation. To carry out this role, platelets go through a series of changes, partially morphologic and partially biochemical. Abnormality may develop at various stages in this process, and platelet function tests have been devised to detect abnormality in certain of these stages. These are special procedures not available in most laboratories and include techniques designed to evaluate platelet factor release (PF-3 or serotonin), platelet aggregation (adenosine diphosphate, thrombin, collagen, epinephrine) and platelet adhesion (glass bead retention). These tests are useful primarily to categorize platelet action abnormality rather than to predict the likelihood of bleeding. The bleeding time test is probably the best procedure to evaluate degree of clinical abnormality.

    Hereditary disorders of defective platelet action with normal platelet count is uncommon, the most famous of this group being Glanzmann’s disease (hereditary thrombasthenia). Platelets in Glanzmann’s disease have abnormal aggregation and glass bead retention. The clot retraction test result is abnormal, whereas the results are normal in other thrombopathic (platelet function abnormality) disorders. Tourniquet test results are variable.

    Defective platelet function has been observed in many patients with uremia and some patients with chronic liver disease, even without the thrombocytopenia that occasionally may develop. Cryoprecipitate or desmopressin can correct the bleeding time elevation in uremia. Many other conditions can sometimes be associated with platelet function test abnormalities, including leukemias and myeloproliferative disorders, dysproteinemias such as myeloma, and systemic lupus. Giant platelets may be found in certain conditions, especially in myeloid metaplasia (less often in chronic myelocytic leukemia), but this does not seem to produce a clinical bleeding tendency.

    Certain drugs may interfere with platelet function, as noted earlier. Aspirin affects platelet factor release and also platelet aggregation. Other drugs may interfere with one or more platelet function stages.

    von Willebrand’s disease

    von Willebrand’s disease combines platelet function abnormalities with deficiency in factor VIII activity. The clinical disease produced has been called “pseudohemophilia.” Although there used to be considerable argument as to just what this disease should include, it is now generally restricted to a hereditary disorder of the von Willebrand factor portion of the factor VIII/vWf complex. As described in the section on factor VIII deficiency, the factor VIII/vWf complex consists of two components, the hemophilia A factor VIII portion and the von Willebrand factor portion. Factor VIII controls factor VIII activity within the coagulation intrinsic pathway system. In hemophilia A, VIII antigenic material is present but is nonfunctional, so that VIII:C activity is decreased. The vWf portion of the complex is normal both in quantity and function. That part of the complex comprising vWf controls at least two aspects of platelet function. In von Willebrand’s disease the vWf antigen is decreased, and platelets display decreased adhesiveness (manifested by decreased retention in glass bead columns) and also decreased platelet agglutination under the stimulus of the antibiotic ristocetin. In addition, the vWf is thought to stabilize factor VIII levels, so that a decrease in vWf leads to a decrease in factor VIII levels, both in the quantity of factor VIII as well as its activity. Thus, the entire factor VIII complex is decreased.

    Classic von Willebrand’s disease (and all but one variant forms) is transmitted as an autosomal dominant trait (in contrast to hemophilia A, which is transmitted as a sex-linked recessive trait). Most patients with von Willebrand’s disease are heterozygous and have a clinically mild hemorrhagic disorder. Most of the serious bleeding episodes are induced by trauma or surgery. Patients homozygous for von Willibrand’s factor deficiency are uncommon; these patients have a severe hemorrhagic disorder. In a third variant, also uncommon, vWf is present in normal quantity but is nonfunctional or only partially functional; these patients have normal factor VIII activity and normal vWf antigen by immunoassay but low ristocetin cofactor and platelet glass bead retention activity and an abnormal bleeding time.

    Acquired von Willebrand’s disease. A few patients have been reported who had clinical and laboratory findings consistent with von Willebrand’s disease but had no evidence of hereditary transmission. These patients had a variety of diseases but most often seemed to have lymphoma, carcinoma, autoimmune disorders, and conditions associated with monoclonal gammopathies.

    Laboratory diagnosis. The various forms of von Willebrand’s disease as well as the expected results of the various diagnostic tests are summarized in Table 8-1. The most commonly used screening tests are the bleeding time and, to a lesser extent, the APTT. As noted previously, there is a wide spectrum of clinical severity and also degree of laboratory test abnormality in patients with von Willebrand’s disease. Therefore, sensitivity of the APTT has varied from 48%-100% in different reports. The bleeding time is more reliable, but also can be normal. In the usual (“classic”) type, factor VIII activity is variably decreased, the bleeding time is prolonged, and platelet function by glass bead column retention and ristocetin aggregation is abnormal. However, even in the classic form, some patients display bleeding times that may intermittently be normal. In some patients factor VIII:C activity is normal but the bleeding time is prolonged. In others the disease is so mild that even the bleeding time is normal, but pretreatment with aspirin can uncover the bleeding time defect. As noted in the section on factor VIII, the factor VIII/vWf complex is one of the so-called acute reaction protein group, so that vWf can be increased to some degree in surgery, infection or noninfectious inflammation, severe exercise, and severe stress of other types and in mild cases may temporarily correct the bleeding time and factor VIII/vWf assay results. Increased estrogens (pregnancy or use of estrogen-containing contraceptives) can increase factor VIII activity and vWf antigen levels in some patients with von Willebrand’s disease even though the bleeding time may continue to be abnormal. In a few cases, especially when laboratory results are conflicting or equivocal, it may be necessary to obtain multimeric analysis (analysis of the factor VIII/vWf complex structure fraction sizes by special gel electrophoresis).

    Table 8-1 von Willebrand’s disease variant forms*
    von Willebrand's disease variant forms

    Therapy. Fresh frozen plasma or cryoprecipitate contain the factor VIII complex and can temporarily correct vWf deficiency. Administration of desmopressin can temporarily stimulate factor VIII/vWf production (to levels about twice baseline) and correct the bleeding time, as was discussed in the section on hemophilia A therapy. However, desmopressin effect on vWf lasts only about 3-6 hours, which is somewhat less prolonged than the effect on factor VIII.

    Thrombocytosis

    Most attention given to blood platelets is focused on disorders of platelet function or decreased platelet number. However, thrombocytosis may sometimes occur. If the platelet count is greater than 900,000 or 1,000,000/mm3, there is concern for the possibility of hypercoagulability leading to venous thrombosis. In one report, about 25% of patients with thrombocytosis (platelet count >900,000/mm3) had a hematologic disorder (myeloproliferative syndrome, idiopathic thrombocythemia, severe hemolytic anemia, posthemorrhage, e.); about 25%-35% had cancer; about 20% were postsplenectomy; about 20% had acute or chronic infection or inflammatory conditions; and about 10% had collagen disease. In most cases there is no clinical problem until the platelet count exceeds 1 million/mm3. When that happens there is an increased tendency to bleed and also to develop thrombosis. The most common diseases associated with very high platelet counts are idiopathic thrombocythemia and the myeloproliferative syndromes.

    Mean platelet volume. Certain automatic particle counters that count platelets also calculate the mean platelet volume (MPV). The reference range apparently varies inversely with the platelet count, unless a wide reference range is established to include all patients with platelet counts within the platelet count reference range. The MPV is said to be increased in ITP and various thrombocytopenias and in conditions associated with increased platelet size such as some of the myeloproliferative syndromes and the May-Hegglin anomaly. It is very typically increased in the Bernard-Soulier syndrome, which has large platelets. The MPV is decreased in the Wiscott-Aldrich syndrome and possibly in some patients with chronic iron deficiency or aplastic anemia. Occasionally, RBC fragments may be counted as platelets, producing artifactual increase in the platelet count. However, MPV is also increased in these patients.

    Vascular defects

    Senile purpura is a frequent nonhereditary type of vascular fragility problem, manifested by localized purpuric lesions or small bruises developing on the extremities of older persons. The only laboratory abnormality is a positive tourniquet test result in some cases. A somewhat similar clinical condition is the easy bruising found in some young adult persons, especially women. All results of standard laboratory tests are usually normal, except for an occasionally positive tourniquet test result. Some of these persons have abnormal platelet function test results; in the majority, however, the reason for abnormality is not known. It should be mentioned that continued or intermittent bleeding from a small localized area is most often due to physical agents (e.g., repeated trauma) or to a local condition (e.g., scar tissue) that prevents normal small-vessel retraction and subsequent closure by thrombosis.

    Allergic (anaphylactoid) purpura is characterized by small blotchy hemorrhages over the extremities, frequently accompanied by ankle edema, produced by an allergic capillary or small-vessel vasculitis. Many patients also have glomerulonephritis. Henoch’s purpura is a subdivision in which the bleeding occurs mainly in the GI tract. Sch?nlein’s purpura features the skin manifestations without GI involvement. The tourniquet test result is usually positive. Platelet counts and other laboratory test results are normal. Diagnosis is made through biopsy of a fresh small purpuric area.

    Embolic purpura can simulate capillary fragility defects, although the skin lesions may resemble petechiae more than the larger lesions of purpura. There may, however, be some component of capillary fragility. These disorders include subacute bacterial endocarditis, fat embolism, and some cases of septicemia (although other cases of septicemia also have thrombocytopenia). The tourniquet test result is often positive and the bleeding time is variable. Other coagulation defect test results are normal (except in septicemia complicated by DIC). When the patient is seriously ill with disease of acute onset, purpura raises the question of meningococcemia.

    Laboratory investigation of purpura

    The etiologic diagnosis of purpura should begin with a platelet count and a complete blood count, with special emphasis on the peripheral blood smear. If the platelet count discloses thrombocytopenia, depending on the clinical circumstances a bone marrow aspiration may be justified, using preferably both a clot section and a smear technique. The clot section affords a better estimate of cellularity. The smear permits better study of morphology. This is true for megakaryocytes as well as for other types of cells. Investigation of purpura without thrombocytopenia should include a bleeding time (for overall platelet function). In a few cases a tourniquet test might be considered to test for abnormal capillary fragility. If necessary, platelet function tests may be done. These tests are not indicated in already known thrombocytopenia, because their results would not add any useful information. The other tests for hemorrhagic disease must have previously ruled out abnormality in other areas. Occasional cases of nonthrombocytopenic purpura are caused by abnormal serum proteins, which may be demonstrated by serum protein electrophoresis (then confirmed by other tests, described in Chapter 22).

    Usually a bleeding tendency does not develop in thrombocytopenia until the platelet count is less than 100,000/mm3 (100 x 109/L) (direct method) and most often does not occur until the platelet count is less than 50,000/mm3. The 20,000/mm3 value is usually considered the critical level. However, some patients do not bleed even with platelet counts near zero, whereas occasionally there may be trouble with patients with counts more than 50,000/mm3. Most likely there is some element of capillary fragility involved, but the actual reason is not known at this time.

  • Anticoagulant Systems

    Coumarin anticoagulants

    Liver cells produce certain coagulation factors that require vitamin K for synthesis into a form that can be activated. Drugs of the coumarin family inhibit vitamin K utilization by liver cells. The vitamin K–dependent factors, listed in order of decreasing sensitivity to coumarin, are factor VII, factor IX, factor X, and prothrombin. Factor VII has the shortest half-life (4-6 hours)and is decreased first and most severely, with activity levels less than 20% of baseline by 24 hours after coumarin intake. The other factor activitiesdecrease somewhat more slowly and less profoundly, reaching their lowest level in 4-5 days. Therefore, some anticoagulation effect is seen in 1.5-2.0 days, with maximum effect in 4-5 days. The early effect can be achieved a few hours earlier, and the degree of effect made greater, by use of a larger (“loading”) initial dose. However, use of a loading dose in some patients may result in complications due to excess anticoagulation. As noted while discussing the PT test earlier in this chapter, vitamin K metabolism may be affected by intake (low intake potentiates the effect of coumarins and unusually high intake antagonizes that effect), absorption (availability of bile salts and necessity for an intact duodenal mucosal cell absorption mechanism), and utilization by hepatic cells (number and health of liver cells).

