Instrumented fusion of thoracolumbar fracture with type I mineralized collagen matrix combined with autogenous bone marrow as a bone graft substitute : a four-case report In order to avoid the morbidity from autogenous bone harvesting , bone graft substitutes are being used more frequently in spinal surgery . There is indirect radiological evidence that bone graft substitutes are efficacious in humans . The purpose of this four-case study was to visually , manually , and histologically assess the quality of a fusion mass produced by a collagen hydroxyapatite scaffold impregnated with autologous bone marrow aspirate for posterolateral fusion . Four patients sustained an acute thoracolumbar fracture and were treated by short posterior segment fusion using the AO fixateur interne . Autologous bone marrow -LRB- iliac crest -RRB- impregnated hydroxyapatite-collagen scaffold was laid on the decorticated posterior elements . Routine implant removal was performed after a mean of 15.3 months -LRB- 12 -- 20 -RRB- . During this second surgery , fusion mass was assessed visually and manually . A bone biopsy was sent for histological analysis of all four cases . Fusion was confirmed in all four patients intraoperatively and sagittal stress testing confirmed mechanical adequacy of the fusion mass . Three out of the four -LRB- cases 2 -- 4 -RRB- had their implants removed between 12 and 15 months after the index surgery . All their histological cuts showed evidence of newly formed bone and presence of active membranous and/or enchondral ossification foci . The last patient -LRB- case 1 -RRB- underwent implant removal at 20 months and his histological cuts showed mature bone , but no active ossification foci . This four-case report suggests that the fusion mass produced by a mineralized collagen matrix graft soaked in aspirated bone marrow is histologically and mechanically adequate in a thoracolumbar fracture model . A larger patient series and/or randomized controlled studies are warranted to confirm these initial results . Introduction Effectiveness of a bone graft can be described as having three core properties : osteoinductivity , osteoconductivity and osteogenicity -LSB- 25 , 28 -RSB- . Autogenous bone graft is the only biological structure simultaneously possessing all three properties . However , the long-term morbidity due to autogenous bone harvesting can be as high as 30 % , although improved technique has probably lowered the incidence -LSB- 1 , 2 , 10 , 26 -RSB- . Allograft bone is the primary alternative to autograft for a number of spinal fusion procedures . However , allograft bone is a poor posterior onlay graft with rates of fusion reported to be consistently lower than autograft except in cases of pediatric deformity -LSB- 4 , 12 , 17 , 18 -RSB- . Therefore , there has been an increasing shift in the past decade to the use of bone graft substitutes for spinal fusion . One commercially available bone graft substitute comprised a Type I mineralized collagen matrix -LRB- MCM -RRB- , coated with hydroxyapatite -LRB- Healos ® Bone Graft Substitute , DePuy Spine , Inc , Raynham , MA , USA -RRB- . Kraiwattanapong et al. -LSB- 21 -RSB- reported a 0 % fusion rate for posterolateral fusion with MCM and bone marrow in rabbits . However , in that study , bone marrow was harvested from the iliac crest which is not a rich source of osteoprogenitor cells in rabbits . Despite these unique findings , the efficacy of this bone graft substitute has been demonstrated in a previous rabbit study of posterolateral fusion preformed by Tay et al. -LSB- 29 -RSB- harvesting bone marrow from rabbit tibiae , with a fusion rate of 100 % . Autogenous bone marrow aspirate has been successfully used in the treatment of congenital and post-traumatic pseudarthrosis -LSB- 7 , 11 , 28 -RSB- . The effectiveness of this method has been further enhanced by the use of a three-dimensional scaffold which avoids wash out of osteogenic bone marrow cells by blood circulation . In humans , Kitchel has recently reported two lumbar posterolateral fusion studies -LSB- 19 , 20 -RSB- demonstrating equivalent rates of fusion for MCM to autograft using CT scans for assessment . The current understanding of fracture healing is primarily based on animal histological and radiological studies . The majority of human studies discussing fusion rates are based on radiological fusion criteria which are not completely reliable , even when CT scans are utilized -LSB- 6 -RSB- . In a 1993 study involving spinal implant removal following fusion , Blumenthal et al. -LSB- 3 -RSB- found an overall agreement between preoperative radiographs and surgical findings in only 69 % of the cases . Brodsky et al. -LSB- 5 -RSB- found a similar overall agreement in their study published in 1991 . The ultimate method to assess the quality of a fusion is