Acquired Inhibitors MLS704 Haematology II 1 1. Factor VIII Inhibitors • The most common autoantibodies directed against, and interfere with, the activity of FVIII, called acquired hemophilia A . • most have been IgG antibodies that do not bind complement. • The reasons for the production of FVIII autoantibodies in a particular individual are not clear but may involve the presence of certain gene polymorphisms (eg, HLA, CTLA4) and/or autoreactive CD4+ T lymphocytes. • Most of these antibodies bind to the C2 or, less often, the A2 domain on factor VIII . • Loss of the C2 domain, which binds to the procoagulant phospholipid phosphatidylserine on activated platelets and endothelial cells and to von Willebrand factor, leads to reduced procoagulant activity MLS704 Haematology II 2 Epidemiology • most of the patients were over age 50, except for women who were pregnant or postpartum. • Also, patients with rheumatoid arthritis, malignancy, systemic lupus erythematosus, and drug reactions (eg, penicillin, sulfonamides, phenytoin, clopidogrel and immunomodulatory agents such as immune checkpoint inhibitors, alemtuzumab, omalizumab, interferon, and fludarabine). • However, in almost one-half of the patient's no underlying disorder was present. MLS704 Haematology II 3 Clinical features • The hallmark of acquired factor VIII antibodies is bleeding that, in some cases, is first noted during or after a surgical procedure. • However, most occur spontaneously without apparent provocation. Symptomatic patients often present with large hematomas, extensive ecchymoses or severe mucosal bleeding, including epistaxis, gastrointestinal bleeding, and gross hematuria. • Spontaneous hemarthroses, which are common in hereditary factor VIII deficiency, are unusual in those with acquired disease. MLS704 Haematology II 4 Diagnosis • The sudden presence of large hematomas or extensive ecchymoses in an older individual without significant trauma or known bleeding disorder should always raise the clinical suspicion of an acquired factor VIII inhibitor. • Prolonged aPTT and normal PT. • The differential diagnosis of this profile in a patient with a bleeding diathesis includes deficiencies of factors XI, IX or VIII, von Willebrand disease, inhibitors of these factors, and the use of heparin. • A prolonged aPTT is also seen in the antiphospholipid antibody syndrome, but these patients typically present with thrombosis rather than bleeding episodes. • First step is to exclude the use of heparin as a cause of the patient's bleeding and/or prolongation of the aPTT. • Redraw blood sample using an uncontaminated peripheral vein. If the repeat aPTT is normal, the original sample was probably contaminated with heparin. • Perform a thrombin time with protamine sulphate and a reptilase time. • Heparin is present if the thrombin time is prolonged and the reptilase time is normal. MLS704 Haematology II 5 • Mixing study: APPT is prolonged. Another mixing study needs to be done after incubation at 37ºC for at least 2 hours. • The next step is adding a source of phospholipid to the mixed plasma. Correction of the aPTT suggests the presence of antiphospholipid antibodies. If the aPTT does not correct, the Bethesda assay is performed. • The Bethesda assay both establishes the diagnosis of a factor VIII inhibitor and quantifies the antibody titer. • By standardizing the strength of factor VIII inhibitors, this assay also permits comparison between different treatment regimens. • Bethesda assay: FVIII activity level is measured as would be done in a patient with hemophilia A. Then, serial dilutions of patient plasma are incubated with pooled normal plasma at 37°C for two hours, and factor VIII activity is again measured, and the dilution at which factor VIII activity is restored. The reciprocal dilution of patient plasma that results in 50 percent factor VIII activity is defined as one Bethesda unit (BU). The stronger the inhibitor, the greater the dilution required to allow for factor VIII activity and the higher the BU titer. • Other testing: ELISA-based immunoassay for anti-factor VIII antibodies has also been investigated. MLS704 Haematology II 6 2. Prothrombin inhibitors — Antibodies to prothrombin are most often detected in patients with antiphospholipid antibodies. The aPL antibody that correlates with antiprothrombin is the antiphosphatidyl serine antibody, which correlates with the lupus anticoagulant phenomenon and likely causes functional interference in the prothrombin assay. • Antiprothrombin antibodies, which can cause significant clinical bleeding, usually bind to a nonactive portion of the molecule, resulting in accelerated clearance of prothrombin . Technically, since these antibodies bind prothrombin and deplete it from the circulation rather than interfering with function, they are not inhibitors in the strict sense of the word. The inhibitor screen will be most consistent with a factor deficiency rather than an inhibitor, since the functional activity of prothrombin is not impaired in a clotting assay when additional prothrombin is added. • The possible presence of prothrombin antibodies should be suspected if a patient with antiphospholipid antibodies develops bleeding rather than the expected thrombotic events. • Specific immunochemical measurement of the prothrombin concentration is required to establish the diagnosis. • Treatment of active bleeding consists of the use of fresh frozen plasma (FFP) to increase prothrombin levels. MLS704 Haematology II 7 3. Thrombin inhibitors Inhibitors specific for human thrombin are quite rare, because of the multiple important functions of this enzyme. • The majority of antibodies directed against thrombin are detected incidentally in the absence of clinical bleeding because of prolongation of the thrombin clotting time and other clot-based anticoagulation assays performed for some other reason. • Thrombin antibodies