    There are several members of the coumarin family derived from the original drug dicumarol; the most commonly used in the United States is warfarin sodium (Coumadin). Warfarin is water soluble, and oral doses are nearly completely absorbed by the small intestine. Maximal plasma concentration is reached 1-9 hours after ingestion. It is 95%-97% bound to albumin after absorption. Because of the very high percentage bound to albumin, substantial decrease in the albumin level increases free warfarin and increases the PT. The warfarin dose may have to be decreased to compensate for this. Warfarin is metabolized by the hepatic cell microsome system. The half-life of warfarin varies greatly among individuals (mean value 35-45 hours; range, 15-60 hours). With an initial dosage of 10 mg once each day, it takes 5-10 days (five half-lives) to reach equilibrium. Coumarin drugs usually take about 36-48 hours (usual range, 24-72 hours) to develop a significant effect on the PT, and between 3 and 5 days to develop maximal anticoagulant effect. Duration of action is 4-5 days. Warfarin effect can be terminated by parenteral vitamin K. The dose needed has some relationship to the degree of PT elevation. Normalization of the PT ordinarily occurs in 6-24 hours.

    Monitoring coumarin anticoagulants. Anticoagulation by coumarin-type drugs is best monitored by the PT, although the PT is not affected by factor IX, one of the four vitamin K–dependent coagulation factors. Until recently, the most commonly accepted PT therapeutic range was 2.0-2.5 times a control value derived either from the midpoint of the reference range or from normal fresh pooled plasma. A recent National Conference on Antithrombotic Therapy recommended that PT therapeutic ranges of 1.3-1.5 times control value be used rather than 2.0-2.5 times control if the PT reagent is derived from rabbit brain or is a rabbit brain-lung combination (these arethe most common PT reagents in the United States). With the usual reference range of 11-13 seconds, this would be roughly equivalent to an anticoagulant rangeof 15-20 seconds. An anticoagulant range of 1.5-2.0 times control was recommended for prosthetic valve prophylaxis against thrombosis. When one considers the large number of patients anticoagulated with coumarin drugs, it is surprising how little information is available from human studies regarding clinical evaluation of different therapeutic levels. Reporting PT results in terms of seconds compared with control is preferred to results in terms of percentage of normal, because percentage must be based on dilution curves, which are frequently inaccurate. Various prestigious organizations and researchers have recommended that PT results be reported as the international normalized ratio (INR). This is thepatient PT divided by the midpoint of the laboratory PT reference range; the result is then adjusted by being multiplied by an exponent derived by comparison of the thromboplastin reagent being used with an international standard. A calculator is needed for this mathematical task. This standardizes all thromboplastin reagents and theoretically permits a patient to have a PT performed with a comparable result in any laboratory using the INR system. However, there are problems regarding use of t he INR. When the PT is performed on an automated instrument, different instruments alter the response of the thromboplastin to some degree, potentially negating some of the uniformity that the INR was designed to created. Most importantly, the INR was designed for—and should only be used for—one condition: long-term warfarin therapy after the patient has been stabilized. Stabilization usually takes at least 1 week, sometimes longer. The INR therefore may produce inaccurate and potentially misleading results when patients are beginning warfarin therapy, ending warfarin therapy, being given heparin in addition to warfarin, or when there is interference by various technical conditions that affect the patient sample, the equipment used, or the patient’s coagulation factors (such as effect of medications or severe liver disease). The INR is not applicable when the PT is being used as a coagulation test rather than a monitor for stabilized warfarin therapy. These other applications are best interpreted by comparing test results to the individual laboratory’s PT reference range (in seconds). In addition, it should be remembered that most laboratories, even if they supply INR values, do not know if individual patients are or are not stabilized on warfarin and in many (or most) cases do not know for what reason the PT is being ordered.

    As of 1994, the usual recommendation for warfarin therapy PT levels on high-risk patients with mechanical heart valves is a target INR range of 2.5-3.5; for all other indications, the target range is 2.0-3.0.

    Warfarin presents increased risk in first trimester pregnancy due to increased congenital malformations and undesirable effects on early fetal bone development and in the last trimester due to increased possibility of fetal or maternal hemorrhage at or near birth. Various medications affecting warfarin are listed.

    Heparin/antithrombin III system

    Antithrombin III. Antithrombin III (AT-III) is a serine protease that requires heparin as an activator to produce effective anticoagulation. It is now believed that AT-III provides most of the anticoagulant effect, whereas heparin serves primarily as the catalyst for AT-III activation. It is thought that AT-III is synthesized by the liver. Antithrombin III concentrations are decreased in the first 6 months of life and in hereditary AT-III deficiency. Concentrations may be secondarily decreased in severe liver disease, extensive malignancy, the nephrotic syndrome, deep venous thrombosis, malnutrition, septicemia, after major surgical operations, and in DIC. Antithrombin III is also decreased 5%-30% by pregnancy or use of estrogen-containing oral contraceptives. Although there is not a good linear correlation between AT-III levels and demonstrated risk of venous thrombosis, in general a considerably decreased AT-III level tends to predispose toward venous thrombosis. This is most evident in hereditary AT-III deficiency, which is uncommon, is transmitted as an autosomal dominant, and is associated with a very high risk of venous thrombosis. A decreased AT-III level also decreases the apparent effect of standard doses of heparin as measured by coagulation tests and thus produces the appearance of “heparin resistance.” In general, patients respond to heparin when AT-III activity levels are more than 60% of normal and frequently do not respond adequately when AT-III activity levels are less than 40% of normal. Warfarin increases AT-III levels somewhat.

    Antithrombin III assay. AT-III can be assayed by several techniques. In general, these can be divided into two groups: those that estimate AT-III activity (“functional” assay) using coagulation factor or synthetic AT-III substrates; and those that measure the quantity of AT-III molecules present (“immunologic” assay) by means of antibody against AT-III. The results of the two assays do notalways correlate in clinical situations, since AT-III normally has little inhibitory effect until it is activated (as with heparin) and since the conditions of AT-III activation and AT-III binding to affected coagulation factors may vary. AT-III assay may not be reliable when thrombosis is present since AT-III may be temporarily depleted. It also may not be reliable when the patient is receiving heparin or warfarin (warfarin tends to increase AT-III levels).

    Heparin. Heparin is an anticoagulant that currently is a biologic substance extractedfrom either porcine gastric mucosa or bovine lung tissue. As a biological extract, heparin contains molecules with molecular weight ranging from 5,000 to 30,000. As noted previously, heparin contains a carbohydrate group that binds to antithrombin III and considerably increases its anticoagulation effect. Although antithrombin III is the actual inhibitor, heparin was discovered before antithrombin III and the actions of this heparin-antithrombin III complex are conventionally worded as though the actions are due to heparin. When standard heparin (extract) activates antithrombin III, the major effect is that of an antithrombin, although it also inhibits most of the other coagulation factors to some degree, with greatest effect on activated factors IX and X.

    Heparin is not consumed during its coagulation activity. It is degraded primarily in reticuloendothelial (RE) cells and blood vessel endothelial cells. About 80% of plasma heparin is metabolized by the RE cell system and 20% is excreted by the kidneys into the urine. Heparin’s half-life averages 90 minutes (range, 30-360 minutes). The half-life is increased with increase indose and shortened in deep vein thrombosis or pulmonary embolization (possibly due to increased platelet factor 4). Alpha-2 acid glycoprotein (an acute reaction protein) and low density lipoprotein (LDL) can bind heparin and decrease sensitivity to heparin’s action. Protamine and polybrene are specific neutralizing agents for heparin (1 mg of protamine for each 100 units of heparin). Platelets contain a heparineutralizing factor, PF-4. Severe thrombocytopenia may increase sensitivity to heparin.

    Heparin is mainly distributed in plasma. Therefore, high hematocrit levels (red cell mass) would tend to increase heparin concentration (in the diminished plasma volume). The anticoagulant effect of heparin is influenced by the antithrombin III level and to a lesser and variable degree by large increases or decreases in platelets (due to PF-4), presence of fibrin (which can bind to thrombin and prevent antithrombin III binding), and by dosage and route of administration. Patient response to heparin is variable, with differences sometimes as much as 300% between individuals within a group of patients. Obese patientsoften have decreased rate of heparin clearance, which may produce elevated plasma heparin levels. Heparin’s major side effect is bleeding; this is influenced by dose, route of administration, presence of renal failure, presence of severe illness, heavy and chronic alcohol intake, use of aspirin, and severe thrombocytopenia. Heparin can itself induce thrombocytopenia in about 10%-15% of patients (range, 1%-31%). Thrombocytopenia generally occurs 5-7 days (range, 2-15 days) after onset of therapy, is usually (but not always) not severe, and is usually reversible a few days after end of therapy. About 2% of patients develop sustained platelet decrease to less than 100,000/mm3 (almost always in patients receiving IV heparin). In about 0.5% of patients there is occurrence of the “white clot” syndrome. In these patients the platelet count falls below 100,000/mm3 6-14 days after heparin is begun, with onset of arterial thrombosis having a partial white color due to masses of platelets. This is due to antibody against platelet membranes. In this type of thrombosis, heparin must be stopped instead of increased.

    More recently, the heparin extract has been fractionated with recovery of molecules having anticoagulant effect and a molecular weight of approximately 5,000. These “low molecular weight” heparins induce antithrombin III action predominantly on activated factor X rather than thrombin. Low molecular weight heparin (LMWH) appears to have about 50% greater effectiveness than standard heparin for prophylaxis against clotting, has a more predictable response to standard doses, and can be given once a day with only initial laboratory monitoring in most cases rather than continued monitoring. In one LMWH study, 10%-15% of patients were hyperresponders (relative to most patients) and 10%-15% were hyporesponders.

    Monitoring of heparin therapy. Anticoagulation by heparin is monitored by the APTT in the majority of U.S. hospitals, although the activated clotting time or other methods can be used satisfactorily. The Lee-White whole blood clotting time was originally used but proved too cumbersome, insensitive, and nonreproducible. The heparin therapeutic range is usually stated as 1.5-2.5 times the APTT upper reference limit, but it is better to perform a pretherapy APTT test and use that value as the reference point for determining the therapeutic range. Heparin is poorly absorbed from the GI tract, so it is usually administered either by continuous IV infusion or by intermittent IV or subcutaneous injection. With constant IV infusion, heparin anticoagulant response is theoretically uniform, and the APTT test theoretically could be performed at any time. However, several studies have reported a heparin circadian rhythm, even with constant IV infusion; the peak effect occurring between 7 P.M. and 7 A.M. with maximum about 4 A.M., and the trough effect occurring between 7 A.M. and 7 P.M. with the maximum effect about 8 A.M. However, other studies have not found a circadian rhythm. When intermittent injection is used, peak heparin response is obtained 30 minutes to 1 hour after IV injection or 3-4 hours after subcutaneous injection. In the average person, using a single small or moderate-sized heparin dose, anticoagulant effect decreases below the threshold of measurement by 3-4 hours after IV injection and by 6 hours after subcutaneous injection. Most believe that the optimum time to monitor intermittent IV therapy is one-half hour before the next dose; and for subcutaneous injection, halfway between doses (i.e., at 6 hours after injection using 12-hour doses). At this time the APTT should be at least 1.5 times the upper normal limit or pretherapy value. There is controversy as to whether the peak of the response should also be measured. If low-dose therapy is used, many clinicians do not monitor blood levels at all. However, in such cases it may be desirable to perform an AT-III assay since there would be no way to know if an unsuspected low AT-III level were present that would prevent any heparin effect.

    So-called low dose heparin has become popular for prophylaxis against thrombosis. This is usually 5,000 units (although the literature range is 3,000-8,000 units) given subcutaneously. In one study, 5,000 units given subcutaneously elevated APTT values above preheparin levels in 80%-85% of patients; 10%-15% of all patients had increase over twice baseline. The frequency and degree of these elevations would depend to some extent on the sensitivity to heparin of the particular thromboplastin used.

