Abstract
The development of clotting factor inhibitor autoantibodies is rarely observed, but can result in a potentially life‐threatening haemorrhagic disorder. These acquired inhibitors are most frequently against factor VIII (FVIII), whilst the detection of inhibitors against other clotting factors is rarer. Inhibitors against FVIII and FIX are mostly observed in patients with classical hereditary haemophilia after receiving factor replacement therapy. We report a rare case of acquired FVIII and factor IX (FIX) inhibitors in a single, non‐haemophilic patient with chronic hepatitis C virus (HCV) infection who was receiving antiviral treatment with pegylated interferon plus ribavirin. The FVIII and FIX activities were <1% and high titres of inhibitors autoantibodies were found in his serum samples. After achieving a sustained virological response, combined immunosuppression with oral corticosteroids (prednisone) and azathioprine was introduced, erradicating the inhibitory autoantibodies. The development of these inhibitors in association with antiviral therapy for chronic hepatitis C is poorly understood, and particular attention must be given to HCV‐infected patients with worsening coagulopathy, particularly if coexistent with treatment related thrombocytopenia.
Introduction
Inhibitors of clotting factors are antibodies directed against a blood coagulation factor, resulting in their inhibition or accelerated clearance from the circulation. Inhibitory antibodies against factor VIII (FVIII) or IX may arise as an acquired autoimmune phenomenon in patients with a previously normal haemostatic state or, most frequently, as an alloimmune reaction in classical haemophilia A or B patients, most commonly severe, after factor replacement therapy.Citation1,Citation2 In acquired haemophilia A (AHA), the inhibitory autoantibodies neutralize endogenous FVIII, and acquired deficiency of FVIII resulting in a potentially severe bleeding tendency. The incidence of AHA is estimated to be only 0·2 to 1·0 case per 1 million persons per year.Citation3 In 46% of cases, this condition is considered idiopathic with no identifiable causative pathology.Citation4 Known predisposing factors include pregnancy, autoimmune diseases, malignancy, and viral hepatitis.Citation5 Acquired FVIII inhibitors can cause a potentially life‐threatening haemorrhagic disorder, with death attributed to its presence occurring in a significant proportion of patients with major bleeding.Citation4–Citation6
Autoantibodies against FVIII occurring in AHA patients have been characterized as polyclonal, mostly of the IgG4 isotype, similar to those arising in classical haemophiliaCitation7 However, AHA differs from classical hereditary haemophilia A in some aspects: patients with AHA do not have past or family history of bleeding, and most of them do not present with haemarthroses. In fact, the clinical picture of patients with AHA is characterized by haemorrhage into the muscles, soft tissues, skin and/or mucous membranes. Moreover, monoclonal autoantibodies (IgA or IgM class) have also been demonstrated in association with AHA.Citation3,Citation8 The detection of inhibitor autoantibodies against other coagulation factors such as FIX, FXI, FXIII, and vWF protein are extremely rare.Citation9
Hepatitis C virus (HCV) infection is recognized to cause chronic liver disease in a majority of infected individuals. A wide variety of extrahepatic manifestations have been reported in association with chronic HCV infection, and for a number of these conditions an underlying immune mediated pathogenic mechanism has been postulated.Citation10,Citation11 Acquisition of factor VIII inhibitors during interferon (IFN)‐alpha therapy for chronic HCV infection was reported in a few studies. Castenskiold et al.Citation12 first described a case of acquired FVIII inhibitor in association with therapy with IFN‐alpha for chronic hepatitis C in a patient diagnosed with haemophilia A.Citation12 Schreiber and Bräu reported a case of FVIII inhibitor development in a patient coinfected with HIV/HCV after prolonged therapy with PEG IFN‐alpha 2a plus ribavirin. Acquired FVIII inhibitors have also rarely arisen in treatment‐naive HCV positive patients.Citation13 Dentale et al. reported a case of severe FVIII deficiency due to an acquired inhibitor in a nonhaemophilic patient with acute HCV infection who developed haemorrhagic manifestations and prolonged activated partial thromboplastin time (aPTT).Citation14 Another study reported severe haemorrhage attributable to an acquired FVII inhibitor in a non‐haemophilic patient with chronic HCV infection.Citation15 In the present report, we describe the development of factors VIII and IX inhibitors in a patient with chronic HCV infection while receiving antiviral treatment with pegylated interferon (PEG IFN)‐alpha 2b plus ribavirin (RBV).