typically arise after exposure to bovine thrombin found in topical thrombin or fibrin-glue preparations used in surgery. • These antibodies do not cause clinical bleeding unless they crossreact with human thrombin or, more often, when antibodies are also present against factor V, a common contaminant in bovine thrombin preparations. • In these patients with antibodies to bovine thrombin, the TT is prolonged when using bovine reagent thrombin and is normal when the thrombin reagent is of human origin. • The aPTT is prolonged and the prothrombin time is increased if factor V antibodies are also present. The availability of human, rather than bovine, thrombin for fibrin sealant and topical thrombin preparations should decrease the incidence of thrombin antibody formation. • Thrombin antibodies have also been described in several patients without exposure to exogenous thrombin. Most of these patients had a comorbid condition such as systemic lupus erythematosus, rheumatoid arthritis, or monoclonal gammopathy • These inhibitors (autoantibodies to prothrombin or thrombin in individuals with rheumatologic or lymphoproliferative disorders) may cause severe bleeding. MLS704 Haematology II 8 4. Factor V inhibitors — As with thrombin inhibitors, the majority of factor V inhibitors are autoantibodies that arise from exposure to topical fibrin glues or bovine thrombin preparations that are contaminated with bovine factor V • Other associated conditions included malignancy, autoimmune disease, the postpartum state, and congenital factor V deficiency. • These antibodies usually recognize the C2 domain of the protein. Loss of this domain, which binds to the procoagulant phospholipid phosphatidylserine on activated platelets and endothelial cells, results in loss of procoagulant activity. MLS704 Haematology II 9 • The hemorrhagic manifestations associated with factor V inhibitors are variable, ranging from asymptomatic to life-threatening bleeding • Bleeding is primarily associated with antibodies directed against the C2 domain of the protein . Second, functionally important factor V is concentrated on platelets. • Platelet factor V may be more important for assembly of the prothrombinase complex than circulating factor V. • The presence of a factor V inhibitor should be suspected in a patient with prolongation of the aPTT and PT but a normal thrombin time and a decreased factor V activity level . • However, the thrombin time may be prolonged in patients with bovine thrombin exposure and the production of antibodies to thrombin as well as to factor V. Similar to factor VIII inhibitors, mixing studies should confirm the diagnosis and the inhibitor titer can be determined using the Bethesda assay. • Acute severe bleeding due to a factor V inhibitor may be life-threatening. For those patients who survive the acute bleeding episode, the prognosis is generally excellent. Most inhibitors disappear within a period of months; it is not clear if immunosuppressive therapy accelerates this response. • Treatment modalities for acutely bleeding patients include platelet transfusions, plasma exchange, and intravenous immune globulin (IVIG). MLS704 Haematology II 10 5.Factor VII inhibitors — Antibodies directed against factor VII are rare and only a small number of cases have been reported. • PT is prolonged but the aPTT is normal, since factor VII is not involved in either the intrinsic coagulation pathway or common pathway . Mixing studies will confirm the presence of an inhibitor; a low level or absence of residual factor VII activity, especially after prolonged incubation (one hour) is necessary to confirm the diagnosis. • The clinical manifestations in the few reported cases have ranged from minor bleeding to life-threatening events such as intracranial hemorrhage. 6.Factor IX inhibitors — Acquired antibodies directed against factor IX in the absence of hemophilia B are also very rare. MLS704 Haematology II 11 7.Factor X inhibitors — very rare • Patients have presented with the sudden onset of bleeding, prolonged PT and aPTT measurements, and transient, severe deficiency of factor X. • Amyloidosis can lead to deficiency of factor X due to binding of the protein to amyloid fibrils . In this disorder, other hemostatic defects probably also contribute to the bleeding. 8.Factor XI inhibitors — Factor XI autoantibodies not occurring in patients with congenital factor XI deficiency are often associated with systemic lupus erythematosus. • Affected patients usually have little or no bleeding despite a prolonged aPTT that does not fully correct in a mixing study. • The relative lack of bleeding, which is also noted in congenital factor XI deficiency, probably reflects the tertiary role for factor XI in factor X activation, although bleeding can occur under certain circumstances. MLS704 Haematology II 12 9. Factor XIII inhibitors —exceedingly rare, can act by one of several mechanisms: inhibition of activation of factor XIII; interference with enzymatic function; or prevention of binding to fibrin . • The clinical hallmark of a factor XIII inhibitor is delayed bleeding after surgery or an invasive procedure, since the initial clot which does form is mechanically weak and unstable. Large spontaneous hematomas or intracranial hemorrhage can occur. • Factor XIII inhibitors can be associated with immune dysregulation. Other associated conditions include other autoimmune disorders, lymphoid malignancies, and some drugs • The PT and aPTT are normal, but the clot stability assay is abnormal. • the D-dimer is normal • D-dimer is formed by the action of XIIIa in crosslinking of the two D domains, which does not occur with factor XIII deficiency. • Management typically involves immunosuppression; immunosuppressive therapies, including prednisone, cyclophosphamide, plasma exchange, IVIG, rituximab, and/or infusion of factor XIII concentrates MLS704 Haematology II 13