    Different commercial APTT reagents differ in their sensitivity and linearity of response to heparin effect. Some APTT reagents have shown rather poor sensitivity, and most are not very linear in response once the upper limit of anticoagulant range is exceeded. Variation in reagent sensitivity is a problem because APTT therapeutic limits obtained with one manufacturer’s reagent might not be applicable to another manufacturer’s reagent and could lead to overdosage or underdosage of heparin. Results of various comparative studies have not unanimously shown any single manufacturer’s product to have clear superiority. Results of comparative studies have also been difficult to interpret because of changes in different manufacturer’s reagents over the years. Various other plasma or whole blood clotting tests are claimed to provide better results in monitoring heparin therapy than the APTT, of which the most frequently used are the thrombin time (TT) or the activated whole blood clotting time (ACT). When large heparin doses are used, as in bypass surgery, the APTT and TT are very nonlinear, prolonged, and poorly sensitive to heparin level changes, while the ACT has better performance characteristics. However, all these tests are influenced to varying degree by many factors that are involved with clotting, not the specific action of heparin. This is shown by the elevation of the APTT by warfarin.

    WHOLE BLOOD RECALCIFICATION TIME. Whole blood is drawn into a tube with citrate anticoagulant. Calcium chloride is added to an aliquot of the whole blood to begin the clotting process. The reference range upper limit for clotting is about 2 minutes at 37°C or 5 minutes at room temperature. Test results are said to be adequately reproducible. The whole blood recalcification time (WBRT) is used primarily to monitor heparin therapy. Its advantage is that whole blood clotting may be a more truthful representation of the varied effects of heparin than tests that circumvent part of the normal clotting process. Only one very cheap reagent is needed. The WBRT is sensitive to platelet variations and is more linear than the APTT at greater heparin effects. The major disadvantage is that the test cannot be automated. One report states that the test must be performed within 1 hour after venipuncture. Equipment is commercially available that enables the test to be performed at the patient’s bedside.

    Thrombin time. One aliquot of diluted thrombin is added to 1 aliquot of patient plasma. Clotting time upper limit is approximately 20 seconds. The test is simple and said to be very sensitive to heparin effect. It is not affected by sodium warfarin (Coumadin). As originally performed, the method was nonlinear and, like the APTT, was greatly prolonged in higher degrees of heparin effect. Certain modifications may have partially corrected this problem. Reagent source and composition may affect results considerably.

    There are, however, some specific assays of heparin activity. One of these is the activated factor X neutralization assay (anti-factor Xa assay) using a synthetic chromogenic substrate to show heparin neutralization of factor Xa action on the substrate. Another is titration with protamine sulfate or polybrene, both of which are specific inhibitors of heparin. The plasma heparin assays are necessary to monitor LMWH therapy, because LMWH has little effect on thrombin (whereas inactivation of thrombin is important in the APTT).

    Protein C/protein S anticoagulant system

    Protein C. Protein C (sometimes called Factor XIV) is a proteolytic enzyme of the serine protease type that produces anticoagulant action by inactivating previously activated factor V and factor VIII. The plasma half-life of protein C is 6-8 hours. Protein C is produced in the liver, is vitamin K–dependent, and circulates as an inactive preenzyme (zymogen). Activation is dependent on a protein called thrombomodulin, which is located on the cell membrane of blood vessel endothelial cells. Thrombomodulin binds thrombin and induces thrombin to attack inactive protein C (rather than the usual thrombin target fibrinogen), converting inactive protein C to active protein C. Activated protein C then attacks both activated factor VIII from the intrinsic factor pathway and activated factor V from the extrinsic factor pathway. Both actions inhibit conversion of prothrombin to thrombin (this end result is similar to the end result of warfarin action, although the mechanism is different). To reach maximum effect, protein C needs a cofactor, called protein S. Protein S is also synthesized in the liver and is also vitamin K dependent. Since protein C is a naturally occurring anticoagulant and protein S is necessary for protein C action, sufficient decrease in either protein C or S may result in a tendency for increased or recurrent vascular thrombosis. Protein C deficiency can be congenital or acquired. The congenital form is transmitted as an autosomal dominant trait. Individuals genetically heterozygous for protein C deficiency have protein C levels about 50% of normal (range, 25%-70%). Some, but not all, of these patients develop recurrent thrombophlebitis or pulmonary embolism. This usually does not occur before adolescence and is often associated with a high-risk condition such as surgery, trauma, or pregnancy. Homozygous protein C deficiency has been reported mostly in newborns, in which case protein C levels are close to zero and symptoms occur soon after birth and are much more severe.

    Acquired protein C deficiency can be associated with parenchymal liver disease, vitamin K deficiency, DIC (especially when DIC occurs with neoplasia), and with certain medications, particularly coumarin anticoagulants. Warfarin reduces protein C activity levels about 40% (and also protein S) in addition to its action on the other vitamin K–dependent coagulation factors. Since protein C has a short plasma half-life, if protein C is already decreased before warfarin is administered, protein C decrease sufficient to induce thrombosis may occur before maximal decrease of factor VII, resulting in thrombosis before the anticoagulant effect of warfarin is manifest.

    Protein C has an additional anticoagulation effect by increasing production of an enzyme called tissue plasminogen activator (t-PA) that initiates activity of the plasmin fibrinolytic system (discussed later).

    It has been estimated that venous thrombosis without disease or drug-induced predisposition in persons less than age 45 can be attributed to protein C, protein S, or antithrombin III deficiency in about 5% of cases for each. About 20%-65% of thrombosis in this type of patient has been attributed to “activated protein C resistance” (APC resistance) that has a hereditary component. It is thought that factor V is responsible due to a genetic alteration.

    Assay for protein C. Protein C can be assayed by quantitative immunologic or immunoelectrophoretic methods or by somewhat complex functional methods. Immunologic methods quantitate total amount of protein C. However, in some cases protein C is normal in quantity but does not function normally. Therefore, some investigators recommend functional assay rather than immunologic assay for screening purposes. If it is desirable to assay protein C (and protein S) quantity or function, it should be assayed before coumarin therapy is started because coumarin drugs interfere with vitamin K and decrease both protein C and S quantity and activity values.

    Protein S. As noted previously, protein S is a vitamin K–dependent cofactor for protein C. Protein S exists about 40% in active form and about 60% bound to a protein belonging to the complement group. Total protein S is measured using quantitative immunologic or immunoelectrophoretic methods similar to those used for protein C.

    Fibrinolysins

    Plasmin/fibrinolysin anticoagulants. Fibrinolysins are naturally occurring or acquired enzymes that attack and destroy either fibrinogen or fibrin or both. Fibrinolysins may be either primary or secondary. Primary fibrinolysins are acquired (not normally present), are rare, attack fibrinogen as their primary target, and are most often seen in association with malignancy or after extensive and lengthy surgery (most often lung operations). Secondary fibrinolysins are a part of normal body response to intravascular clotting, which consists of an attempt to dissolve the clot. The fibrinolysin produced is called plasmin, which is generated from an inactive precursor called plasminogen. Certain enzymes, such as streptokinase or urokinase, catalyze the reaction. Plasminogen also can be activated by tissue plasminogen activator (t-PA) which is regulated by another enzyme called t-PA inhibitor. Protein C exerts its action on this system by inhibiting t-PA inhibitor, which, in turn, releases t-PA to activate more plasminogen to become plasmin. Plasmin attacks the fibrin clot and splits the fibrin into various fragments (“split products,” or “fibrin degradation products” [FDPs]). The FDP can interfere with polymerization of fibrin monomer (which ordinarily becomes fibrin polymer) and thus complement the fibrin-dissolving action of plasmin by helping to retard further clotting. Secondary fibrinolysin usually does not attack fibrinogen, although it may do so if the concentration of fibrinolysin (plasmin) is high enough. Primary fibrinolysin seems to be very similar, if not identical, to plasmin. Primary fibrinolysins circulate without clotting being present, so that primary fibrinolysins ordinarily attack fibrinogen rather than fibrin.

    In addition to plasminogen activator inhibitor, there is an enzyme that directly inhibits plasmin called alpha-2 antiplasmin. This substance prevents plasmin from prematurely dissolving vessel thrombi that are preventing injured vessels from bleeding. Alpha-2 antiplasmin forms a covalent complex with plasmin that inactivates plasmin and thereby prevents fibrin clot dissolution and formation of FDP.

    Tests for fibrinolysins

    Various tests available to screen for fibrinolysins include the clot lysis test, fibrinogen assay, thrombin time, PT, APTT, and tests for FDPs. The clot lysis test is the oldest. Patient plasma is added to the blood clot of a normal person, and the clot is observed for 6-24 hours for signs of clot breakdown. This procedure is slow and fails to detect weak fibrinolysins. Thrombin time, PT, and PTT depend on formation of fibrin from fibrinogen as the test end point and thus will display abnormal results if there is sufficient decrease in fibrinogen or sufficient interference with the transformation of fibrinogen to fibrin. Fibrinogen assay was discussed previously; fibrinogen can be measured either directly, by immunologic methods, or indirectly, by measuring fibrin after inducing transformation of fibrinogen to fibrin. These methods are all moderately sensitive to fibrinolysins but still fail to detect some low-grade circulating anticoagulants. The most sensitive tests currently available are those that detect FDP; these will be discussed in detail next.

    Fibrinogen/fibrin split products (FDPs). Normally, thrombin catalyzes conversion of fibrinogen to fibrin by splitting two fibrinopeptide molecules, known as fibrinopeptide A and B, from the central portion of fibrinogen. This action exposes polymerization sites on the remaining portion of the fibrinogen molecule, which is now called fibrin monomer. Fibrin monomers spontaneously aggregate together in side-to-side and end-to-end configurations to form a fibrin gel. Thrombin also activates factor XIII, which helps introduce cross-linking isopeptide bonds between the fibrin monomers to form stabilized insoluble fibrin polymer. Fibrin polymer forms the scaffolding for blood clots. Fibrinolysins may attack either fibrinogen or fibrin, splitting off FDPs, which, in turn, are broken into smaller pieces. These split products may form a complex with fibrin monomers and interfere with polymerization. Fibrinogen degradation products can be produced by action of primary fibrinolysin on fibrinogen and also by action of plasmin on fibrinogen and fibrin monomers or fibrin clots formed in a variety of conditions, normal and abnormal. Plasmin attacks intravascular blood clots formed as part of normal hemostasis (e.g., trauma or surgery) as well as blood clots that produce disease (e.g., thrombosis or embolization). In addition, FDPs can be associated with intravascular clots composed of fibrin (e.g., DIC) and some partially extravascular conditions such as extensive malignancy, tissue necrosis and infarcts, infection, and inflammation.

    Protamine sulfate test. Protamine sulfate in low concentration is thought to release fibrin monomers from the split-product complex and allow the monomers to polymerize. The test result is positive in conditions producing secondary fibrinolysin and is negative with primary fibrinolysin. Primary fibrinolysin will not induce a protamine sulfate reaction because the end point of this test depends on the presence of fibrin monomers produced by the action of thrombin. Primary fibrinolysin is not ordinarily associated with activation of the thrombin clotting mechanism.

    Since DIC is the major condition associated with substantial production of secondary fibrinolysin, various studies endorse protamine sulfate as a good screening test for DIC. Negative results are strong evidence against DIC. Various modifications of the protamine sulfate method have been introduced. These vary in sensitivity, so that it is difficult to compare results in diseases other than DIC. Any condition leading to intravascular clotting may conceivably produce secondary fibrinolysin; these conditions include pulmonary embolization, venous thrombosis, infarcts, and either normal or abnormal postoperative blood vessel clots in the operative area. Occasional positive results have been reported without good explanation.

