If, after an adequate infusion of VII, VIII, IX, XI, and V, bleeding continues, a factor-inhibitor assay is indicated. Although nearly all procoagulants have an inhibitor, the inhibitor to factor VIII is the most common.
Under normal circumstances, no factor inhibitors are present.
Factor inhibitors are reported in Bethesda units (BU).
They are present in factor VIII inhibitors, which are IgG antibodies against factor VIII, occur in hemophilia A (alloantibodies). As autoantibodies they usually arise spontaneously, particularly in the elderly population, but they are also found in pregnancy and autoimmune disease.
Collection and Panels
Collection details areas follows:
Specimen: Blood (0.5 mL frozen plasma X4 or two 3.2% sodium citrated tubes, 5 mL each)
Container: Blue-top vacuum tube
Collection method: Routine venipuncture
To comply with Occupational Safety and Health Administration (OSHA) safety standards, all samples must be sent in a sealed, leak-proof container with a biohazard sticker attached.
An inhibitor exists for nearly every procoagulant, with the inhibitor to factor VIII being the most common. Detection of factor VIII inhibitor is accomplished by mixing the test plasma with a known amount of factor VIII. Following a 2-hour incubation period at 37°C, a factor VIII assay is used to determine residual factor VIII activity. The presence or absence of a factor VIII inhibitor can be determined through comparison of the difference in factor VIII activity between the incubation mixture and a control mixture. The partial thromboplastin time (PTT) is prolonged by some antibodies only after incubation. 
Among the many protease inhibitors in plasma, tissue factor pathway inhibitor (TFPI) and antithrombin are the ones that are most specifically involved in inhibition of coagulation factors. Another potentially important regulator of the coagulation system, the protein Z/protein Z–dependent protease inhibitor (ZPI) system, is also emerging. 
Tissue factor pathway inhibitor
Depending on the degree of proteolysis of the carboxy-terminal region, TFPI, a single-chain polypeptide, has an Mr of 34,000-40,000. Unlike other coagulation protease inhibitors, TFPI has inhibitory sites for factor Xa and for the factor VIIa/tissue factor (TF) complex, and it cannot inhibit the factor VIIa/TF complex without being bound to factor Xa. [2, 3]
Endothelial cells are the primary site of plasma TFPI synthesis.  Most circulating TFPI is bound to lipoproteins, but on the surface of endothelial cells, a second pool of TFPI is bound to heparan sulfates. When heparin is administered, the endothelial cell–bound TFPI is released and the plasma level rises severalfold. 
TFPI exists in the plasma at only about 2.5 nM, while antithrombin occurs at about 2 µmol. However, TFPI and antithrombin have a similar rate of reaction with factor Xa in plasma. Therefore, TFPI makes a significant contribution to factor Xa inhibition in vivo.  The human TFPI gene is found on chromosome 2.
A deficiency of antithrombin increases the risk of thrombosis, revealing its importance as a functional inhibitor of the blood coagulation proteases. Thrombin, factor Xa, and factor IXa are the primary proteases that antithrombin targets, [7, 8] with heparin increasing its inhibition of coagulation factors. Unless it is complexed to TF in the presence of heparin or cell surface glycosaminoglycans, factor VIIa resists being inhibited by antithrombin. 
The protease-serpin complex is cleared from the circulation by receptor-mediated endocytosis in the liver.  The antithrombin gene is found on the long arm of chromosome 1.
Protein Z/protein Z–dependent protease inhibitor
Coagulation factors XIa and Xa are inhibited by ZPI, an Mr 72,000 serine protease inhibitor. The presence of protein Z, a vitamin K–dependent plasma protein, increases its inhibition of factor Xa over 1000-fold.  Protein Z/ZPI's physiologic activity in the coagulation system is unclear.
In a mouse model, protein Z deficiency does not cause thrombosis, but it does significantly increase the thrombotic tendency of mice who simultaneously express the factor V Leiden genotype, a known thrombotic risk factor. 
The protein Z gene exists on the long arm of chromosome 13, where it lies in close proximity to the genes for factor X and factor VII. 
Factor VIII inhibitors
Factor VIII inhibitors are the inhibitors that occur most commonly in the factor inhibitor assay. Although they are found most often in individuals with hemophilia who are receiving factor replacement, they can also occur in patients with autoimmune diseases and lymphoma and in pregnant (postpartal) or elderly patients.
Factor VIII inhibitors are measured in BU; highly potent inhibitors have levels of more than 10 BU.
In order to predict the response of a patient with hemophilia to factor replacement therapy, factor VIII inhibitor should be measured before the patient undergoes any significant invasive procedure. It is important to confirm the patient's response to factor administration before the procedure is begun.
In the short term, the inhibitors for low titers may be overridden by infusion of factor VIII concentrate, although in some patients, such infusion may increase the inhibitor titer. Infusion of prothrombin complex concentrate (PCC), activated PCC, porcine factor VIII concentrates, and VIIa have been found to be useful for high-titer inhibitors.  To manage inhibitors, it is efficacious to use combined immunosuppressant programs that include steroids, cytotoxic agents, plasma processing over columns that remove IgG and IgG-containing immune complexes,  and high-dose intravenous immunoglobulin.