perioperative manual palpation and histological analysis of a bone biopsy following implant removal . There are no reports in the literature of the histological behavior of this bone graft substitute in humans . We present here our findings of radiological and histological results on four patients that had surgery performed for an acute thoracolumbar fracture without neurologic deficit . Materials and methods Five consecutive patients underwent surgical management for an acute vertebral fracture of the thoracolumbar spine without neurologic deficit . There were three females and two male patients , and the mean age was 47 years -LRB- range 30 -- 64 -RRB- . Three patients sustained their fracture after a fall from a horse , one patient after a fall from a scaffold , the last after a fall from a tree . All patients were initially admitted to our institution , which is a level one trauma facility . Initial management was applied according to Advanced Trauma Life Support -LRB- ATLS ® -RRB- guidelines . Four out of the five patients were diagnosed with no other lesion than the vertebral fracture . The patient who fell from a scaffold was also diagnosed with pulmonary contusions and severe tibial pilon fractures , open type IIIA according to Gustilo 's classification -LSB- 13 , 14 -RSB- . All patients were worked-up under standard hospital protocol which included plain anteroposterior and lateral radiographs as well as a CT scan of the spine . The AO classification was used to characterize the type of fracture in each patient -LSB- 23 -RSB- . Three patients sustained an L1 fracture : two patients had a type A2 .3 -LRB- Burst-split -RRB- , one patient had a type B1 .2 fracture -LRB- flexion-distraction -RRB- . One patient sustained a type C2 .1 fracture -LRB- flexion-distraction with rotation -RRB- of T12 . The fifth patient sustained a type A3 .1 fracture of L2 -LRB- lateral burst -RRB- . All patients were conscious and given the choice between non-surgical and surgical management of their fracture after discussing with each of them the advantages and disadvantages of each treatment method , and alternatives . All patients signed an informed written consent to be included in this pilot study . All surgeries were performed by two fellowship trained surgeons -LRB- AAF and AJK -RRB- . Surgical technique The short segment fusion-stabilization technique as described originally by Lindsey and Dick -LSB- 9 , 22 -RSB- was utilized . The spine was approached posteriorly with a midline incision . Musculature was detached subperiosteally and the fracture was exposed posteriorly , both inferiorly and superiorly , to immediately adjacent vertebrae , taking care to preserve the facet joints of the intact segment . Following this , pedicle screws were placed in one vertebra above and one vertebra below the fracture under fluoroscopic control . Whenever possible , the authors attempted to stabilize -LRB- and fuse -RRB- only one segment if pedicle screws could be placed in the fractured vertebra , typically hemiburst fractures -LRB- AO classification type A3 .1 -RRB- where the inferior part of the vertebral body is primarily intact . This was not possible in either of the patients with type A fractures in this study . Following pedicle screw placement , careful decortication of the facet joints and posterior arch of the fractured vertebra was performed . Typically , transverse processes are not decorticated unless a laminectomy to achieve decompression is necessary . Percutaneous aspiration of 10 cc of autogenous bone marrow from the posterior iliac crest was performed . Two 5 cc rectangles of the MCM graft were saturated with the bone marrow aspirate and laid bilaterally on the posterior elements previously decorticated . No other bone , bone graft extender , or substitute was placed as part of the graft . In the patient who sustained a lateral burst fracture , we performed a transpedicular bone grafting of the vertebral body , but no other graft substitute other than Healos ® was used for the posterior fusion stabilization . Rods were connected to the pedicle screws and fracture reduction in the sagittal plane , and when necessary coronal plane , was achieved by means of ligamentotaxis through the `` fixateur interne '' -LRB- USS ® : Universal Spine System , Synthes Spine , Paoli , PA , USA -RRB- as described by Walter Dick -LSB- 9 , 22 -RSB- . Standard procedure with this instrumentation is to remove it at 12 months following surgery when radiological and clinical healing is evident . There are two major reasons for this : -LRB- 1 -RRB- implant removal allows the fixed but non-fused segment to recover some mobility which may protect the adjacent segment from accelerated degeneration ; -LRB- 2 -RRB- in general , after the fracture is radiologically healed , patients experience some discomfort when