    Immunologic fibrin degradation product assay. When fibrinogen or fibrin monomers are attacked by plasmin, a large fragment called X is broken off. In turn, X is split into a larger fragment Y, a smaller fragment D, and the smallest fragment of all, E. Therefore, intermediate products are fragments X and Y, and final products are two fragment Ds and one fragment E. The split products retain some antigenic determinants of the parent fibrinogen molecule. Antibody to certain of these fragments can be produced and, as part of an immunologic test, can detect and quantitate these fragments. Since the antibodies will react with fibrinogen, fibrinogen must be removed before the test, usually by clotting the blood with thrombin. Heparin may interfere with the action of thrombin, which, in turn, leads to residual fibrinogen, producing false apparent FDP elevation. The earliest immunologic FDP test in general use was the “Fi test,” a slide latex agglutination procedure detecting mainly intermediate fragments X and Y, which was shown to be insufficiently sensitive. Another test, the tanned RBC hemagglutination-inhibition test (TRCHII), detected fragments X, Y, and D and was sensitive enough but too complicated for most laboratories. A newer 2-minute latex agglutination slide procedure (Thrombo-Wellcotest) has immunologic reactivity against fragments D and E and seems to have adequate sensitivity and reliability. The Thrombo-Wellcotest is replacing protamine sulfate in many laboratories. All of the FDP tests are usually abnormal in the various conditions that affect the protamine sulfate test. Titers of 1:10 or less on the Thrombo-Wellcotest are considered normal. Occasional clinically normal persons have titers between 1:10 and 1:40. Patients with venous thrombosis develop a secondary fibrinolysin body response that can produce an abnormal Thrombo-Wellcotest result. In most of these cases the titer is greater than 1:10 but less than 1:40, but a minority of patients may have a titer of 1:40 or greater. Classic DIC induces a strong secondary fibrinolysin response producing large quantities of split products with a Thrombo-Wellcotest titer over 1:40. However, mild cases of DIC may have a titer in the equivocal zone between 1:10 and 1:40. Thus, there is overlap between normal and abnormal between 1:10 and 1:40. In addition, it is necessary to add a plasmin inhibitor (e.g., aprotinin [Trasylol]) to the patient sample soon after collection to prevent continued breakdown of fibrinogen or fibrin, which could result in falsely elevated FDP levels.

    D-dimer test. Another type of FDP test detects breakdown products of plasmin action on fibrin clots. Since cross-linkage has already occurred, the degradation fragments also have some degree of residual cross-linkage and are called cross-linked FDP, or D-dimer (since fragment D is the major constituent). D-dimer assay is abnormal in nearly all of the same conditions as protamine sulfate or Thrombo-Wellcotest. However, D-dimer assay (like protamine sulfate) is normal with primary fibrinolysins, since D-dimers can be produced only after fibrin formation and cross-linking.

    Disseminated intravascular coagulation (DIC)

    The most common serious condition associated with fibrinolysin is DIC. Tissue thromboplastin or unknown substances with similar effect are liberated into the bloodstream and result in deposition of fibrin and fibrin clots in many small blood vessels, thus depleting plasma of the fibrin precursor substance fibrinogen (defibrination syndrome). Originally considered an obstetric disease associated with premature separation of the placenta and amniotic fluid embolism, DIC is now attributed to a growing list of etiologies, among which are septicemia, surgery complicated by intraoperative or postoperative shock, severe burns or trauma, extensive cancer, and newborn respiratory distress syndrome; and it is occasionally seen in many other conditions such as virus infection. Shock is the best common denominator but is not always present, nor has it been definitely proved to be either a cause or an effect.

    Clinically, DIC is manifested by coagulation problems, which may be severe. Blood oozing from tissues during surgery is a frequent warning sign. Shock, acute renal failure, and acute respiratory failure are common in severe cases. Vascular thrombi and focal tissue infarction are also relatively frequent. Purpura fulminans refers to the rare cases in which bleeding into the skin dominates the clinical picture. Hemoglobin levels may be within reference range initially or may be decreased. Peripheral smear in classic cases displays many schistocytes, producing the picture of microangiopathic hemolytic anemia. Platelets are usually, although not always, decreased. Differential diagnosis is discussed later in the section on thrombotic thrombocytopenic purpura.

    Laboratory diagnosis. Various laboratory tests have been advocated for the diagnosis of DIC, many of which have been discarded as newer tests are announced or older ones reevaluated. At present, hypofibrinogenemia and thrombocytopenia are the two best-established findings, which, occurring together, are considered strongly suggestive of DIC. The PT and APTT are usually both abnormal. Milder forms of DIC have been reported, however, in which one or all of these tests may be normal. The most sensitive and widely used tests for DIC screening are the protamine sulfate test and the immunologic FDP tests (e.g., the Thrombo-Wellcotest and D-dimer). The Thrombo-Wellcotest has been more reliable than protamine sulfate in my experience, but there are not sufficient comparison studies in the literature to permit definitive judgment between the two procedures. Since the two procedures give similar information, there is no need to use both for the diagnosis of DIC. D-dimer assay should give results similar to the Thrombo-Wellcotest.

    Primary vs. secondary fibrinolysin. Occasionally it is necessary to distinguish between primary and secondary etiologies for a fibrinolysin. The protamine sulfate test and D-dimer assay are normal with primary fibrinolysins and abnormal with secondary fibrinolysins. This occurs because the protamine sulfate test needs fibrin monomers (and the D-dimer assay needs cross-linked fibrin) to produce a positive reaction, whereas with primary fibrinolysins, since the clotting (thrombin) mechanism is not ordinarily activated, no fibrin monomers or cross-linked fibrin molecules are present and FDPs are generated from fibrinogen rather than fibrin. Primary fibrinolysins can be treated with e-aminocaproic acid, or EACA (Amicar), but EACA is contraindicated in DIC, since inhibition of secondary fibrinolysin generation would aggravate the clotting disorder.

    Immunologic-type coagulation factor inhibitors

    There are several antibody-type inhibitors of coagulation factors. The most common are antiphospholipid antibodies (APA) and the antibodies that may develop against factor VIII:C. Nomenclature of the APAs is confusing because they were originally discovered in patients with systemic lupus erythematosis (SLE) and therefore were called “lupus anticoagulants.” Later, it was found that APAs did not act as anticoagulants, were found more often without than with SLE, and often did not react in the screening procedure traditionally used to detect the “lupus anticoagulant” antibodies. There are also some other differences between these antibodies that suggest two subdivisions of APAs, currently being named “lupus anticoagulants” (LAC) and “anticardiolipin antibodies” (ACA) and differentiated on the basis of certain laboratory tests, discussed later. In about 40% of patients with APAs, only one or the other type is present; in the other 60% of cases, both are present concurrently. APAs may be IgG, IgM, or IgA type. IgG is the type most often associated with complications; of these, most are thrombosis, either venous (70%) or arterial (30%). Thrombocytopenia also occurs and possibly hemolytic anemia. Increased spontaneous abortion or fetal loss has been reported in some studies but not in others. The antibodies vary considerably in titer, activity, and frequency. A considerable number are transient, and whether they are transient or longer-lasting seem to be a response to infection, acquired immunodeficiency syndrome (AIDS), inflammation, autoimmune diseases, malignancy, certain medications (especially chlorpromazine and procainamide; also dilantin, penicillin, and various antibiotics; hydralazine, and quinidine), and various or unknown antigens. The category known as LAC are IgG or IgM antibodies originally found in SLE (about 10%-15% of cases; range, 0.4%-65%), but have subsequently been reported in some patients with various conditions previously mentioned. In some cases, no cause is found. Clinical bleeding is uncommon unless some other coagulation abnormality is present (e.g., thrombocytopenia, present in about 25% of cases [range, 14%-63%]; and in about 50%-60% of cases when LAC occurs in SLE). Instead, vascular thrombosis is associated with LACs in about 30%-40% of cases (range, 14%-60%).

    Laboratory Diagnosis.—The trademark of LACs is interference with coagulation factor assays that use phospholipid reagents or phospholipid-dependent procedures such as the APTT as part of the test method, even though the antiphospholipid LAC does not directly affect the actual coagulation factor that the test is supposed to be measuring. The most commonly used screening test is the APTT, since it is fast, inexpensive, and easily automated. The kaolin clotting time and the dilute Russell viper venom time have also been used; they are claimed to be a little more sensitive than the APTT but are more expensive and not automated. The APTT is elevated in about 90% of patients with LACs. In fact, unexpected APTT elevation is the usual way the LAC is detected (of course, other etiologies of elevated APTT must be considered, especially heparin contamination, which several reports found to be the most common cause for unexpected APTT elevation). Factor assays that use the APTT as part of the assay method are also abnormal. Different APTT reagents have varying phospholipid composition and quantity and therefore do not all have the same sensitivity for the LAC. This partially accounts for reports of APTT detection of the LAC ranging from 45% to 90%. A technical factor of importance is necessity for platelet-poor plasma when testing for LAC. Sensitivity for LAC improves as the number of platelets in the plasma specimen decreases. When LACs are present the PT is typically normal, although the PT can be elevated in about 20% (range 10%-27%) of the patients. The thrombin time is normal. The cardiolipin tests for syphilis (rapid plasma reagin, VDRL) are positive in about 20%-40% (range 0%-87%) of cases. The easiest diagnostic test for the lupus inhibitor consists of diluting the patient’s plasma with an equal portion of fresh normal plasma. The mixture should correct the APTT back to reference range (or nearly so) when the problem is a factor deficiency or antifactor antibody, but the APTT should not decrease substantially in the presence of a lupus inhibitor. Unfortunately, there are varying criteria in the literature as to what constitutes substantial correction. Also, there may be interpretive difficulty when the APTT elevation is less than 8-10 seconds. In this situation, some investigators prefer 1 part fresh normal plasma to 4 parts patient plasma, which is considered to be more sensitive than a 1:1 mixture. To further complicate matters, about 15% of LAC cases (range, 10%-40%) are reported to demonstrate substantial or complete correction. In many of these cases there may be time-dependent inhibition; that is, the APTT corrects but slowly becomes abnormal again 1-2 hours later during incubation at 37°C (this behavior is more typical of a factor VIII inhibitor). Equivocal or time-dependent results should probably be confirmed by a more specific test. Whether clear-cut (noncorrected) results need to be confirmed is not clear. Tests reported to be more sensitive and reliable for definitive diagnosis, although not widely available, include the tissue thromboplastin inhibition test and the platelet neutralization procedure. The platelet neutralization procedure appears to be more reliable and is commercially available in kit form. A phospholipid neutralization test has been reported that is very promising.

    Tests for the ACA category include a solid-phase radioimmunoassay (the first reliable procedure developed) and nonradioactive immunoassays like enzyme-linked immunosorbent assay (ELISA). These generally would only be available in large commercial laboratories or university medical centers. Most are now using the ELISA method. These tests are necessary to detect non-LAC antiphospholipid antibodies (i.e., ACAs) and demonstrate that mixtures of LAC (shown by positive LAC tests discussed previously) and non-LAC ACAs are present.

    Factor VIII antibody. Antibodies against factor VIII develop in approximately 15% of patients with hemophilia A (literature range, 5%-25%). Similar antibodies occasionally have been reported in nonhemophiliacs, where the presence and severity of clinical disease resembling hemophilia depends on the strength or titer of its antibody. This subject is discussed in the section on hemophilia A. The most commonly used screening test is the same as that for anticardiolipin antibodies.

    Miscellaneous anticoagulants

    Medications. Various drugs may affect coagulation. Aspirin and certain others have direct effects on clotting. Aspirin inhibits platelet adhesion and thus may potentiate a tendency to hemorrhage when a patient is receiving anticoagulant therapy. Other medications may act indirectly by enhancing or inhibiting the effects of anticoagulants.

    Dysproteinemia. Some persons with abnormal serum proteins, most frequently persons with myeloma or one of the macroglobulinemias, have interference with conversion of fibrinogen to fibrin despite normal fibrinogen levels. This is manifested by poor clot stability and failure of clot retraction and may cause a hemorrhagic tendency or purpura.

    Thrombolytic therapy. Various conditions predispose to thrombosis (see box on this page). Antithrombin III, protein C and S, and t-PA were discussed earlier. Many of the other conditions are discussed elsewhere. Stasis, vessel narrowing, and hyperviscosity are the most common etiologic problems. Cancer is a surprisingly important cause of thrombosis and embolization, especially in persons without evidence of the usual predisposing conditions. According to several studies about 5%-15% (range, 1%-34%) of cases of pulmonary embolization or deep vein thrombosis are associated with cancer, the cancer usually becoming manifest in less than 2 years. GI tract adenocarcinoma (especially pancreas), lung, and ovary are the most common primary tumors.