Data Sources and Search Strategy
We performed a computer‐generated search in the English language literature using electronic databases such as MEDLINE (1966–1996 and 1997–2010), PubMed, and ISI Web of Knowledge. The following search terms were used: Factor VIII AND/OR Factor IX AND/OR Inhibitor AND/OR HCV; Factor VIII AND/OR Factor IX AND/OR Inhibitor AND/OR IFN, and relevant literature identified through these databases was selected. In addition, we searched for additional references in the reference lists of all selected studies.
Case Report
Description
A previously healthy 52‐year‐old man presented as an outpatient at Gaffrée & Guinle University Hospital Liver Disease Unit, in August 2001, for therapeutic evaluation after testing positive for serum anti‐HCV antibodies and HCV‐RNA. He had a history of recreational intravenousl drug abuse over 30 years as an identifiable risk factor for HCV infection. His past medical history was unremarkable, except for a few skin lesions attributable to scalp discoid lupus erythematosus (DLE) and patches of vitiligo. On physical examination, the patient was a well‐nourished man with no stigmata of chronic liver disease, nor occult malignancy. Complete blood cell counts, prothrombin time (PT), aPTT, clotting time, bleeding time, bilirubin, albumin, thyroid tests (free thyroxine [FT4], thyroid‐stimulating hormone [TSH]), and creatinine were normal, as determined by routine laboratory methods. Serum aminotransferase values were as follows: alanine aminotransferase (ALT), 65·2 U/l (normal range, ⩽46 U/l), and aspartate aminotransferase (AST), 36·0 U/l (normal range, ⩽50 U/l).
An abdominal ultrasound excluded portal hypertension and advanced chronic liver disease with no evidence of varicies upon oesophageal‐gastroduodenoscopy. HCV‐RNA was detected by reverse transcription‐nested polymerase chain reaction (RT‐nested PCR) with primers against the 5’ noncoding region in serum, with a lower limit of detection estimated as 50 IU/ml.Citation16 The patient was demonstrated to be infected with HCV genotype 1b, as determined by using the Versant HCV Genotype 2·0 Assay (LiPA 2·0; Innogenetics, Ghent, Belgium).Citation17 From 16 August 2001 to 22 November 2003, the patient was under clinical follow‐up only, as he initially presented with fluctuating aminotransferase (ALT and AST) levels and did not give informed consent to undergo a liver biopsy as part of the pretreatment investigation for chronic HCV infection until week 144 post‐diagnosis. Liver biopsy at that time revealed mild necroinflammatory (histological activity) and fibrosis (portal fibrosis without septa) lesions, besides mild and focal steatosis (A1 F1, according to the METAVIR scoring system).Citation18 No bleeding or other signs of coagulopathy were reported during the pretreatment follow‐up. The patient’s laboratory evaluation as part of the pretreatment investigation for chronic HCV infection is shown in .
Table 1. Patient’s laboratory characteristics during pretreatment evaluation
At week 168 post‐diagnosis, the patient was started on a treatment with PEG IFN‐alpha 2b administered subcutaneously at a total dose of 112 μg weekly plus RBV administered orally at a dose of 1000 mg/day in divided doses (500 mg twice daily) for 48 weeks. Predictable, mild to moderate drug‐related side effects were reported, including fever, headache, malaise, myalgia (flu‐like), – all of them ameliorated by acetaminophen –, poor appetite, weight loss, fatigue and apathy. At week 24 of antiviral therapy, ALT and AST levels were normal and serum HCV‐RNA had become undetectable by using our qualitative RT‐PCR assay. The antiviral therapy had been relatively well tolerated, despite some degree of worsening of DLE skin lesions. At week 28 of antiviral therapy, his routine coagulation tests showed a prolonged aPTT (60 s; normal, 25–35 s) as well as abnormal thyroid tests (FT4 0·29 ng/dl, normal range 0·8–1·9 ng/dl; TSH 47·27 μIU/ml, normal range 0·3–5·0 μIU/ml; antithyroperoxydase antibodies [TPO‐Ab] 234 U/ml, normal range ⩽34·0 U/ml; and antithyroglobulin antibodies [Tg‐Ab]>2000 U/ml, normal range ⩽115·0 U/ml), indicating an autoimmune thyroid disorder, and short‐term thyroid replacement was required. After 34 weeks into antiviral therapy at his last visit with the hepatologist, the patient reported the development of a large (4–5 cm) soft‐tissue haematoma covering his left lower limb, in association with a greater prolongation in his aPTT (113·6 s), and was referred for haematological evaluation.