trying to increase their physical activity . Patients clearly differentiate this discomfort from the pain they had from the fracture and during the healing period . The USS system is in fact a very bulky implant , which the authors believe , is eventually responsible for this discomfort . Due to surgeon and patient availability , the time to instrumentation removal is often variable , but always performed at a minimum of 12 months following the index surgery at our institution . When radiological and clinical healing was achieved , implant removal was performed in four patients . The fifth patient declined implant removal following healing of the fracture because she did not experience any discomfort . Technically , the second surgery required a very short general anesthesia and lasted approximately 30 min from skin incision to skin suture . During the second surgery , the mechanical stability of the fused segment was tested using thin curettes introduced in the pedicle screw tracts and manual stress was applied sagittally . A bone biopsy was performed in the heart of the fusion mass which was clearly distinguishable from the lamina and sent for histological analysis . Biopsies were sent in formalin to the institution 's pathology department . They were then decalcified in formic acid , embedded in paraffin and cut in 3-μm slices with a microtome . They were further colored with hematoxylin -- eosin and analyzed under a microscope at magnifications ranging from 20 × to 400 × . Radiological assessment Anteroposterior and lateral radiographs were taken in the supine position immediately after surgery , in the standing position a few days after surgery , then at approximately 6 weeks , 3 , 6 , and 12 months following the index surgery , as well as following implant removal . Assessment of healing radiographically was based only on the remodeling of the vertebral body . Overall fusion assessment was based on radiographic healing of the vertebral body as well as patient pain , function , and activity level . Fusion of the posterior elements could not be assessed radiologically . CT scan was not used because it would not have been possible to assess fusion status of the posterior elements due to metallic artifacts from implants . When smoothening of bony edges and some sclerosis of the fractured vertebral body became visible , the fracture was considered to be healed . This was also based on the patient 's subjective appreciation . When fracture and surgery pain regressed and the patient felt he or she could go back to almost all previous all day activity , the fracture was considered clinically healed . No specific clinical outcome scale was used in this limited patient sample . Results The four patients who had the secondary surgery underwent implant removal after a mean period of 15.3 months from the index surgery -LRB- 12 -- 20 -RRB- . All patients had returned to their previous activity at latest follow-up , except one who was involved in worker 's compensation litigation . During the second surgery , we visually confirmed that the resorbable matrix had been replaced by new bone -LRB- Fig. 1 -RRB- and that this new bone had formed at the location where the MCM graft had been laid . Manual palpation and sagittal stress through curettes placed in the pedicle tracts demonstrated that no movement could be detected visually in any of the four patients . Fig. 1This 30-year-old female sustained an AO type C2 .1 fracture of T12 after a fall from a horse . Initial work-up in the ER did not show any other lesion . She was neurologically intact . We performed a T11-L1 posterior short segment fusion as previously described . Implant removal was performed 14 months following the index surgery . The biopsy demonstrated irregular bony trabeculae , foci of enchondral ossification and numerous osteoblasts , all characteristic elements of ongoing bone remodeling . Several aggregates of lymphoplasmocytes were observed and have been attributed to a foreign body inflammatory type of response . a Preoperative sagittal CT. b Preoperative lateral radiograph . c Lateral radiograph at 5 months postop . d Perioperative image of extensive new bone growth during the secondary surgery . e AP radiograph 15 months following the index surgery after implant removal . f Histology at 100 × magnification . g Histology at 400 × magnification Histological analysis of these four cases showed clear evidence of newly formed bone . Patients 1 , 3 , and 4 had implant removal performed between 12 and 15 months follow-up . Their histological cuts also showed several foci of active membranous and/or enchondral ossification . Interestingly , patient 2 had implant removal performed at 20 months of follow-up and though his histology cuts showed newly formed bone , he had no visible active ossification focus . Patient 3 had