    Various forms of therapy have been used to dissolve thrombi. Heparin has been used the longest, but heparin therapy has several problems (discussed earlier) and is better in preventing clot propagation than in dissolving the clot. Fibrinolysin (plasmin) can be used therapeutically to lyse intravascular thrombi and has produced better results than heparin. The most common indications are coronary artery thrombi, deep vein thrombosis (femoral, iliac, or popliteal veins), and large pulmonary emboli. A plasminogen activator can be administered to generate endogenous plasmin. To date, these activators include streptokinase (SK), urokinase (UK), tissue plasminogen activator (t-PA), anisoylated plasminogen-streptokinase activator complex (AP-SAC), and pro-urokinase (P-UK). SK is a protein that is produced by beta-hemolytic streptococci. It forms a complex with plasminogen; this complex then activates noncomplexed plasminogen (either circulating or fibrin bound) to form plasmin. However, this process has the potential of overactivating the system, depleting plasminogen, and producing hemorrhage. Also, there is a tendency to form anti-SK antibodies. UK produces therapeutic results at least as good as those of SK and is less antigenic. It directly activates plasminogen. However, it is very expensive. t-PA activator acts on plasminogen by forming a complex with plasminogen that is bound to fibrin. Therefore, t-PA preferentially acts on plasminogen at the site of clotting rather than circulating plasminogen, which is a major advantage. Disadvantages of t-PA include a very short half-life (2-5 minutes), which necessitates substantial doses for best results; an extremely high cost per dose at present; and reports of some systemic fibrinolytic effect when administered in large doses. Better therapeutic results are being reported for t-PA (at least for coronary artery thrombus dissolution) than results for SK and UK. AP-SAC is a modified SK-t-PA complex that can circulate without activating plasminogen. However, when the complex meets fibrin, such as a blood clot, the complex binds to the fibrin and then activates plasminogen that is also bound to the fibrin. P-UK is the inactive precursor of urokinase and circulates in the blood. When clotting occurs, it attaches to fibrin and forms (activated) urokinase there. Action is specific for fibrin, and the half-life of P-UK is considerably longer than t-PA, so that a smaller dose is needed. Once the decision is made to use a thrombolytic agent, current practice is to obtain a baseline blood specimen for either the APTT or the thrombin time. A second specimen is obtained 3-4 hours following administration of the thrombolytic agent. The desired result is a value sufficiently elevated above baseline so that there is no question of a true increase. The reason for testing is to prove that a thrombolytic effect has been obtained; other than this, the degree of test abnormality is not helpful since it does not correlate well with the actual amount of fibrinolysis taking place.

    Some Conditions That Predispose to Thrombosis
    Vessel lumen narrowing (e.g., atherosclerosis)
    Vessel wall damage (e.g., vasculitis)
    Stasis (e.g., congestive heart failure, immobile extremity)
    Increased platelets (thrombocythemia)
    Heparin-induced thrombocytopenia
    Polycythemia
    Serum hyperviscosity (e.g., Waldenstrцm’s macroglobulinemia)
    Deficiency of AT-III, protein C, or protein S
    Pregnancy
    Oral (estrogen-containing) contraceptives
    Antiphospholipid antibodies
    Fibrin thrombi (e.g., DIC)
    Platelet thrombi (thrombotic thrombocytopenic purpura [TTP])
    Paroxysmal nocturnal hemoglobinuria
    Malignancy (e.g., pancreatic carcinoma)

  • Coagulation Tests in Newborns

    The APTT is elevated in the newborn compared to the adult reference range. At least in part this is due to reduction of 30%-50% in activity of factors XI, XII, HMWK, and Fleher Factor. Activity is even lower in premature infants. Adult values are reached in 3-6 months. Vitamin K–dependent factors are reduced to 20%-60% of adult reference range at birth. Antithrombin III and Protein C are also reduced. If the infant’s hematocrit level is very high (over 65%), the specimen plasma volume is reduced relative to the amount of anticoagulant in the tube and elevated APTT or PT may result.

  • Coagulation Pathway Factors

    In this section the various coagulation pathway factors are discussed individually, with emphasis on abnormalities for which laboratory tests are useful.

    Factor I (fibrinogen)

    Fibrinogen is a glycoprotein that is synthesized in the liver, the liver being a major source of many coagulation factors. Conversion of fibrinogen to fibrin under the influence of thrombin is the final major step in coagulation. Besides its role in coagulation, fibrinogen is a risk factor for coronary heart disease and stroke. Increased plasma fibrinogen most often is temporary and involves the important role it plays as part of the body’s “acute-reaction” response to trauma or to onset of a variety of severe illnesses. This initial response to illness results in fibrinogen production increase and therefore serum level increase. Fibrinogen levels also can be increased by cigarette smoking and by genetic influences. Decreased plasma fibrinogen levels may occurfrom decreased liver production, from the action of fibrinolysins (enzymes thatdestroy fibrin and may attack fibrinogen), and from conversion of fibrinogen tofibrin that is too extensive to permit adequate replacement of the fibrinogen. Decreased fibrinogen production is usually due to liver cell damage, as occurs in acute hepatitis or cirrhosis. However, production mechanisms are efficient, and a severe degree of liver damage is required before significant hypofibrinogenemia develops. Fibrinolysins may be primary or secondary. Primary fibrinolysins are rare and usually attack only fibrinogen. Secondary fibrinolysins are more common and attack primarily fibrin but can attack fibrinogen. Fibrinolysins are discussed in more detail in the section on anticoagulants. The major etiology of hypofibrinogenemia other than severe liver disease is fibrinogen depletion caused by DIC.

    Current therapeutic sources of fibrinogen are fresh frozen plasma or cryoprecipitate (Chapter 10).

    Factor II (prothrombin)

    Prothrombin is a proenzyme synthesized by liver cells. That portion of the prothrombin molecule that permits the molecule to function in the coagulation pathway is formed with the help of vitamin K. Without vitamin K a variety of prothrombin molecules are produced that are inactive. Vitamin K exists in two forms, one of which is preformed in green vegetables and certain other foods and oneof which is synthesized by GI tract bacteria. Both forms are fat soluble and depend on bile salts for absorption. A deficiency of vitamin K may thus result from malabsorption of fat (as seen with lack of bile salts in obstructive jaundice, or primary malabsorption from sprue; see Chapter 26) or from failure of GI tract bacteria to synthesize this substance (due to prolonged oral antibiotic therapy). It usually takes more than 3 weeks before the body vitamin K stores are exhausted and the deficiency of available vitamin K becomes manifest. Dietary lack may be important but ordinarily does not causea severe enough deficiency to produce clinical abnormality unless other factors(e.g., the anticoagulant vitamin K inhibitors) are present.

    Neonatal hemorrhage due to vitamin K deficiency may occur during the first 24 hours of life, from 1-7 days after birth, and uncommonly, 1-3 months later. Neonatal deficiency may be idiopathic or associated with certain maternal medications (anticonvulsants, warfarin, antibiotic therapy). It has been reported that up to 3% of infants at birth are vitamin K deficient. Thereafter, dietis important. Breast milk and certain infant milk formulas are relatively low in vitamin K and predispose to clinical deficiency. One dose of vitamin K at birth is frequently used as a prophylactic measure.

    Assuming that normal supplies of vitamin K are available, the other main limiting factor in prothrombin formation is the ability of the liver to synthesizeit. In severe liver disease (most often far-advanced cirrhosis), enough parenchyma is destroyed to decrease prothrombin formation in a measurable way, eventually leading to a clinical coagulation defect. Whereas a deficiency of availablevitamin K responds promptly to administration of parenteral vitamin K, hypoprothrombinemia due to liver parenchymal disease responds poorly, if at all, to parenteral vitamin K therapy.

    Vitamin K is also necessary for synthesis of factors VII, IX, and X. The greatest effect seems to be on prothrombin and factor VII.

    Factor III (tissue thromboplastin)

    Tissue thromboplastin is composed of phospholipids and lipoproteins and can be extracted from most tissues. Tissue thromboplastin forms a complex with factor VII and calcium ions that initiates the extrinsic coagulation pathway. This complex is used as the reagent for the PT test.

    Factor IV (calcium ions)

    Calcium is necessary as a cofactor in several steps of the coagulation pathway. The common hematology and blood bank anticoagulants oxalate, ethylenediamine tetraacetic acid (EDTA), and citrate exert their anticoagulant effect by blocking calcium function through chelation of the calcium ions.

    Factor V (labile factor)

    Factor V is synthesized by the liver and is decreased in severe liver disease. Factor V is found in plasma but not in serum. Factor V is unstable and becomes markedly reduced in 2-3 days when refrigerated anticoagulated blood is stored in the blood bank. Refrigeration helps preserve factor V in laboratory specimen plasma, but even at 4°C factor V is considerably reduced within 24 hours, and freezing soon after the specimen is obtained is necessary to maintain activity. In factor V deficiency the PT is abnormal; the APTT test is abnormal in severe deficiency but may be normal in mild deficiency.

    Factor VII (stable factor)

    Factor VII is synthesized in the liver and is dependent on vitamin K for itsactivity. The biologic half-life of factor VII is only 4-6 hours, making it theshortest lived of the vitamin K–dependent coagulation factors. The coumarin vitamin K antagonists produce a more rapid and profound depressive effect onfactor VII than on any of the other vitamin K–dependent factors (discussed at greater length in the section on Coumadin anticoagulants). Factor VII is present in plasma and in serum and is stable in both. In factor VII deficiency the PT is abnormal; the APTT and bleeding time are normal.

    Factor VIII/von Willebrand factor complex

    Nomenclature of Factor VIII/von Willebrand factor (factor VIII/vWF) complex has been confusing. Until recently, the entire complex was known as “Factor VIII.” In 1985 the International Committee on Thrombosis and Haemostasis proposed a new nomenclature system that is adopted here. Research onFactor VIII over many years demonstrated that the factor was not a single molecule but a complex with at least two components. One is the substance whose deficiency produces classic hemophilia. This component previously was called antihemophilic globulin (AHG) and is now called factor VIII (or antihemophilic factor). For coagulation test purposes, factor VIII (antihemophilic factor) is separated into (1) the factor protein molecule, and (2) its coagulant activity. Antibodies have been produced against the factor VIII protein, and this antigen is now called factor VIII antigen, or VIII:Ag (formerly this was designated VIII C:Ag). The coagulant activity of factor VIII is now designated VIII:C. The secondcomponent of the complex is associated with von Willebrand’s disease and is called von Willebrand factor, or vWf (formerly, this was called factor VIII-related antigen, or VIIIR). Antibodies have been produced against von Willebrandfactor protein, and this antigen is now called von Willebrand factor antigen, or vWf:Ag (formerly, this was called VIII R:Ag). In addition, the von Willebrandfactor has at least two coagulant actions or activities: an effect on platelet adhesion demonstrated by the glass bead column retention test and an effect on platelet aggregation demonstrated by the ristocetin test. The von Willebrand–ristocetin effect on platelets was formerly called the von Willebrand ristocetin cofactor and designated VIIIR:RC; but in the new nomenclature system no formal name or abbreviation is assigned to any vWf function such as the ristocetin cofactor or the vWf effect on platelet adhesion. The site where factor VIII is synthesized in humans is not known, but the von Willebrand factor is apparently synthesized in endothelial cells and megakaryocytes.

    The hemorrhagic disease known as hemophilia is usually associated with factor VIIIC deficiency. A similar disease may result from factor IX deficiency (anda similar, but milder, disease from factor XI (deficiency), so factor VIIIC deficiency is sometimes referred to as hemophilia A and factor IX deficiency as hemophilia B. About 90% of clinical hemophilia cases are due to hemophiliaA and about 9% to hemophilia B.

    The gene for factor VIII is located on the long arm of the female sex chromosome (Xq28) and is transmitted as a sex-linked recessive trait. Females with the hemophilia A gene are usually carriers (heterozygous, xX), and males with thegene have clinical disease (xY, the hemophilic x gene not being suppressed by anormal X gene as it is in a carrier female). However, about one third of cases are due to fetal gene mutation, not gene inheritance. The gene for vWf is usually transmitted as an autosomal incomplete dominant trait and uncommonly as an autosomal recessive trait. Rare cases of x-linked recessive inheritance have also been reported.

    The VIII/vWf complex is found in plasma but not in serum. Both VIII:C and vWf activity (or vWf:RC) are unstable. Refrigeration helps preserve factor VIII activity (VIII:C), but even at 4°C the coagulant activity decreases considerably within 24 hours in laboratory plasma specimens. Freezing maintains factor VIII:C activity if it is done within a short time after the specimen is obtained.