Haematological evaluation
While waiting for haematological evaluation the patient went to the otorhinolaringologist who had diagnosed a spontaneous, nontraumatic oropharangeal blood clot. The patient was reviewed at our clinic at week 37 of anti‐viral therapy. He denied any personal or family history of bleeding. On physical examination, the patient had generalized ecchymoses. At that time, his laboratory findings were: haemoglobin 11·2 g/dl, haematocrit 34·3%, white blood cell (WBC) count 3800/mm3, platelet count 278 000/mm3, fibrinogen 448 mg/dl, PT 9·8 s (128·7%, INR 0·86), and a prolonged aPTT (74 s; patient/control plasma ratio, 2·47), partially corrected with normal plasma. Further laboratory investigation was negative for cryoglobulins, rheumatoid factor, anti‐cardiolipin antibodies, lupus anticoagulant (coagulometric assay), and antinuclear antibodies. Serum immunoglobulin levels and protein electrophoresis, erythrocyte sedimentation rate (Westergreen), bleeding time, and platelet aggregation were normal. FVIII, FIX, and vWF antigen activities were at 2% (normal, 60–150%), 10% (normal, 60–150%), and 327% (normal, 50–150%), respectively. Determination of FVIII and FIX inhibitors levels was performed using the Bethesda assay,Citation19 and showed the following values: FVIII 4096·0 Bethesda Units/dl, and FIX 62·9 Bethesda Units/dl (negative or <0·5 as a reference value). Additional clotting factors assays were not performed.
Therapy and outcome
Based on these findings, the diagnosis of FVIII and FIX deficiencies due to the presence of acquired inhibitors was made. At that time (41th week of antiviral therapy), the dose of PEG IFN‐alpha 2b was reduced to 80 μg weekly upon haematologist’s recommendation. Despite counselling, the patient declined immunosuppressive therapy. Thus, antifibrinolytic therapy (oral tranexamic acid 250 mg every six hours) was initiated along with additional supportive care to control his haemorrhagic manifestations while on treatment for chronic hepatitis C. Skin and mucosal lesions as well as his pain had slightly improved after week 5 of antifibrinolytic therapy. At the end of 48 weeks of antiviral therapy and six months after its completion, serum HCV‐RNA was undetectable. Skin lesions enhancement associated with DLE had also diminished with time. The patient’s hypothyroidism resolved; however, antithyroid antibodies were still detected in high titres even 10 months after stopping antiviral therapy. At week 30 after stopping antiviral therapy, our patient presented with multiple haemorrhagic manifestations including epistaxis, macroscopic haematuria, haematoma in limbs, petechiae and abdominal wall ecchymosis. He consented to start immunosuppressive therapy with prednisone 60 mg/day and azathioprine 100 mg/day. The six months follow‐up thereafter had revealed the patient in good health, without any clinical sign of the acquired bleeding disorder, and immunosuppressive therapy tapering was started. FIX and FVIII levels start to normalize at week 48 and 240 after completion of antiviral therapy, respectively. In remission from the inhibitory antibodies, 17 months from the beginning of immunosuppressive therapy, the immunosuppressive drugs were tapered and stopped. Review 27 months after cessation of immunosuppression revealed the patient was asymptomatic and his screening coagulation tests were as follows: PT 9·0 s (100·0%, INR 1·0); aPTT, 31·1 s (patient/control plasma ratio, 0·95); PLT, 253 000/mm3, normal levels of FVIII and FIX, and FVIII and FIX inhibitors levels <0·5 Bethesda U/dl. The main clinical and laboratory features of the patient before, during, and after completion of antiviral therapy are summarized in Table 2.
Table 2. Patient’s laboratory characteristics during and after completion of antiviral therapy with pegylated interferon plus ribavirin
Discussion
Inhibitors against FVIII and FIX are mostly observed in patients with classical hereditary haemophilia after receiving replacement factor therapy, with an estimated frequency of 15–30 and 3–5%, respectively.Citation20 However, the development of acquired inhibitors against FVIII in nonhaemophilic patients is a rare condition, and inhibitors against other blood clotting factors such as FIX even rarer.Citation9 In this report, we describe a case of a man with chronic HCV infection who developed FVIII and FIX deficiencies due to the presence of inhibitors against these two clotting factors while being treated with PEG IFN‐alpha 2b plus RBV. Although acquisition of a factor VIII inhibitor during the course of acute HCV infection or during IFN‐alpha therapy for chronic HCV infection has been previously reported in a few studies,Citation12–Citation15 to the best of our knowledge, this is the first report of simultaneous occurrence of acquired FVIII and FIX inhibitors in a nonhaemophilic patient receiving antiviral therapy for chronic hepatitis C.
