Dorsal root ganglionectomy for pseudotumor of the L3 dorsal root ganglion : a rare case and a rare treatment Dorsal root ganglia are oval enlargements on the dorsal nerve roots and contain the cell bodies of sensory neurons . Asymmetry of dorsal root ganglia may occur naturally , yet natural occurrence of gigantic dorsal root ganglion -LRB- DRG -RRB- is rare . The patient was 61-year-old woman who presented with atypical symptoms like neuropathic pain and urinary distention . Neuroimaging has shown left L3-4 far-lateral disc herniation and a gigantic L3 DRG . At surgery , the dural sheath of the ganglion had to be opened and a firm , yellow-colored abnormal tissue was exposed . The abnormal tissue considered to be a tumor of neural origin was gross totally excised and the patient 's symptoms ceased immediately after surgery . Histopathological examination of the specimen revealed nothing more than normal DRG morphology . At 4 months postoperatively , the patient is well with mild L3 hyperesthesia and hyperalgesia . Dural sheath opening in neurosurgery is not a routine practice . The sheath may need to be opened when surgeon suspects of a tumor , a free disc fragment and any inflammation within the ganglion . Operative morphology of a severely edematous but non-tumoral -LRB- pseudotumor -RRB- ganglion has not previously been documented . Introduction Small neoplastic lesions of the vertebral column and the spinal canal are often misdiagnosed as intervertebral disc disease owing to the commoner occurrence of the latter -LSB- 1 -RSB- . Rarely , a neoplastic lesion may coincide with a herniated intervertebral disc usually without a topographic relationship in between -LSB- 8 -RSB- . Concomittant appearance of a disk herniation and a neoplastic lesion compromising the same nerve root represent a unique situation -LSB- 1 -RSB- . We herein report the unique case of a pseudotumor of the L3 dorsal root ganglion -LRB- DRG -RRB- adjacent to a far-lateral L3-4 disc herniation . Case history A 61-year-old physician presented with excruciating pain in her left leg . The mild leg pain that has been around the left knee and around the posterior hip for some weeks suddenly became unbearable one afternoon . The patient came to emergency room 8 h after the onset of the pain . Upon further questioning , the patient confessed that she had not emptied her bladder since the pain started . Past medical history was positive for a distant episode of trigeminal neuralgia , cluster headache , and an episode of significant back and right leg pain 7 years ago and a recent pulmonary embolus . Past surgical history included total hysterectomy and bilateral salpingo-oopherectomy after a serious pelvic inflammatory disease and abscess formation within the uterine tube . In the emergency room , the patient was in extreme pain . Any movement or touch would exacerbate the pain . Right leg-raising test was positive at 60 and left leg-raising test was positive at 20 ° . Power was normal in both legs , including knee extension . Patellar and the ankle reflexes were normoactive bilaterally . Abdominal examination for a distended bladder was fruitless because of softened subcutaneous fat . Lumbar MR showed disc degeneration and mild bulging at L3-4 , L4-5 and L5-S1 levels yet at L3-4 level there was foraminal and a round extraforaminal disc herniation -LRB- Fig. 1a , b -RRB- . Lumbar MR scan also verified a fully distended bladder -LRB- Fig. 1c -RRB- . Considering vaque radiological findings in contrast to serious pain and urinary retention , the MR examination was extended to cover the whole cervico-thoracic and thoracic levels . There was no cord compression . Fig. 1Axial T1-weighted -LRB- a -RRB- and T2-weighted -LRB- b -RRB- MR scans through L3-4 interspace show an extraforaminal lesion with questionable continuity with the disc space . The lesion is isointense on both T1 - and T2-images . Please note that on T2-weighted axial image -LRB- b -RRB- , the contralateral dorsal root ganglion -LRB- DRG -RRB- shows intermediate to high signal . c Parasagittal MR scan show L3-4 disk herniation obliterating the left neural foramen anteroinferiorly.Yet the left L3 nerve is of same width as the roots one above or below . d Parasagittal MR scan 5 mm lateral to section c shows that the enlarged DRG -LRB- arrows -RRB- appears to have been stuck in the distal neural foramen . Please note that the fat plane around the DRG is totally obliterated The next morning , urinary retention re-occurred and an indwelling