Rituximab therapy, while effective in patients with high titers of acquired factor VIII, may not produce a sustained response on its own. It may therefore be better to combine rituximab with other therapies in high-risk individuals. [15, 16, 17, 18]
In persons without hemophilia, inhibitors are more responsive to simple immunosuppressive agents such as prednisone and azathioprine. However, inhibitors must be controlled in such patients to avoid an increase in the risk of bleeding. Owing to the high risk of hemorrhage, an emphasis has been placed on meticulous perioperative hemostatic correction. Following major surgery, it is necessary to maintain the correction for at least 10 days to prevent delayed hemorrhage. 
Thrombosis (hypercoagulable state), rather than bleeding, is caused by lupus anticoagulant (phospholipid inhibitor), despite the fact that it is usually associated with a prolonged PTT. This inhibitor may, however, cause bleeding diathesis in very rare cases; such a case may be indicated by prolongation of the prothrombin time (PT) in addition to the PTT. 
Laboratory results typically consist of a prolonged PTT and/or Russell viper venom time (RVVT) with evidence of an inhibitor and interference on several of the coagulation factor assays. Characteristically, via the tissue thromboplastin inhibition [TTI] test, an abnormally steep rise in the PT with serial dilution of thromboplastin in vitro is found.
This test should be carried out prior to anticoagulation, since heparin and warfarin anticoagulation may demonstrate similar phenomena. By removing the inhibitor, absorption of the patient's plasma with platelet phospholipid in vitro may normalize the PTT, TTI, and RVVT test results.
Clinically important hypercoagulability exists in approximately 25% of patients with lupus anticoagulants. To reduce the risk of venous thromboembolism, perioperative prophylactic heparin is recommended. Because PT in these patients is unpredictable, the degree of warfarin effect may be more reliably monitored using chromogenic factor X levels. The risk of arterial thrombosis is also greater in these patients.
Factor V inhibitors
An inhibitor to factor V (a contaminant of the bovine product) may develop in some individuals who have been exposed to surgical sealant containing topical bovine thrombin. In rare cases, cross-reactivity may cause a significant reduction in human factor V levels. Although these antibodies eventually disappear in weeks, patients with significant bleeding may require treatment with plasma and platelets.
Continuing bleeding after adequate infusion of VII, VIII, IX, XI, and V is an indication for the use of the factor-inhibitor assay.
If a prolonged PT or aPTT performed on a 1:1 mixture of the patient's plasma and normal plasma leads to suspicion that an inhibitor is present, additional studies can help to determine the inhibitor's nature and titer.
Among immediate-type inhibitors, that is, those that do not require incubation, the existence of heparin in the sample may be the most common cause. This cause can be verified by finding a prolonged thrombin time on a test of the patient's plasma that is corrected with toluidine blue or other agents that neutralize heparin.
Several methods are available for the detection of the lupus anticoagulant, which also requires no incubation. However, lupus anticoagulant is usually associated with a PT that is less prolonged than the aPTT. Moreover, depending on how much phosphatidyl serine exists in each reagent, aPTT reagents differ markedly in their sensitivity to lupus-type anticoagulant. 
The development of immunoglobulin inhibitors to specific coagulation factors may occur either (1) after patients with inherited coagulation factor deficiencies have undergone factor replacement therapy or (2) spontaneously in patients with no factor deficiencies. By incubating the patient's plasma with normal plasma, usually for 2 hours at 37°C (98.6°F), and then assaying the specific factor, it is frequently possible to detect antibodies that neutralize factor activity. Although originally designed to quantify factor VIII inhibitors, the Bethesda assay can be modified to detect other coagulation factor inhibitors. 
Rather than directly neutralizing clotting activity, some inhibitors instead form complexes with coagulation factors as a means of reducing factor levels, with the complexes rapidly being cleared from the circulation. Such plasmas may be confused with inherited deficiency states because they do not produce prolonged clotting times when mixed 1:1 with normal plasma. Identification of this type of inhibitor requires more elaborate assays; such inhibitors may, for example, cause severe prothrombin deficiency in some patients with antiphospholipid syndrome and deficiency of von Willebrand factor in patients with some acquired forms of von Willebrand disease. 
Coagulation factor inhibitors may lead to potentially fatal situations, particularly in patients who are otherwise critically ill. The inhibitor's potency can be measured by titrating the effect of plasma dilutions on the result of mixing the patient's plasma with pooled normal plasma.
Among coagulation factor inhibitors, those directed against factor VIII are the most commonly found. Lupus anticoagulant has been encountered not only in patients with systemic lupus erythematosus (SLE) and drug-induced SLE syndromes, but also in patients with other autoimmune diseases, as well as in otherwise normal persons. A spontaneous reduction in lupus anticoagulants may occur after an offending drug, such as procainamide or a phenothiazine, is discontinued, or may result from the use of immunosuppressive agents such as prednisone.