    Differentiation of hemophilia A and von Willebrand’s disease. In hemophilia A, male patients have decreased factor VIII coagulant activity, whereas vWf coagulant activity, as measured by platelet function tests, is normal. Factor VIII:Ag is decreased by antibody techniques, whereas vWf: Ag levels are normal. The majority of female hemophilia A carriers have also been shownto have reduced VIII:Ag levels, although usually not depressed to a marked degree (since one chromosome has a normal X gene and apparently can ensure some production of factor VIII). In contrast to hemophilia, patients with classic von Willebrand’s disease have reduced VIII:C activity and vWf activity, both reduced about equal in degree, and demonstrate corresponding decreases in both antigens using antibody techniques. Further discussion of von Willebrand’s disease will be deferred to the section on platelet disorders.

    CLINICAL ASPECTS OF HEMOPHILIA A. Hemophilia A exists in varying degrees of factor VIII:C activity deficiency that roughly correlate with clinical severity. Severe (classic) hemophiliacs have 1% or less of normal factor VIII:C activity levels on assay, moderate hemophiliacs have 2%-5%, and mild hemophiliacs have 5%-25% of normal levels. Hemophilia with levels between 25% and 50% of normal is sometimes called subhemophilia or borderline hemophilia. In one study, 55% (literature range, 40%-70%) of factor VIII:C–deficient male patients had classic (severe) hemophilia, about 25% had moderately severe disease, and about 20% had mild disease. Most carrier females have about 50% of normal factor VIII:C activity. About one third of carrier females have factor VIII:C activitylevels similar to those in the borderline decreased group, although usually thecarriers are asymptomatic. About 5% of female carriers have been reported to have mild clinical disease with factor VIII:C activity also in the mildly decreased range; (both X chromosomes affected) and rare homozygous females withclassic disease have been reported.

    The major symptom of hemophilia is excessive bleeding. Most bleeding episodes follow trauma. In classic cases the trauma may be very slight. In mild cases the trauma must usually be more significant, such as a surgical operation or dental extraction. Bleeding into joints (hemarthrosis) is a characteristic finding in classic and moderate factor VIII:C activity deficiency, with the more repeated and severe hemorrhagic episodes being found in the more severely factor-deficient patients. Bleeding from the mouth, urinary tract, and GI tract and intracranial hemorrhage are also relatively common in severe cases. In mild cases there may be only an equivocal history of excessive bleeding or no history at all before severe trauma or a surgical procedure brings the condition to light. Another complication of severe hemophilia is hepatitis virus infection from factor replacement therapy. Before laboratory screening of donor blood was possible, transmission of human immunodeficiency virus-I (HIV-I) infection through blood product transfusion affected many hemophiliacs.

    The biologic half-life of factor VIII:C activity is approximately 8-12 hours. Factor VIII:C activity is unstable, and in freshly drawn and anticoagulated blood stored in the refrigerator the activity decreases to 40% by 24 hours. The current therapeutic sources of factor VIII:C include fresh frozen plasma, factor VIII:C concentrate, and cryoprecipitate (Chapter 10). Factor VIII:C in therapeutic material is measured in terms of units that equal the amount of factor VIII:C activity in 1 ml of normal plasma. As a rule of thumb, one factor VIII unit in therapeutic material per kilogram of body weight should increase factor VIII activity levels by 2% of normal with a half-life of 12 hours.

    About 15% (range, 5%-25%) of patients with hemophilia Adevelop antibodies (acquired inhibitors) against transfused factor VIII, whether obtained from pooled or synthetic (recombinant) plasma. In addition, some persons without factor deficiency develop inhibitors against a coagulation factor (usually factor VIII) for unknown reasons. The most common associated conditions are rheumatoid-collagen diseases, medications, pregnancy or postpartum, malignancy, and Crohn’s disease. Whereas hemophilics typically hemorrhage into joints, factor VIII inhibitors in nonhemophiliacs tend to produce hemorrhage in nonjoint areas.

    In either hemophiliacs or nonhemophiliacs, factor VIII inhibitors elevate the APTT just as factor VIII does; and similar to factor VIII deficiency, a 1:1 mixture of patient plasma and fresh normal plasma should decrease the APTT into or nearly into APTT reference range. Incubation of the mixture at 37°C for 1 hour shows relatively little change (less than 8 seconds) in factor VIII deficiency but a significant increase (8 seconds or more) when an inhibitor is present. A 2-hour incubation is necessary with weak inhibitors. About 10%-15% of patients with antiphospholipid antibodies (“lupus anticoagulant,” discussed later in detail) have the same response as a factor VIII inhibitor. The strength of the inhibitor can be quantitated by testing its effect on a standardized preparation of factor VIII, with the result reported in Bethesda units. This test is available mostly in large reference laboratories, university medical centers, and hemophiliac care centers. Factor VIII inhibitors may interfere with those factor VIII assays and other factor assays that use theAPTT as the assay endpoint. Elevation of the APTT due to action of the inhibitor similates the result due to factor deficiency.

    Factor IX (prothrombin complex) concentrate has been used with some success in factor inhibitor patients on an empirical basis.

    Mild hemophilia A (factor VIII levels >5%) can also be treated with desmopressin (DDAVP), which was found to act as a stimulant to factor VIII and to vWf production. Intravenous injection of desmopressin produces peak action in about 30 minutes, resulting in average twofold increases in factor VIII that last about as long as the effect of cryoprecipitate. Since desmopressin is synthetic, the possible infectious complications of blood products are avoided. However, patients with severe hemophilia A cannot respond sufficiently.

    Laboratory tests in hemophilia A.— The PT test results are normal in all factor VIII:C activity–deficient groups. The bleeding time is usually normal; but it will be elevated in about 20% of patients, more often those with severe deficiency. The major screening test for hemophilia A is the APTT. The APTT becomes abnormal when factor VIII:C activity is <30%-35% of normal (range, <20%-<49%). The APTT detects 99%-100% of patients with severe and moderate factor VIII:C activity deficiency. In mild cases, reported detection rate varies from 48%-100% (depending on the particular reagents used and the population selected). Therefore, some of the milder (“subhemophilia”) cases produce borderline or normal results. Definitive diagnosis of hemophilia A usually requires assay of factor VIII:C activity. This used to require the thromboplastin generation test but now is ordinarily carried out with the APTT test and commercially obtained factor VIII-deficient plasma reagent. There is a substantial variation in results in average laboratories, and the reference range is usually considered to be 50%-150% of a normal pooled plasma. The APTT alone detects patients with factor VIII:C less than 30%-35% of normal. This will miss some mild and borderline hemophiliacs and most carriers. Factor VIII:C assay can detect severe, moderate, and mild male hemophiliacs; a considerable number of borderline male hemophiliacs; and about 35% of female carriers. Studies using a combination of factor VIII:C activity assay and vWf protein assay (obtaining a ratio of the two results) are more sensitive than standard factor VIII activity assays alone in detection of hemophilia A, with results of the combined studies (ratio) positive in approximately 50%-75% of female carriers (literature range, 48%-94%). Deoxyribonucleic acid (DNA) probes using multiple targets have been reported to detect 90%-95% of female carriers, making it the most sensitive screening or diagnostic method.

    Increased estrogen levels (pregnancy or estrogen-containing contraceptive medication) increase factor VIII:C activity and could mask a mild deficiency. Factor VIII protein is one of the so-called “acute reaction” proteins (Chapter 22) whose synthesis is increased in response to acute illness or stress. Surgery, acute bleeding, pregnancy, severe exercise, inflammation (infectious or noninfectious), and severe stress of other types may raise factor VIII:C activity levels somewhat (within 1-2 days after stress onset) and may temporarily mask a mild factor deficiency state if testing is delayed that long. Another problem is therapy given before test specimens areobtained. There are some technical details that affect tests for factor VIII:C activity. The specimen must be fresh or preserved by short-term refrigeration or by freezing. Most methods quantitate results by comparing the patient resultsagainst those of a reference plasma. Commercially available lyophilized reference plasma gives more reliable results than fresh “normal” plasma. Photooptical instruments in general give better results than mechanical clot timers.

    Factor IX (plasma thromboplastin component)

    Factor IX deficiency is inherited as an X-linked recessive trait. Deficiencyoccurs in variable degree, and clinical severity varies accordingly, similar tohemophilia due to factor VIII deficiency. Factor IX deficiency produces a hemophilia syndrome comparable to that of factor VII:C deficiency, but hemorrhage into joints is not as frequent as in factor VII:C deficiency, even in the severe form of factor IX disease. The factor IX deficiency syndrome is also called hemophilia B, or Christmas disease (named after the first patient studied in detail). Factor IX is found in plasma or serum and is stable in either.

    The APTT test is the standard screening method; however, some reagents only detect deficiency when <20% of normal. It is also used for diagnosis and quantitative activity assay with factor IX-deficient plasma reagent. As in factor VIII deficiency, the PT is normal. Quick differentiation of factor IX deficiency from factor VIII deficiency can be provided by addition of normal serum or normal fresh plasma to patient serum; the APTT abnormality is corrected byserum in factor IX deficiency, whereas fresh plasma but not serum is required to correct a factor VIII deficiency. Increased estrogens may falsely increase factor IX. Diagnosis can also be made by DNA probe methods, similar to hemophilia A.

    Current therapeutic sources of factor IX include ordinary blood bank plasma, fresh frozen plasma, and factor IX (prothrombin complex) concentrate. Cryoprecipitate is not useful because it does not contain any factor IX. As a rule of thumb, one unit of factor IX in therapeutic material per kilogram of body weightshould increase factor IX levels by 1% of normal with a half-life of 24 hours. Occasionally, patients develop antibodies against transfused factor IX.

    Factor X (Stuart factor)

    This factor is a part of both the extrinsic and the intrinsic coagulation pathways. Deficiency is rare and is inherited as an autosomal recessive trait. The clinical syndrome produced is usually mild compared with classic hemophilia A. Factor X is present in plasma and in serum and is stable in both. Both the APTT and the PT are abnormal; the bleeding time is normal.

    Factor XI (plasma thromboplastin antecedent)

    This factor deficiency is uncommon and is inherited as an autosomal incompletely recessive trait. Deficiency varies in degree according to whether the patient is homozygous or heterozygous. Clinical symptoms from either degree of deficiency are milder than those of comparable cases of hemophilia A or B. Factor XI is present in plasma and serum and is stable in both. The APTT is abnormal; the PT and bleeding time are normal.

    Factor XII (Hageman factor)

    Factor XII deficiency is very uncommon and is inherited as an autosomal recessive trait. Clinical manifestations (bleeding) are rare, and most of the interest in this disorder derives from the role of factor XII as a surface contact activator of the intrinsic coagulation pathway. The extrinsic pathway is not affected. Factor XII deficiency produces clotting deficiency in laboratory tests that is particularly marked when activation enhancement materials are included in coagulation test reagents (such as the APTT) or glass tubes are used (glass ordinarily would strongly activate factor XII). The APTT and whole blood clotting time are abnormal; the PT and the bleeding time are normal. Diagnosis can be confirmed by means of the APTT with commercial factor XII–deficient plasma reagent. It has been reported that patients with factor XII deficiency have an increased incidence of myocardial infarction and thrombosis.

    High molecular weight kininogen (HMWK; Fitzgerald factor)

    This factor is a part of the kallikrein system, which affects factor XII in the coagulation intrinsic pathway and also plays a role in the body inflammatory response. Deficiency of HMWK is rare, and no clinical bleeding is induced. The APTT and whole blood clotting time are abnormal; the PT and bleeding time are normal.

    Prekallikrein (Fleher factor)

    This proenzyme is the central part of the kallikrein system. Prekallikrein deficiency does not predispose to abnormal bleeding. The whole blood clotting time is abnormal; the PT and bleeding time are normal. The APTT is abnormal if particulate activators such as silica or kaolin are used but is normal if solubleactivators such as ellagic acid are used. The APTT abnormality with particle activators may be corrected by prolonged incubation (10-15 minutes vs. the usual 1-3 minutes).