urinary catheter had to be placed . An ultrasound of the abdomen and pelvis was not contributory except identification of few benign adhesions secondary to previous pelvic surgery and a distended gall bladder . Meanwhile the left knee pain and the left sciatica got only a bit better but the patient described additional occasional electric shock like pain radiating medially from the left groin to the left knee . Lumbar computerized tomography -LRB- CT -RRB- to rule out a small bone chip originating from end-plate attached to a free disc fragment was done next . Axial CT scans showed the presence of a left-sided round hyperdense extraforaminal lesion at L3-4 level that was reported to be consistent with far-lateral disc herniation -LRB- Fig. 2a -RRB- . Coronal reformatted CT images better demonstrated the lateral extent of the disc herniation -LRB- Fig. 2b -RRB- . After an unsatisfactory 2-day narcotic treatment with codein 60 mg q6h , gabapentin 300 mg twice a day was started . On the third day of admission , a new pain emerged around the left inguinal area . By this time , the left patellar reflex diminished and left knee extension got 20 % weaker . IV steroid -LRB- 80 mg methyl prednisolon as the loading and 40 mg twice a day as the maintenance dose -RRB- was then started . Steroid alleviated a significant portion of the pain . Tapering steroid dose immediately caused return of intense pain , so surgery was considered as the last resort . Fig. 2Axial CT image through the lower end-plate of L3 -LRB- a -RRB- and coronal reformatted CT -LRB- b -RRB- image show a round hyperdense extraforaminal lesion consistent with far-lateral disc herniation The patient was informed to consent for the removal of far-lateral disc herniation at L3-4 level . After medial facetectomy and ligamentous removal , the left L4 nerve and the L3-4 disc space were exposed . The bulging disc was compressing the L4 root from its shoulder . After standard discectomy , the L4 root became mobile . Next , lateral disc space was emptied by angling the rongeur laterally . Yet , disc compression alone was far from explaining the dramatic clinical picture . After total removal of left L3 inferior facet and L4 superior facet and undercutting the L3 pedicle , the left L3 root was identified and traced laterally . L3 nerve was more or less normal sized yet L3 DRG was extremely different than normal . The DRG was 3 -- 4 times bigger than a normal DRG . The L3 root was totally immobile -LRB- Fig. 3a -RRB- . The DRG felt extremely gritty . The dural sheath of nerve root and the DRG was opened . A yellow-colored firm tissue came into view -LRB- Fig. 3b , c -RRB- . There was good cleavage from the dural sheath and the underlying septum . The healthy ventral rootlets were pushed anteriorly and inferiorly by the mass -LRB- Fig. 3d -RRB- . Overall , the mass lesion within the root sleeve was totally extirpated . Although no cerebrospinal fluid -LRB- CSF -RRB- was seen in the operative field , the dural sheath was sutured with interrupted prolene sutures and the suture was reinforced with tissue fibrin glue . On postoperative day 1 , the urinary catheter was removed and the patient emptied her bladder spontaneously . The original pain was totally gone . The left knee function returned to normal . On third postoperative day , she complained of mild hyperesthesia and hyperalgesia over the left knee which did not affect her daily life . At 4 months postoperatively , she is leading a normal life with gabapentin 300 mg twice a day . Fig. 3Operative microphotographs show that : a the left L3 root and the DRG are extremely swollen , b after opening the dural nerve sheath , the upper end of a yellow colored tumor is being dissected , c the lower end of the tumor is being dissected with better cleavage , d after the removal of the tumor normal appearing ventral motor rootlets come into view , e root size return to normal after suture closure of the dural sheath -LRB- black arrows -RRB- The histopathological specimen was extensively studied and did not reveal anything other than normal DRG tissue with mature ganglion cells , axons and Schwannian stroma supporting the axons -LRB- Fig. 4 -RRB- . Fig. 4Photomicrographs a with hematoxylin and eosin and b with neurofilament -LRB- NF -RRB- protein immuno-stain demonstrate axons -LRB- left -RRB- and ganglion cells dispersed in a cellular stroma consisting of bundles of elongated spindle cells -LRB- right -RRB- -LRB- × 200 original magnification -RRB- . This represents