implant removal performed at 14 months . His histology showed newly formed bone and active ossification foci . Discussion Autogenous graft harvesting site morbidity is a significant issue , and the incidence may be underestimated -LSB- 15 -RSB- . Other substitutes such as hydroxyapatite -LSB- 30 -- 33 -RSB- and other ceramics have had variable results when used for spinal fusion or other orthopedic applications . Disease transmission through allograft though remote , still remains a possibility . The effectiveness of bone morphogenetic proteins -LRB- BMPs -RRB- has been shown in human clinical trials . But high cost , unknown potential long-term biological effects , and issues with carriers and dosing may alter widespread clinical use , at least in the short term -LSB- 16 , 27 -RSB- . Autogenous bone marrow aspirate has been successfully used in the treatment of congenital and post-traumatic pseudarthrosis -LSB- 7 , 8 , 11 , 25 , 28 -RSB- . In 1997 , Muschler et al. -LSB- 24 -RSB- studied the prevalence and concentration of osteoblastic progenitors in marrow aspirates from the anterior iliac crest of 32 patients without systemic disease . They determined the number of alkaline phosphatase-positive colony-forming units that grew after placing the bone-marrow derived cells into tissue-culture medium . Three important conclusions have been drawn from this study : -LRB- 1 -RRB- the authors estimated that 80 % of the cells found in the first 2 ml of aspirated bone marrow have a prevalence of alkaline phosphatase-positive colony-forming of one for 35,000 nucleated cells ; -LRB- 2 -RRB- the bone marrow derived cells concentration decreases with the volume of aspirate due to dilution ; -LRB- 3 -RRB- the cellularity of bone marrow and the prevalence of osteoblastic progenitor cells significantly differ between humans . The prevalence of osteogenic precursors cells , can be increased by centrifugation -LSB- 7 -RSB- or using a three-dimensional structure to which these cells can attach -LSB- 25 -RSB- . MCM is such a three-dimensional matrix . As described previously , implant removal was performed usually as soon as there was radiological evidence of vertebral body fracture healing and the patient could return to all-day activity , but not earlier than 12 months after surgery . Patient 2 , who suffered from a polytrauma , underwent repeat surgery for his open pilon fractures and was bound to a wheelchair for a long period . This is the reason why implant removal has been done later than in other patients , almost 2 years after index surgery -LRB- 20 months -RRB- . Conclusions We conclude that this study shows some evidence that this MCM graft may be a valid bone substitute for posterior or posterolateral spinal fusion under favorable conditions : presence of bone marrow cells , presence of healthy bleeding bone and mechanical stability -LRB- provided by the AO fixateur interne in these cases -RRB- . A larger patient series and/or randomized controlled studies are warranted to confirm these initial results .

O B O O B O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I I O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

thoracolumbar spine
fracture
fusion
bone graft substitute


Hepatitis C virus infection acquired in childhood Hepatitis C virus -LRB- HCV -RRB- infection occurs less frequently in children than in adult patients , and the natural history , prognosis , and clinical significance of HCV infection in children are poorly defined . We report here a descriptive follow-up of the clinical course , biochemical data , and viral markers observed in 37 children with anti-HCV . Ten patients included in the study tested persistently negative for serum HCV-RNA -LRB- group 1 -RRB- and 27 patients tested persistently positive -LRB- group 2 -RRB- . In group 1 , serum alanine aminotransferase -LRB- ALT -RRB- was normal in all patients , while two patients had non-organ-specific autoantibodies . In group 2 , serum ALT was elevated in 13 of 27 patients , and five patients had non-organ-specific autoantibodies . HCV genotype 1a and 1b were the most prevalent among HCV-RNA-positive patients . Twenty liver biopsies were carried out on 17 patients in our series -LRB- mean evolution time , 11.2 years ; range , 3 -- 21 years -RRB- . The liver specimens showed mild necroinflammatory changes in most patients , and fibrosis was absent or low grade . Two HCV-RNA-positive patients became persistently HCV-RNA negative . Of the 26 children investigated , 7 -LRB- one in group 1 , six in group 2 -RRB- had a co-infection with hepatitis G virus . Conclusion Most children chronically infected with HCV were asymptomatic and presented only mild biochemical evidence of hepatic injury . Autoimmunity in the form of non-organ-specific autoantibodies was common . HCV in children induced mild changes in the