    Factor XIII (fibrin stabilizing factor)

    Factor XIII deficiency is transmitted as an autosomal recessive trait. Bleeding frequently is first noted in newborns. In later life bleeding episodes are usually mild except when related to severe trauma or surgery. Secondary deficiency may occur in a variety of conditions, especially malignancy or severe liverdisease, but abnormality is usually subclinical. All of the usual coagulation tests (PT, APTT, and bleeding time) are normal, even in congenital deficiency. Diagnosis can be made because fibrin clots from factor XIII–deficient persons are soluble in certain concentrations of urea, whereas clots from normal persons are not. Several other assay methods have been reported.

  • Basic Tests in Hemorrhagic Disorders

    History. A history of easy bleeding or easy bruising should lead to further investigation.

    Platelet count. Platelet disorders will be discussed later. Using the direct count (reference values 150,000-400,000/mm3; 150-400 x 109/L), a platelet count less than 100,000/mm3 indicates moderate thrombocytopenia, and one less than 50,000/mm3 indicates severe thrombocytopenia. Platelet number can be estimated with a reasonable degree of reliability from a well-made peripheral blood smear.

    Clot retraction. Platelets have a major role in clot retraction. The clot shrinks and pushes out serum that was trapped within as the blood clotted. The shrunken clot is much firmer than it was originally. Normally, clot retraction begins at approximately 1 hour and usually is complete (a firm clot with about 50% serum and 50% clot) by 24 hours. There is deficient clot retraction in all typesof thrombocytopenia and in Glanzmann’s thrombasthenia, but clot retractionis usually normal in other varieties of platelet function abnormality. Clot retraction is rarely used today because of wider availability of accurate plateletcounts and also platelet function tests.

    Tourniquet test. This test demonstrates capillary abnormality due either to an intrinsic defect in the capillary walls (capillary fragility) or to some types of thrombocytopenia. The tourniquet test is usually abnormal in immunologic thrombocytopenias such as idiopathic thrombocytopenic purpura (ITP) and drug-induced thrombocytopenia. It produces variable results, more often normal, in thrombocytopenia of other etiologies. It is usually normal in disorders that donot entail increased capillary fragility or thrombocytopenia, but occasionally it may be abnormal in patients with hemophilia or vitamin K disorders. The testis abnormal in hereditary telangiectasia. The tourniquet test is another procedure that is rarely used today since platelet counts are readily available, but it could be helpful in some instances of nonthrombocytopenic purpura if capillary fragility is suspected.

    Bleeding time. The template (Mielke) modification of the Ivy technique is now the standard procedure for the bleeding time test. The template procedure, however, requiresthat a 9-mm long, 1-mm deep incision be made, which may leave a scar. Many laboratories (including my own) use commercial modifications of this technique suchas Surgicutt or Simplate, a disposable spring-driven small lancet in a plastic case, which makes a uniform and reproducible incision 5 mm long and 1 mm deep with minimal pain or sequelae. There are some technical factors that influence results; for example, an incision parallel to the antecubital fossa produces a longer bleeding time than one oriented perpendicular to that line. The bleeding time is most helpful as an indicator of platelet abnormality, either in number or function. The bleeding time is usually normal when the platelet count is decreased but still more than 100,000/mm3 (100 Ч 109/L). With platelet counts less than 100,000/mm3, there is a rough correlation between severity of thrombocytopenia and degree of bleeding time prolongation. The bleeding time is usually abnormal in congenital defects of platelet function such as Glanzmann’s thrombasthenia. The bleeding time is frequently abnormal in acquired platelet function abnormality such as that seen in uremia and the myeloproliferative syndromes. In uremia, there frequently aredemonstrable abnormalities in platelet function tests but not sufficient to entirely explain bleeding problems. In addition, up to 50% of uremic patients develop some degree of thrombocytopenia. In one study, about 45% of uremic patients had elevated bleeding times; the occurrence and degree of bleeding time elevation did not correlate well with the blood urea nitrogen (BUN) level since elevated bleeding times occurred at BUN levels as low as 50 mg/100ml (18 mmol/L) (although more often >100 mg/100 ml), whereas patients with normal bleeding times had BUN values as high as 180 mg/100 ml (64 mmol/L). The majority of studies report that actual bleeding in uremia is infrequent unless the bleeding time is elevated, but some studies do not agree. Reports indicate that the uremic bleeding tendency can be corrected by fresh frozen plasma, cryoprecipitate, or a synthetic analog of vasopressin (antidiuretic hormone, ADH) named 1-deamino-8-D-arginine vasopressin, or desmopressin (DDAVP).

    Certain drugs interfere with platelet function and can produce a prolonged bleeding time, the most common and important being aspirin. After asingle small dose of aspirin, prolongation of the bleeding time over baseline is present by 2 hours or less, with maximum effect at about 24 hours. Whether the bleeding time exceeds population normal limits depends on whether the preaspirin value was in the lower or the upper half of the reference range and on individual susceptibility to aspirin. In one study, bleeding times that exceeded the reference range upper limit occurred in about 50% of clinically normalpersons even with a relatively small aspirin dose such as 10 grains (two adult tablets); sometimes even with one 5-grain tablet. About 5% of the population appear to be hyperresponders, producing relatively large and prolonged bleeding time elevations. Aspirin permanently affects all platelets in circulation; platelet life span is 7-10 days, and about 10% of the circulating platelets are replaced each day. After aspirin is stopped, it takes 2-3 days (range, 1-8 days) for sufficient production of new (nonaffected) platelets to reduce an elevated bleeding time to normal. Ethyl alcohol ingested with aspirin is reported to increase the magnitude and duration of the bleeding time prolongation. Pretreatment with aspirin (aspirin tolerance test) increases the sensitivity ofthe bleeding time to platelet function defects such as occur in von Willebrand’s disease. Even without aspirin the bleeding time is usually, although notalways, abnormal in von Willebrand’s disease (discussed later). The bleeding time may be abnormal in a variable number of patients with capillary fragility problems. The bleeding time typically is normal in the hemophilias and the vitamin K or fibrinogen deficiencies but may be abnormal in severe (or sometimes even in moderately severe) cases. The bleeding time is elevated in about 20% (range, 0%-65%) of hemophilia A patients. In one study, theIvy bleeding time was consistentlynormal even though the Simplate method was abnormal in a significant number of patients. Heparin can increase the bleeding time.

    There is disagreement in the literature as to the clinical significance of aprolonged bleeding time as a predictor of hemorrhage during surgery. There definitely is not a good linear correlation be- tween degree of elevated values andprobability of hemorrhage. In general, however, when the bleeding time is 1.5 times the upper limit of the reference range, the possibility of excessive bleeding during surgery is somewhat increased. When the bleeding time is more than twice the upper reference limit, there is definitely an increased risk of excess bleeding. Others found no correlation between bleeding time elevation (at least, up to moderate elevation) and hemorrhage during surgery, with approximately the same numberof patients bleeding with and without elevated bleeding time. There is also disagreement as to the surgical risk after ingestion of aspirin or aspirin-containing compounds. In the majority of patients there does not seem to be a greatly increased risk. However, some patients develop a greater degree of platelet dysfunction than the average, so that the bleeding time is useful in uncovering these cases.

    Prothrombin time (PT). The prothrombin time (PT) is used in three ways: (1) to monitor anticoagulant therapy with Coumadin, (2) as part of a general screen for coagulation systemdisorders, (3) as a liver function test. A “complete” tissue thromboplastin plus calcium is added to the patient’s plasma (complete thromboplastin contains tissue-derived material that activates the extrinsic coagulationsystem plus phospholipid that acts as a platelet substitute). Formation of a fibrin clot is the end point. The PT mainly indicates defects in the extrinsic coagulation system (prothrombin and factors V, VII, and X). If defect in fibrinogen is severe, it also will produce abnormal PT test results, since the test depends on an intact fibrin mechanism to generate the clot end point. However, thefibrinogen level usually must be less than 100 mg/100 ml (reference range, 200-400 mg/100 ml; 2-4 g/L) before hypofibrinogenemia affects the PT. Platelet or intrinsic system factor defects before the prothrombin to thrombin stage do not affect the PT because a complete thromboplastin reagent activates the extrinsic coagulation system and bypasses the intrinsic system.

    Because the coagulation theory designates conversion of prothrombin to thrombin as the major reaction directly affected by thromboplastin activity, prothrombin is commonly considered the principal agent measured by the PT. Actually, the test is much more sensitive to factor VII than to prothrombin. Clinically this makes little difference, since both factor VII and prothrombin are altered by the same two major conditions affecting the extrinsic system—liver parenchymal disease (most often cirrhosis) and vitamin K deficiency. Vitamin K is discussed in the section on prothrombin. Interference with vitamin K metabolism is most often drug-induced (coumarin anticoagulation or, less frequently, use of certain cephalosporin antibiotics (cefamandole, cefoperazone, and moxalactam)or secondary to malabsorption. In some cases low vitamin K intake due to anorexia or use of prolonged intravenous (IV) feeding because of serious illness may accentuate previous subclinical degrees of vitamin K deficiency or the action of medications that affect vitamin K metabolism.

    The effect of sodium warfarin on the PT can be influenced by the amount of vitamin K in the diet. Some other factors include the effect of previous low tissue vitamin K stores (potentiated by low dietary intake), impaired vitamin K absorption, or action of broad-spectrum antibiotics on gastrointestinal (GI) tract bacteria. In one report, antibiotic therapy superimposed on IV feeding was said to precipitate vitamin K deficiency in as little as 48 hours after admission.

    The PT can be affected by heparin if the blood level is high enough. Levels ordinarily associated with continuous IV heparin usually do not prolong the PT significantly. With intermittent bolus (rather than continuous) full-dose IV heparin, heparin blood levels are relatively high (sufficiently high to prolong the PT shortly after administration; they decline thereafter with minimal (1-2 seconds) PT elevation at 2 hours. Subcutaneous heparin peak occurs later than with IV bolus and the heparin effect lasts longer. Coagulation monitoring may notadequately demonstrate this heparin effect since the tests are customarily not done until approximately 1 hour or less before the next heparin dose, thereby not reflecting peak heparin sodium activity. When heparin and warfarin sodium (Coumadin) are given together, heparin (even continuous IV heparin) affects the PT in addition to the usual effect of Coumadin. To obtain a valid PT, one must wait until the heparin effect has mostly disappeared (5 hours after IV bolus injection and 12-24 hours after subcutaneous injection). Heparin blood levels are increased in patients with a high hematocrit level (the same dose in a smaller plasma volume). This is especially important in neonates, who normally have a relatively high hematocrit level.

    Also, it should be remembered that neonates and young children have higher reference-range values than older children and adults. The effect of heparin canbe neutralized in vivo and in laboratory specimens with protamine (1 mg of protamine sulfate given intravenously for every 100 mg of heparin in the last dose). Excess protamine should be avoided since protamine itself may elevate the PT. Other ways to eliminate heparin from laboratory specimens are neutralization with hexadimethrine bromide (Polybrene) or absorption onto a special cellulose material (Heparsorb). Warfarin effect can be treated by parenteral administration of vitamin K, after which the PT should return to normal within 6-24 hours. In bleeding emergencies, fresh frozen plasma can be used.

    Certain technical problems are discussed after the following section on the activated partial thromboplastin time. Coumadin and heparin anticoagulation is discussed in a later section on Anticoagulant Systems.

    Activated partial thromboplastin time. The precursor of the activated partial thromboplastin time (APTT) was the partial thromboplastin time (PTT). An “incomplete” thromboplastin reagent plus calcium is added to patient plasma, and the time necessary to form a fibrin clot is measured. The partial thromboplastin reagent is only a phospholipid platelet substitute without any of the other components of thromboplastin. The PTT was useful in detecting intrinsic factor abnormalities but was relatively insensitive to effects of heparin. Adding certain “contact activators” (usually chemicals or particulate matter, such as kaolin) to the PTT reagent was found to activate factor XII (contact factor) swiftly and uniformly and thus eliminate another variable in the clotting process. In addition, the activated APTT was found to be sensitive to heparin. The APTT is very sensitive tocoagulation factor deficiencies within the intrinsic system before the prothrombin to thrombin stage. It may also be abnormal in prothrombin or fibrinogen deficiencies but only if the defect is relatively severe (prothrombin or fibrinogen/fibrin abnormalities may affect the test because the test depends on fibrin clot formation as the reaction end point). The APTT is not as sensitive to prothrombin abnormalities as the PT because the extrinsic thromboplastin used inthe PT test is more powerful than the intrinsic system prothrombin activator complex generated by the APTT, thus enabling the PT to demonstrate relatively smaller defects in prothrombin. Platelet abnormalities do not influence the APTT.