normal DRG morphology Discussion Dorsal root ganglia are oval enlargements on the dorsal nerve roots and contain the cell bodies of sensory neurons . Although both the DRG and the ventral motor root are surrounded by a common dural sheath , a thin fibrous septum separates both . The DRG are surrounded by fat within the intervertebral foramen . According to the baseline anatomic information that Hasegawa et al. -LSB- 5 -RSB- obtained from male volenteers using MRI , the mean width , length and height is 5.7 × 7.1 × 7.3 mm3 for L3 DRG , 6.2 × 8.4 × 8.2 mm3 for L4 DRG , 5.9 × 9.4 × 8.3 mm3 for L5 DRG and 6.2 × 11.2 mm2 for S1 DRG , respectively -LRB- data on the height of S1 DRG was not given in this study -RRB- . The lower was the spinal level , the bigger was the ganglion size . In that study , the DRGs were located in lumbar intervertebral foramen in 92 % of L1 , 98 % of L2 , 100 % of L3 and L4 and 95 % of L5 levels . The S1 DRG was intraspinal in 79 % . However analyzing patients presenting with low-back pain or sciatica , Hamanishi and Tanaka -LSB- 4 -RSB- found that 100 % of L2 DRG , 48 % of L3 DRG , 27 % of L4 DRG and 12 % of L5 DRG were located extraforaminally while 52 % of L3 DRG , 72 % of L4 DRG and 75 % of L5 DRG were located intraforaminally . Preoperative radiological diagnosis of spinal nerve tumors producing radiculopathy or masquerading as spinal and/or root pain has been facilitated by combined use of CT and MR. Intradural extramedullary tumors like schwannoma or extradural tumors like neurofibroma frequently affect the DRG and appear as dumbbell-shaped in 15 % of cases -LSB- 10 , 14 -RSB- . Diagnosis of tumors that had already enlarged the neural foramen is usually straightforward . However , a smaller pathology intrinsic to the root or the DRG will first be expected to cause an asymmetry as also depicted by MR scans in our patient . Yet asymmetry of DRGs may also occur naturally -LSB- 4 -RSB- . Hamanishi and Tanaka -LSB- 4 -RSB- found asymmetrical locations and sizes in 11 % of the reviewed DRGs . Asymmetry at L3-4 level is rarer . Out of 104 patients that they reviewed , only one had DRG asymmetry at L3-4 level . Two cases who had asymmetrically gigantic DRGs similar to our case underwent surgery at different spinal levels -LSB- 4 -RSB- . In the first case , the lowered pedicle appeared to push down and kink the intraspinal portion of the L5 DRG . In the second case , the mass mimicking the gigantic DRG was histologically confirmed to be very old sequesterated nucleus pulposus at L5-S1 level . However , anteriorly compressed DRG could not be detected by MR scan at all -LSB- 4 -RSB- . As for MR signal intensity , schwannomas and neurofibromas will often appear iso - to hypointense on T1 - and hyperintense on T2-weighted MR images -LSB- 14 -RSB- . The left L3 DRG in our case appeared isointense on both T1 - and T2-weighted MR images . Intermediate signal on T2-weighted MR image usually suggests the presence of a tumor with numerous cellular and fibrous components and very little myxoid stroma . Unfortunately , paramagnetic contrast was not given in our case . One can argue that the preoperative radiological diagnosis of a tumor could have been possible if intravenous gadolinium was given . Although gadolinium enhanced MR imaging allows for better detection of intraneural edema with diffuse enhancement along the involved nerve roots , this characteristic probably would not have helped us in diagnosing a DRG tumor since DRG always enhance with contrast medium because of a less developed blood -- nerve barrier anyway . Recently , a French group studying enhancement pattern on 180 healthy DRG also concluded that 100 % demonstrated significant and homogenous enhancement -LSB- 3 -RSB- . Therefore , even if we had used intravenous paramagnetic contrast preoperatively , it would probably be impossible to differentiate between an intraradicular tumor and a healthy DRG . It was only at surgery that the left L3 DRG was found to be extremely swollen and had to be opened . A nerve root and a DRG may become swollen because of both biochemical irritation of nucleus pulposus and the mechanical compression of the disc materal -LSB- 2 , 6 -RSB- . The mechanical compression of the DRG can increase the endoneural fluid pressure within the DRG almost threefolds -LSB- 11 -RSB- . Yet since the dural sheath is not an elastic tissue , a DRG can hardly ever reach the gigantic size that we have observed . Morever , the far-lateral disc protrusion at L3-4 level observed