liver with a low level of fibrosis and at a low rate of progression . Introduction HCV -LRB- hepatitis C virus -RRB- infection occurs less frequently in children than in adult patients . The natural history , prognosis and clinical significance of HCV infection are poorly defined in childhood -LSB- 11 -RSB- ; in contrast , HCV infection in adults presents a high degree of chronicity , with up to 50 % of all HCV-infected adults developing progressive liver disease -LSB- 21 -RSB- . Results from prospective studies show that 20 -- 30 % of chronically infected adults develop compensated and eventually decompensated cirrhosis or hepatocellular carcinoma , or both , within 20 years of the initial infection -LSB- 1 -RSB- . The lower prevalence of HCV infection in children and the fact that most patients undergo antiviral drugs treatment result in very limited knowledge of the natural outcome of chronic HCV infection acquired at early ages of life . Thus , immediate goals in the investigation of HCV infection should be to characterize current epidemiology and to describe the pathogenesis and course of hepatitis C in children and adults -LSB- 20 -RSB- . To understand the evolution of HCV infection could be itself an important surrogate end-point for the evaluation of infected young patients with prognostic implications and therapeutical consequences . The aim of this study was to determine the clinical features and long-term evolution of HCV infection in a group of children who had never received treatment with antiviral drugs . Patients and methods Thirty-seven children -LRB- 16 females , 21 males -RRB- with positive antibodies to hepatitis C -LRB- anti-HCV -RRB- were investigated retrospectively . These patients were followed-up for a period of 5 years . None had received treatment with antiviral drugs for viral hepatitis or had a history of intravenous drug abuse . All subjects made regular visits to our outpatient clinic , and the serum levels of alanine aminotransferase -LRB- ALT -RRB- , albumin , prothrombin time , antinuclear antibodies -LRB- ANA -RRB- , anti-mitochondrial antibodies -LRB- AMA -RRB- , anti-smooth muscle antibodies -LRB- SMA -RRB- , liver-kidney anti-microsomal antibodies type I -LRB- LKM -RRB- , anti-gastric parietal cells antibodies -LRB- GPCA -RRB- , rheumatoid factor , thyroxine -LRB- T4 -RRB- , thyroid stimulating hormone -LRB- TSH -RRB- , anti-thyroid antibodies , anti-HCV and HCV-RNA were determined at least on five consecutive occasions at 1-year intervals . Hepatitis B virus -LRB- HBV -RRB- surface antigen -LRB- HBsAg -RRB- , antibodies to HBV surface antigen -LRB- anti-HBs -RRB- , antibodies to HBV core antigen -LRB- Anti-HBc -RRB- , human immunodeficiency virus -LRB- HIV -RRB- and antibodies to hepatitis E -LRB- anti-HEV -RRB- were investigated in all patients during one visit . Hepatitis G virus-RNA -LRB- HGV-RNA -RRB- and antibodies to HGV -LRB- anti-HGV -RRB- were determined in 26 patients . Genotypes of HCV were performed in 21 viremic children . The duration of infection was calculated as the interval between the presumed date of infection and the date of the last visit to the clinic . Liver biopsies were obtained in 17 patients . Repeated biopsies were performed in three patients . Viral markers for HBV , HCV and HIV were tested by third generation ELISA -LRB- Axsym ; Abbott Diagnostics , Chicago , Ill. -RRB- . Anti-HEV was detected by a commercially available ELISA -LRB- Bioelisa HEV IgG ; Biokit , Barcelona , Spain -RRB- . HGV-RNA and anti-HEV antibodies were detected using commercial tests -LRB- Roche Diagnostics , Mannheim , Germany -RRB- . HCV-RNA was detected by PCR -LRB- Amplicor HCV PCR test , Roche Diagnostics -RRB- , and HCV genotyping was performed by a second generation line probe assay -LRB- INNO-LIPA HCV ; Innogenetics , Ghent , Belgium -RRB- . Liver biopsies were performed percutaneously and under the ultrasound guidance of an experienced operator . All liver biopsy specimens -LRB- > 12 mm long -RRB- were fixed in formalin , embedded in paraffin stained with hematoxylin-eosin and Masson 's trichrome and then observed by a single experienced pathologist -LRB- AM -RRB- . Histological necroinflammatory activity and fibrosis were scored separately . The degree of histological necrosis with inflammatory activity was scored using the three parameters of the histological activity index -LRB- HAI -RRB- developed by Knodell et al. -LSB- 12 -RSB- ; these consist of : -LRB- 1 -RRB- piecemeal necrosis and bridging necrosis -LRB- score : 0 -- 10 -RRB- , -LRB- 2 -RRB- lobular cytolysis -LRB- score : 0 -- 4 -RRB- and -LRB- 3 -RRB- portal inflammation -LRB- score : 0 -- 4 -RRB- . Fibrosis was scored independently using the following system : F0 , absence of fibrosis ; F1 , fibrous expansion of portal areas ; F2 , portal to portal bridging fibrous