    Advantages of the APTT are adequate reproducibility (<10% variation), speed (reaction time of about 30-50 seconds), ease of performance, and suitability for automation. Disadvantages are the following:

    1. Blood levels of heparin that are very much above anticoagulant range cause the APTT to become nonlinear, excessively prolonged, and unreliable.
    2. Various techniques and equipment are used for the APTT readout (clot detection); the different machines as well as different companies’ reagents may produce results that deviate significantly on the same specimen. This may cause problems in comparing values from different laboratories.
    3. The APTT is not affected by platelets, whereas platelets do influence heparin activity in vivo (platelets contain platelet factor 4 [PF-4], which inhibits the anticoagulant activity of heparin).
    4. The APTT is affected by warfarin. When the PT is in the warfarin therapeutic range, the APTT is also prolonged and may even be above the APTT therapeutic range for heparin.

    Technical problems that affect interpretation of PT and APTT test results. The labile factors are preserved better in citrate than in oxalate anticoagulant. Once exposed to air, citrated plasma is stable for only 2 hours at room temperature (22°-25°) and 4 hours in the refrigerator at 4°C. If collected in a vacuum tube and if the top has not been opened, the plasma is stable for a longer time (6 hours at room temperature in one study and 14-16 hours at 4°C in another study). Excess anticoagulant relative to the amount of plasma may affect results. Insufficient blood drawn in the tube means that less plasma is available for the amount of anticoagulant present, and the test resultsmay be falsely prolonged. The same thing may occur in blood that has a high hematocrit value, since in that case the excess red blood cells (RBCs) are replacing some of the plasma. When 3.8% sodium citrate (the most commonly used anticoagulant) is used to prepare plasma, both the PT and APTT may be falsely prolonged when the hematocrit value reaches 50%, and prolongation may become marked with a hematocrit value more than 60%. The APTT is affected more than the PT. The hematocrit effect is especially troublesome in neonates, since a newborn normally has a relatively high hematocrit value. A hematocrit value of 20% or less may produce a false decrease in the PT and APTT. If 3.2% sodium citrate is used, the effect of hematocrit level is reported to be considerably less. One of the most frequent causes for falsely elevated APTT (and sometimes PT) arises from attempts to keep IV lines open by heparin flushes. Usually this is not known to the phlebotomist. One report indicates that heparin tends to adhere to the wall of catheters (depending to some extent on the chemical composition of the catheter), which can affect results from specimens drawn from the catheter. In patients on constant-infusion heparin therapy, faulty or improperly calibrated delivery systems may be the reason for otherwiseinexplicable APTT fluctuations. Technical considerations are important even in patients not known to be receiving anticoagulants; according to one report, 40% of abnormal APTT results in these patients were eventually interpreted as being false elevations due to technical factors. Another report found a 2% incidence of false elevations in clinically normal persons and 11% inpatients being evaluated for bleeding. Recollection by an experienced technologist has solved many problems. Other possible causes for unexpected APTT elevation are circulating factor inhibitors or the lupus anticoagulant (discussed later). One study found 3% of routine preoperative APTT results elevated in patients below 12 years of age and 0.5% elevated in patients over age 12. All had inhibitors (acquired anticoagulants), most of which disappeared in less than 7 months (range, 1 day to 7 months). A few persisted. No patients bled excessively in surgery even though no therapy was given. The reference range for the APTT is higher in young children than in adults.

    Activated whole blood clotting time. Whole blood is drawn into a vacuum tube containing a contact factor activator. The tube is incubated in a heat block at 37°C and tilted every 5 seconds. The normal upper limit is about 2 minutes. The test is claimed to be very reproducible. It can be used for coagulation defect screening and to monitor heparin therapy. It is said to have approximately the same sensitivity as the APTT in coagulation defects, and in heparin assay it is more linear than APTT at greater heparin effects. Many coagulation experts prefer this test to the APTT whenmonitoring heparin therapy. Disadvantages are the need for a portable heating unit and inability to automate the procedure (that the test is done at the bedside is considered by its proponents to be an advantage). The test is not sensitive to platelet variations.

    Venous clotting time. The Lee-White method is preferred for venous clotting time (VCT). Technique is extremely important. When the usual three test tubes are used, number 3 is filled first, since the last blood to enter the syringe is probably least contaminated with tissue juice. If glass syringes are used, the test timing should bestarted as soon as blood enters the syringe. If plastic syringes are used, one can wait until blood enters tube number 3 (the first tube filled) to start the test timing, because clotting time in plastic material is prolonged. The tubes should be incubated at 37°C. The VCT is affected mainly by defects in the intrinsic pathway factors before prothrombin and by defects in fibrinogen/fibrin. It is not sensitive to platelet abnormalities and is relatively insensitive to abnormalities of prothrombin or factor VII (in which severe deficiency isrequired to produce significant VCT abnormality). In deficiencies of the intrinsic pathway factors or fibrinogen, the test is only moderately sensitive, requiring considerable deficiency to cause abnormal test results. The VCT is reasonably sensitive to heparin effect and was the original test used to monitor heparin therapy.

    Disadvantages of the VCT are relative lack of reproducibility (>15% variation in most laboratories when the test is repeated on the same patient), necessity for 37°C incubation and careful technique (hardly ever observed by the average technologist), relatively long reaction time (5-15 minutes), the fact that each test must be done separately at the patient’s bedside, and the fact that platelets do not affect the test. The VCT has been replaced in most laboratories by the APTT or some other method that is more reproducible and more easily controlled.

    Thrombin time. If commercially supplied thrombin is added to patient plasma, the time required to form a clot can be used to estimate the rate of fibrin formation. Variables include the amount of patient fibrinogen available and whether any inhibitors are present. The inhibitors could either be fibrin split products (which interfere with fibrin monomer polymerization) or antithrombins (e.g., heparin). Some manufacturers market reagents based on certain snake venoms that directly convert fibrinogen to fibrin without being affected by heparin. Fibrinolysins canalso produce abnormality both by destroying fibrin or fibrinogen and by producing fibrin split products. The thrombin time is therefore used as a test for abnormalities in the fibrinogen/fibrin stage of coagulation. The test is not as widely used as some of the other coagulation procedures.

    Plasma fibrinogen. There are several methods for measuring plasma fibrinogen levels. The oldestmethods (“clottable protein”) involved precipitating a fibrin clot from plasma either by adding thrombin or by recalcifying the plasma (adding enough calcium to neutralize the chelating effect of the laboratory anticoagulant used to obtain the plasma). The fibrin clot was then assayed chemically by one of several indirect methods, or the change in optical density of the clot was measured. Newer techniques consist of immunologic methods using antibodies against fibrinogen and methods based on a modified thrombin time principle. At present, the most widely used is the thrombin time technique. Most of the techniquesare not reliable when high titers of fibrinolysins are present or when heparin is present. Fibrinogen can be measured using certain snake venom reagents (Reptilase or Ancrod) instead of thrombin; these venoms convert fibrinogen to fibrinand are not affected by heparin. True low plasma fibrinogen levels may be due to high titers of fibrinolysin (when fibrinolysins are present in high titer, fibrinogen may be attacked as well as fibrin) or to the disseminated intravascular coagulation (DIC) syndrome. Disseminated intravascular coagulation is by far the more common cause. A thromboplastin tissue substance or substance with equivalent action is liberated in the bloodstream and causes fibrin deposition (clots) in small blood vessels.

    It might be useful to compare the results of some laboratory tests just discussed in the various phases of blood coagulation:

    Test result* affected by abnormality in:

    Test result affected by abnormality
    * VS = very sensitive; Ins = insensitive; Mod S = moderately sensitive; No = not affected.
    †Factors XIII, IX, X, XI, and XII (not including platelets or common pathway.

    In hemophilia (factor VIII defect), the various tests have approximately the following sensitivity:
    PT—normal at all levels of factor VIII C deficiency.
    VCT—normal until factor VIIIC activity levels are less than 2% of normal.
    APTT—normal until factor VIII activity levels are less than 30%-35% of normal.

    Note that normal persons may have 50%-150% of “normal” levels on factor VIII activity assay.

  • Blood Coagulation Theory

    According to current theories of blood coagulation, the clotting mechanism is activated in two ways. The first activation pathway begins either when the endothelial lining of a blood vessel is damaged or when blood comes into contact with certain types of foreign surfaces. This activating sequence is begun by substances normally present within blood and is therefore called the intrinsic system pathway. The second trigger is a substance, tissue thromboplastin, which is not present in blood but which can be released from endothelial cells or other body tissues, usually when the tissue cells are injured. Tissue thromboplastin, sometimes called factor III, initiates the extrinsic system pathway. Activation of the clotting sequence by extrinsic system thromboplastin bypasses the first half of the intrinsic system pathway, although both systems share several final pathway coagulation factor reaction sequences.

    Extrinsic system

    The extrinsic system tissue thromboplastin forms a complex with calcium ionsand a proenzyme known as factor VII. Factor VII is normally inactive but now becomes activated by tissue thromboplastin. The thromboplastin complex with activated factor VII converts another inactive enzyme (proenzyme), factor X, to an active form. Activated factor X with two cofactors (phospholipid from tissue thromboplastin and activated factor V) in the presence of calcium ions converts prothrombin to thrombin. Thrombin, in turn, converts fibrinogen to fibrin monomer. Fibrin monomer polymerizes; the polymer then becomes “stabilized” (resistant to dissociation) by adding cross-linkagesbetween molecules with the assistance of activated factor XIII. The stabilized fibrin also becomes insoluble in certain substances such as urea.

    Intrinsic system

    The intrinsic system is triggered by contact between blood and a suitable foreign surface. Within vessels, this usually occurs at a break in the vascular endothelial lining where collagen is exposed. Platelets adhere to the exposed collagen and release a phospholipid called platelet factor 3, or PF-3. In addition, a proenzyme in serum called factor XII becomes activated by exposure to the collagen. Activated factor XII initiates a side reaction involving high molecular weight kininogen (HMWK) as a cofactor, which converts a proenzyme called prekallikrein, to kallikrein which, in turn, helps convert more factor XII to its active form. The major consequence of activated factor XII is conversion of inactive factor XI to its active form, which, in turn, converts factor IX to its active form. Activated factor IX convertsfactor X to activated factor X with the assistance of activated factor VIII andPF-3 in the presence of calcium ions. (Thus, activated factors VIII and IX plusPF-3 produce the same effect as the tissue thromboplastin complex and activatedfactor VII. Tissue thromboplastin supplies phospholipid to the extrinsic systemand PF-3 does so for the intrinsic system.) Activated factor X plus activated factor V and PF-3 convert factor II (prothrombin) to thrombin, leading to conversion of fibrin to fibrinogen. The steps subsequent to formation of active factor X are the same in both the extrinsic and the intrinsic pathway (“final common pathway”), except that in the intrinsic system PF-3 supplies phospholipid cofactor for conversion of prothrombin to thrombin, whereas the phospholipid component of tissue thromboplastin performs this function for the extrinsic system.

    Nine of the 13 coagulation factors are proenzymes that must be activated. Exceptions are factor I (fibrinogen), which is not an enzyme; factor III (tissue thromboplastin), which is a complex rather than a single protein; factor IV (calcium); and factor VI, which currently is a number that is not in use.

  • Blood Coagulation

    Normally, blood remains fluid within a closed vascular system. Abnormalitiesof blood coagulation take two main forms: failure to clot normally (and thus toprevent abnormal degrees of leakage from the vascular system) and failure to prevent excessive clotting (and thus to maintain the patency of the blood vessels). Most emphasis in clinical medicine has been on the diagnosis and treatment of clotting deficiency. To understand the various laboratory tests designed to pinpoint defects in the coagulation mechanism, we must outline the most currently accepted theory of blood coagulation (Fig. 8-1).

    Blood coagulation pathways

    Fig. 8-1 Blood coagulation pathways. a, activated.