at surgery -LRB- in contrast to the extent of the herniation as seen on CT and MR images -RRB- could not have exerted the force necessary to cause a significant endoneural fluid pressure rise within the left L3 DRG . Therefore , the adjacent far-lateral disc herniation probably just coexisted with the gigantic left L3 DRG of unknown origin and served a better purpose by attracting our surgical attention to this part of the spine . Not only the size of the involved DRG , but also its firm feeling suggested the presence of something unusual inside it . Although intraradicular disc herniations occur more commonly at L5-S1 and L4-5 levels -LSB- 12 -RSB- , the surgeon 's impression before the microsurgical opening of the dural sheath was more inclined to a rare intradicular disc herniation rather than a tumor . Dural opening in similar cases totally depend on surgeon 's experience and/or attitude or on the medicolegal restrictions of the preoperative consenting procedure . By opening the dural sheath of a nerve root or a DRG the surgeon not only will prolong the operative procedure but also will take the morbidity risk associated with CSF leak and difficulty with wound healing . Moreover , extra expense of tissue fibrin glue and/or external lumbar drainage may also trigger an hesitation before dural opening in the currently managed health care systems . If a gross pathology like a tumor or a disc fragment is not readily encountered between the rootlets , chances are histopathology will be the only diagnostic tool . In addition to a real mass lesion like tumor or disc material , inflammatory reactions within a DRG -- although very rare -- may also cause a tumor-like lesion . A team from Tübingen , Germany recently has reported a very similar case presenting with low back pain and progressive hypesthesia of the thigh -LSB- 10 -RSB- . MR imaging with contrast enhancement revealed an intraspinal -- extradural mass at L1-2 level compressing the right L2 root . The surgical part of their case is almost identical to ours . The surgical team emptied the disc and the nerve was still compressed and appeared to be enlarged . In the end , they had to open the dural sheath and resect the indurated and swollen part of the ganglion . Histopathological diagnosis was ganglionitis characterized by loss of ganglion cells , increased number of nodules of Nagcotte and diffuse infiltration of T cells . Ganglionectomy as performed for cancer pain or non-cancer pain -LRB- for failed back surgery syndrome or chronic lumbar radiculopathy -RRB- was originally described by Osgood et al. -LSB- 9 -RSB- and by definition is an extradural operation -LSB- 13 -RSB- . Wilkinson and Chan -LSB- 15 -RSB- in their recent publication advocated the use of microscope for the procedure . After exposure of the ganglion and surrounding plexus and compression of the venous plexus with cottonoids , the longitudinal incision path along the dural sheath is coagulated using low-current bipolar . Once the dural sheath is incised dorsally using a no. 11 knife , ganglion tissue will come into view . Young -LSB- 16 -RSB- described the DRG as a round , yellow , irregular structure . Wilkinson and Chan -LSB- 15 -RSB- described the ganglion as having an appearance similar to fat , with tiny `` sparkles '' within . The latter description is for ganglia from patients with failed back surgery syndrome or chronic lumbar radiculopathy . In rare circumstances like we had , differentiation between an healthy and a tumoral ganglionic tissue may be extremely difficult even with the operating microscope . At this stage , the surgeon has to decide between a biopsy or total removal of the abnormal looking ganglionic tissue . Biopsy may be safer but then there is the possibility of sampling error . If total removal of the DRG is aimed -LRB- sensory ganglionectomy -RRB- , then the afferents and efferents of the ganglion are elevated first and then the ganglion is separated from the underlying septum . To lessen the likelihood of neuroma formation , the proximal end of the ganglion is either hemoclipped or cut with CO2 laser -LSB- 7 -RSB- . If leakage of CSF is not encountered -LRB- which usually is the case -RRB- a watertight closure of the perineurium is not necessary . Some even advocated to leave an autogenous fat graft in the surgically created defect -LSB- 15 -RSB- . As for complication of the procedure , deafferentiation pain has been exaggerated because it is usually transient and never reaches to the point of anesthesia