tracts ; F3 , portal-central bridging fibrous septa ; F4 , cirrhosis -LRB- bridging fibrous septa with parenchymal nodules -RRB- . This score is a modification of the fibrosis scoring system of Knodell 's method and introduces one additional degree of fibrosis . Macrovesicular steatosis was graded semiquantitatively using a modified score from Brunt et al. -LSB- 5 -RSB- . Alternative and additional diagnoses identified in the liver biopsy were also recorded . Results Mean chronological age for the 37 patients -LRB- 21 males and 16 females -RRB- at the last follow-up evaluation was 20.1 years -LRB- range : 9 -- 30 years -RRB- . Most of the subjects were diagnosed among patients who were screened for HCV infection after receiving transfusions of blood products for heart surgery , hematological diseases or casual injuries before 1992 . Thus , most of the patients included in the study presented with other diseases that were unrelated to HCV infection : 16 patients had a congenital heart disease , seven had various hematological disorders , three had neurological diseases , one had vesicoureteral reflux and one had Turner syndrome associated to IgA deficiency . Ten patients were anti-HCV-positive and HCV-RNA-negative -LRB- Group 1 -RRB- . The presence of HCV-RNA was demonstrated in the other 27 patients -LRB- Group 2 -RRB- . Demographic characteristics and the main results obtained in these children are shown in Table 1 . Table 1Characteristics and biologic data of children with HCV infection Patients negative for HCV-RNA -LRB- n = 10 -RRB- Patients positive for HCV-RNA -LRB- n = 27 -RRB- Age -LRB- years -RRB- at the follow-up evaluationa18 -LRB- 9 -- 26 -RRB- 20 -LRB- 9 -- 30 -RRB- Duration of the HCV infection -LRB- years -RRB- in 36 patients at the follow-up evaluationa14 -LRB- 6 -- 23 -RRB- 16 -LRB- 5 -- 30 -RRB- Route of infection Parenteral1024 Vertical02 Unknown01Alanine aminotransferase -LRB- U/l -RRB- First visitLast visit Normal -LRB- n -RRB- 10714 ≤ 2 × UNL -LRB- n -RRB- b067 2 -- 4 × UNL -LRB- n -RRB- 055 > 4 × UNL -LRB- n -RRB- 091Non-organ specific antibodies Antinuclear antibody02 Anti-mitochondrial antibodies10 Anti-smooth muscle antibodies01 Liver-kidney anti-microsomal antibody type 111 Gastric parietal cell antibodies01HCV genotype 1a8 1b12 2a1 Unknown6HBV00HIV10HGV-RNA/Anti-HGV -LRB- n = 26 -RRB- 1/04/2Anti-HEV 00aValues are given as the mean , with the range presented in parenthesisbUNL , Upper normal level Group 1 The once-yearly PCR analysis revealed that ten patients had positive anti-HCV and negative viremia , which reflected an ongoing or past infection with HCV . These patients were asymptomatic , and the liver function tests remained normal in all but two who had been diagnosed with congestive heart failure ; the liver abnormalities of these two patients were normalized after their cardiac condition had improved . One female patient presented persistent positive AMA for 3 years , which then normalized and remained negative during the subsequent 9-year follow-up period . Throughout this period the patient was asymptomatic , and liver tests were normal . Two years after the laboratory tests were negative for antibodies , the patient presented with psoriasis . Another patient had persistent high titers -LRB- 1:620 -RRB- of anti-LKM . Fifteen years after acquiring the infection this patient lost the anti-HCV but the anti-LKM remained positive for two more years . During the follow-up this patient did not present liver abnormalities or other diseases . During the follow-up span of this study , another female patient became persistently anti-HCV negative 12 years after she was infected . At the time of writing , eight patients remain anti-HCV-positive and HCV-RNA-negative , and they show no evidence of liver disorders . Group 2 HCV-RNA was detectable in the blood of 27 patients , two of whom became HCV-RNA-negative . The age of exposure to the infection and the type of exposure could not be established in one patient ; following the detection of HCV in the blood serum , this patient was followed for 11 years and was positive for HCV up to and including the last follow-up visit . All patients , with one exception , were asymptomatic . The exception had been diagnosed with leukemia in 1987 and received transfusions of blood products on several occasions . She underwent chemotherapy and had a cholestatic hepatitis ; liver tests revealed persistent abnormalities . Anti-HCV and HCV-RNA were detected in 1991 . The patient presented portal hypertension and progressive liver failure , and when she was 8 years old , in 1993 , she received a liver transplant . The evolution of serum ALT levels is shown in Table 1 . Serum levels of albumin and prothrombin time were abnormal only in the patient who developed hepatic failure . The presence of non-organ-specific autoantibodies -LRB- NOSAs -RRB- was detected in five patients . One patient -LRB- genotype1a -RRB- diagnosed with a single ventricle and Fontan surgery developed persistent anti-LKM -LRB- titers 1:160 -- 1:620 -RRB- and the rheumatoid factor throughout the evolution of the disease . Liver biopsy could not be obtained in this patient . Another male patient -LRB- genotype 1b -RRB- had positive ANA for 2 years , which subsequently normalized . This patient had increased ALT , and the liver biopsy showed minimal necrosis with inflammatory changes -LRB- HAI : 2 -RRB- and no fibrosis . A third patient -LRB- genotype 1b -RRB- had persistent PGCA during her follow-up period -LRB- 10 years -RRB- , with slightly elevated ALT levels , mild necrosis with inflammatory changes -LRB- HAI : 4 -RRB- and minimal fibrosis -LRB- stage 1 -RRB- . A fourth patient -LRB- genotype1b -RRB- with AST levels greater than fourfold the upper normal level presented SMA during the last 2 years of her follow-up -LRB- HAI : 6 -RRB- . The patient diagnosed with Turner syndrome and IgA deficiency -LRB- genotype 1b -RRB- developed throughout the years a seronegative poli-articular rheumatoid arthritis , psoriasis and celiac disease . She had intermittently positive ANA . The levels of T4 and TSH were normal in all patients , and no patient developed antithyroid antibodies . Abdominal ultrasounds did not show significant abnormalities . Only the patient who presented cirrhosis had changes associated to portal hypertension . Liver biopsies were performed in viremic patients who presented altered hepatic function tests and for whom parental consent had been obtained . Seventeen patients underwent 20 liver biopsies . Mean evolution time of the infection at the time of biopsy was established in 16 patients -LRB- mean : 11.2 years ; range : 3 -- 21 years -RRB- . The results on necroinflammatory activity and fibrosis are shown in Table 2 . Table 2Histopathological features of patients positive for HCV-RNALiver biopsyPatients -LRB- n -RRB- Necroinflammatory activity 1 -- 26 3 -- 47 5 -- 64 6 -- 180Fibrosis F08 F16 F22 F30 F4 -LRB- Cirrhosis -RRB- 1Steatosis0 A second hepatic biopsy was performed in three patients after 5 , 12 and 14 years , respectively . The minimal necroinflammatory activity observed in the first biopsy specimens of these three children remained unchanged in the second biopsy . One of the patients progressed from F0 to F1 fibrosis . A second patient never presented with fibrosis , and both samples of the third patient had minimal fibrosis -LRB- F1 -RRB- . During the follow-up period , 2/27 patients became persistently HCV-RNA-negative -LRB- for 9 and 10 years , respectively -RRB- . Both patients showed minimal necrosis and inflammatory activity with minimal fibrosis -LRB- F1 -RRB- in the biopsy performed before HCV-RNA clearance . One patient of Group 1 was HIV-positive , while one patient of Group 1 and six patients of Group 2 were HGV-positive . Viral coinfections observed in both groups are expressed in Table 1 . Discussion In this study , chronic hepatitis C infection was silent in most of the children , and there was little biochemical evidence of liver disease in these patients . Only one girl with portal hypertension and liver failure developed symptoms related to the infection . Consequently , our data suggest that HCV infection may be currently underdiagnosed in children and , moreover , that young patients could become a potential source of infection . We therefore strongly recommend that children falling in risk groups be screened for HCV infection . Of the 37 pediatric patients in this series , seven -LRB- 19 % -RRB- had NOSAs . These antibodies are highly prevalent in subjects exposed to the HCV , and a positive test result for SMA , GPCA and ANA is part of the natural course of chronic HCV infection in adults and children -LSB- 7 -RSB- . Lenzi et al. -LSB- 13 -RSB- reported a higher prevalence of NOSAs in anti-HCV-positive adult patients than in normal controls -LRB- 25 vs. 6 % -RRB- . Stroffolini et al. -LSB- 22 -RSB- found positive NOSAs in 36.9 % of 502 subjects with HCV-RNA-positive chronic hepatitis . Muratori et al. -LSB- 16 -RSB- detected positive NOSAs in 16 of 47 children -LRB- 34 % -RRB- with chronic hepatitis C , and anti-LKM was present in 11 % of the children . Of the 37 children participating in our study who manifested past or active HCV infection , two had serum anti-LKM . This incidence is lower than that observed by Muratori et al. -LRB- 11 % -RRB- -LSB- 16 -RSB- and Bortolotti et al. -LRB- 10.3 % -RRB- -LSB- 4 -RSB- in children with HCV infection . A possible explanation for this finding is the higher age and longer mean duration of HCV infection in the patients included in our series in comparison to the children in the other two studies . The prevalence reported by Lenzi et al. -LRB- 