Patients with acquired haemophilia classically present with a different bleeding pattern to inhibitors of classical haemophilia. Although muscle bleeds occur, joints are generally spared, and skin and/or mucous membranes of gastrointestinal and genitourinary tracts predominate.Citation3 The presented case was characterized by such a pattern. The haemorrhagic syndrome related to the presence of acquired inhibitors developing in patients with HCV‐related hepatic disease may represent a diagnostic and therapeutic challenge to practising clinicians, particularly if concurrent thrombocytopenia relating to treatment or underlying liver disease.
The interplay between the immune mediated effects on host cells and the effects of the virus itself may lead to consequences affecting the host immune response, which are not completely known.Citation21 The immunological dysregulation in hepatitis C and associated treatment effects, may result in autoimmune phenomenon, but the mechanisms involved in the development of inhibitors against FVIII/FIX are not well understood. FVIII inhibitors are known to bind specifically to highly antigenic regions located on the FVIII molecule, mainly on its A2, A3, and C2 domains. Moreover, the synthesis of these inhibitors was demonstrated to require CD4+ (helper) T‐cells specific for FVIII.Citation22,Citation23 The diagnosis of FVIII/FIX inhibitors is often a challenging problem in the clinical laboratory investigation, since problems by using standard assays, including lack of adequate assay accuracy, are still unresolved issues.Citation24 In our case report, we have used normal plasma buffering with Owren’s Veronal buffer (Biomerieux/Organon Teknika Corp., Durham, NC, USA) to pH 7·35 in both patient and control mixtures, besides adding factor VIII or IX deficient substrate plasmas (Biomerieux/Organon Teknika Corp.) after 2 hours incubation at 37°C in an attempt to circumvent these issues. Autoantibodies against FVIII are recognized to inhibit FVIII in the FIX assay, and could result in an artefactual, borderline FIX deficiency.Citation1 The presence of inhibitors to FVIII and/or FIX typically results in an isolated prolonged aPTT with no effect on other screening coagulation tests.Citation25 To date, combination therapy with IFN‐alpha or pegylated IFN‐alpha plus ribavirin is the standard therapy for chronic HCV infection, and new therapeutic approaches to HCV infection are ongoing with the development of novel compounds known as specifically targeted antiviral therapy for HCV (STAT‐C).Citation26,Citation27 A myriad of side effects of both IFNs have been described, including the induction of autoantibodies and/or exacerbation of autoimmune disorders.Citation28,Citation29 Some side effects of IFN‐alpha usually do not require dose modification, and once established, discontinuation of antiviral therapy is not always associated with disappearance of the autoimmune maniffestations.Citation30–Citation32 In our patient, there have been no alterations in screening haemostasis tests before and over the first six months of the treatment with PEG IFN‐alpha 2b, thus suggesting that this drug is involved, at least in part, in the process that leads to the development of this autoimmune abnormality.
Therapy with immunosuppressive agents such as corticosteroids and/or cytotoxic drugs remains the mainstay of eradication therapy for the inhibitory autoantibodies in patients with acquired haemophilia.Citation1 Our patient was treated with antifibrinolytic agents to control his haemorrhagic manifestations while on treatment for chronic hepatitis C, as he did not give consent to undergo immunosuppressive therapy. Because the latter could influence the rate of achieving sustained virological response,Citation33 the patient received oral corticosteroids (prednisone) added to azathioprine just six months after completion of antiviral therapy, when he presented with HCV‐RNA undetectable in serum, as a means of decreasing the titres of inhibitor autoantibodies.
Inhibitory antibodies confer refractoriness to replacement therapy with FVIII and FIX. Bypassing agents, such as activated recombinant human factor VII (rFVIIa)Citation6,Citation34 and FVIII inhibitor bypassing activity (FEIBA), have haemostatic activity in the presence of inhibitory antibodiesCitation35 However, according to Brazilian Ministry of Healthy guidelines, the use of rFVIIa was not available for our patient, being a non‐haemophilic patient attending a Public Health Unit. Concerning FEIBA, physicians, patients, and medical care units are first registered in a Brazilian database, while waiting for receiving this product. This patient did not receive any FEIBA.
In conclusion, the development of these inhibitors in association with antiviral therapy for chronic hepatitis C is poorly understood, and particular attention must be given to HCV‐infected patients with this complication because of its seriousness. This report suggests the possibility that, in this case, the treatment with pegylated IFN could have induced the development of FVIII and FIX inhibitors.
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