dolorosa -LSB- 13 , 15 -RSB- . Taha et al. -LSB- 13 -RSB- states that there is no uniformly beneficial treatment for deaffarentiation pain after ganglionectomy but our patient definitely benefited from brief use of gabapentin . In conclusion , we reported the unique case of a pseudotumor of L3 DRG . The patient presented with atypical symptoms like neuropathic pain and urinary distention . Preoperative MR scans showed a gigantic left L3 DRG yet this asymmetry was masked by the presence of an adjacent ipsilateral far-lateral disc herniation . Attention to foraminal and extraforaminal part of the spine is of utmost importance in the evaluation of lumbar MR scans . Surgery was both diagnostic and therapeutic in this case . Before opening the dural sheath of a nerve root or DRG , a surgeon should realize that if a tumor or a disc fragment does not come out at once , then it may only be through histopathology that a proper diagnosis can be reached .

O O B 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 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 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 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What is new in surgical treatment of vesicoureteric reflux ? In addition to conventional open surgery and endoscopic techniques , laparoscopic correction of vesicoureteric reflux , sometimes even robot-assisted , is becoming an alternative surgical treatment modality for this condition in a number of centres around the world . At least for a subgroup of patients laparoscopists are trying to develop new techniques in an effort to combine the best of both worlds : the minimal invasiveness of the STING and the same lasting effectiveness as in open surgery . The efficacy and potential advantages or disadvantages of these techniques are still under investigation . The different laparoscopic techniques and available data are presented . Introduction When confronted with the title `` What is new in surgical treatment of vesicoureteric reflux ? '' many readers will automatically think of endoscopic techniques with subureteric injection of bulking agents , also known as STING -LRB- Subureteral Teflon INjection -RRB- . Over the years several substances have been advocated as bulking agents , but the original Teflon is no longer in use . -LRB- The most commonly used substance nowadays is DefluxR , a dextranomer/hyaluronic acid copolymer . -RRB- However , a technique that has been around for a quarter of a century can hardly be considered for a text on surgical novelties . Instead , this review concentrates on the use of laparoscopic techniques in this setting . General considerations about vesicoureteric reflux Vesicoureteric reflux -LRB- VUR -RRB- remains one of the most frequent conditions in paediatric urology , although the exact prevalence is largely unknown . VUR can be primary , secondary -LRB- e.g. to elevated bladder pressures in neurogenic bladders or dysfunctional voiding -RRB- and sometimes intermittent in nature , only disclosing itself when infection has possibly induced a degree of insufficiency of the ureterovesical junction . It is generally assumed that VUR predisposes to urinary tract infections and that surgical treatment of reflux and prophylactic antibiotics are equivalent in terms of preventing infections and renal scarring . The relative merit of these interventions in the natural course of these conditions remains to some extent controversial -LSB- 32 -RSB- . The importance of voiding dysfunction with detrusor overactivity , underactivity or dysfunctional elimination disorder in the aetiology of VUR should not be underestimated -LSB- 4 , 30 -RSB- and this has its implications in the treatment offered to these children . Hence bladder training and minimally invasive techniques have acquired a prominent role over the years . Children with VUR and concomitant voiding dysfunction are likely to suffer more breakthrough infections and have lower spontaneous resolution rates and therefore represent a large proportion of the patients undergoing surgical intervention -LSB- 31 -RSB- . Antimicrobials form the mainstay in the treatment of VUR , in combination with other conservative measures , because VUR will spontaneously disappear in a majority of children and rarely gives rise to serious long-term complications -LSB- 2 -RSB- . Increasingly however the exact role of prophylaxis is being questioned as well-designed prospective trials are rare -LSB- 10 -RSB- . Nevertheless , a small subgroup of