1.3 % -LSB- 13 -RSB- -RRB- , Stroffolini et al. -LRB- 2.25 % -LSB- 22 -RSB- -RRB- and Reddy et al. -LRB- 0 % -LSB- 18 -RSB- -RRB- in adults support this hypothesis . Anti-LKM is an immunomarker of type 2 autoimmune hepatitis -LSB- 2 -RSB- that tends to present at younger ages and to affect mainly children . Our data suggest that age may be a factor contributing to the presence of anti-LKM1 in children infected with HCV . Moreover , we observed that one of our non-viremic patients had persistent antibodies after the test for anti-HCV became negative . Liver specimens from the children with hepatitis C showed mild necroinflammatory changes and a low level of fibrosis . In most patients , fibrosis was absent or low grade . Only one patient , as mentioned above , developed cirrhosis and liver failure over a period of 6 years . This leukemic patient had other additional risk factors for liver damage that may have affected this outcome . In this respect , Hoshiyama et al. -LSB- 9 -RSB- observed that chronic hepatitis C is more frequent among children with hepatitis C infection following blood transfusions for malignant disease than in patients with hepatitis C following blood transfusions for open heart surgery . We have compared our results with those obtained from other pediatric series -LSB- 6 , 8 , 9 , 23 -RSB- . Table 3 summarizes the data on fibrosis obtained from liver biopsies of 210 pediatric patients -LRB- above-mentioned studies -RRB- . Fibrosis was not detected or was low grade in 200 patients -LRB- 95 % -RRB- , and severe fibrosis or cirrhosis was detected in ten patients -LRB- 5 % -RRB- . Minola et al. -LSB- 15 -RSB- and Poynard et al. -LSB- 17 -RSB- demonstrated an inverse correlation between age at HCV infection and progression to cirrhosis that suggests that chronic HCV infection in childhood induces mild changes in the liver with a low level of fibrosis and a low rate of progression . However , it has been observed that mild chronic hepatitis C can be a progressive disease in adult patients -LSB- 3 , 19 -RSB- and that the long evolution of infection acquired in childhood could increase the risk of fibrosis . Thus , some children infected early in life , in whom chronic disease has a mild liver expression , might develop fibrosis in adulthood . Table 3Hepatic fibrosis in biopsies from children with HCV infection Vogt et al. -LSB- 23 -RSB- Hoshiyama et al. -LSB- 9 -RSB- Guido et al. -LSB- 8 -RSB- El-Raziky et al. -LSB- 6 -RSB- This reportNumber of patients173811226a17Mean duration of the HCV infection -LRB- year -RRB- 21.2 ± 4.67.1 ± 2.8 -LRB- n = 23 -RRB- 12.9 ± 3.1 -LRB- n = 15 -RRB- 8.04 ± 5.3Non-determined11.2 ± 5,6 FibrosisNo/low grade14/2b231525/8120 / 47/9Severe/cirrhosis0 / 1c005/11/1d0 / 1eaSome patients were co-infected with HBV and / or had secondary iron overloadbTwo patients with congestive heart failurecPatient with Anti-HBs , anti-HBc and anti-HEV antibodiesdThis 12-year-old cirrhotic patient was not thalassaemic and HBV markers were negativeeThis leukemic patient was infected with HCV and was also receiving chemotherapy concurrently It is noteworthy that 6/27 of the children with HCV infection in our series had a co-infection with HGV -LRB- HGV-RNA-positive -RRB- or had a past HGV infection -LRB- anti-HGV-positive -RRB- . This prevalence of HGV infection is higher than that found in healthy children -LRB- 6 % -RRB- in Spain -LSB- 10 -RSB- . These data suggest that HCV-infected children must be considered to be a risk group for HGV infection since HCV and HGV are both parenterally transmitted . Moreover , no differences were observed in biochemical and histological findings in HCV-infected children with and without a concomitant infection with HGV , which suggests that superimposed HGV infection does not influence the course of HCV infection . Conversely , our data do not support that HEV , which can be transmitted by a parenteral route -LSB- 24 -RSB- , is prevalent in children who had undergone blood transfusions -LRB- prevalence rate : 0 % -RRB- . However , this low value may result from the low prevalence of HEV infection in Spain -LRB- 2.8 % -RRB- -LSB- 14 -RSB- . Conclusion Most children chronically infected with HCV are asymptomatic and present only mild biochemical evidence of hepatic injury . Spontaneous clearing of the virus occurs occasionally . Autoantibodies are common in HCV patients . The natural history of chronic hepatitis C in children differs from that in adults since HCV infection is relatively benign , induces mild changes in the liver with a low level of fibrosis and a low rate of progression and is rarely associated with severe or decompensate liver disease .

B I I O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

anti-liver/kidney microsomal antibody
infectious hepatitis
liver fibrosis
nonorgan-specific autoantibodies