patients does pose problems of break-through infections despite all conservative measures and in fact some of them seem prone to renal scarring leading to hypertension and exceptionally even end stage renal failure -LSB- 16 -RSB- . Traditional surgical techniques in the treatment of VUR Since the 1950s several surgical techniques have been developed for the correction of VUR . All techniques share the same basic principle of creating an anti-reflux mechanism by increasing the portion of the distal ureter lying in a submucosal tunnel between the detrusor muscle and the bladder mucosa . They offer comparable and very high success rates with few complications -LSB- 12 -RSB- . From a purely technical standpoint , these open techniques can basically be divided into two groups . There are those that involve mainly or entirely intravesical ureteral dissection -LRB- and hence a need for postoperative bladder drainage -RRB- and those that use a purely extravesical approach to the ureter without disconnecting it from the bladder . To the former group belong the techniques of Politano and Leadbetter -LRB- 1958 -RRB- , Glenn and Anderson -LRB- 1967 -RRB- , the psoas-hitch technique and the -LRB- most widely used -RRB- Cohen technique -LRB- 1975 -RRB- -LSB- 6 , 11 , 13 , 25 -RSB- . In these techniques the ureter is disconnected from the bladder and reimplanted in a new and longer submucosal tunnel from the luminal side of the bladder . In the Cohen technique , a cross-trigonal tunnel is created bringing the ureter to the contralateral side , the other techniques result in a more natural course of the ureter , but are somewhat more prone to complications such as bowel injury or kinking of the ureter . The psoas-hitch technique is generally reserved for more complex situations as in mega-ureters or re-do surgery and is helpful in creating a longer tunnel . The conceptually different extravesical approach was popularized by Lich and Gregoir , reducing postoperative bladder irritation to insignificance , but predisposing to temporary bladder retention when performed bilaterally -LSB- 14 , 21 -RSB- . The more recent and certainly minimally invasive STING technique where bulking agents are injected submucosally has gained wide acceptance . Undoubtedly this is technically a very easy , relatively cheap and patient-friendly treatment modality , tempting many doctors into an increasingly pre-emptive approach to VUR , using it as first-line treatment in cases of -LRB- antenatally detected -RRB- high-grade reflux even in infants -LSB- 27 -RSB- . Success rates , even in low-grade reflux , are clearly lower than in open surgery and a second injection of bulking agent is often necessary -LSB- 8 -RSB- . Moreover , prospective randomised trials and long-term results are still not available . The tendency to use this endoscopic technique as an alternative to medical treatment is underscored by the fact that since the Food and Drug Administration -LRB- FDA -RRB- approval of DefluxR the total number of procedures for reflux has increased , while open surgery rates have remained stable -LSB- 20 -RSB- . All these facts and tendencies mentioned above in turn suggest that , at least for the foreseeable future , there will remain a group of patients in whom STING is deemed-or proves to be-insufficient . Open surgery on the other hand has its drawbacks as well due to its invasiveness . In an ideal world physicians would be able to define very precisely and at the earliest possible point in time which group of patients with VUR is at increased risk for the complication of pyelonephritic scarring and which group is not . This would in turn allow a very tailored approach to each individual child with pre-emptive surgical measures in the group at risk . Failing this knowledge , the next best thing to aim for is to combine the superior results of time-honoured open procedures like a Cohen reimplantation or Lich-Gregoir operation with the much sought after minimal invasiveness of laparoscopy , possibly with the added ultra-precise tissue handling and dexterity of robotic surgery . These considerations are the driving force of the developments described in this text . Conventional laparoscopic techniques Both intra - and extravesical laparoscopic treatments have been described in a great variety of techniques . Most series however remain small and follow-up is very limited . Ehrlich et al. and Janetschek et al. were the first to report in 1994 and 1995 on two and six children undergoing laparoscopic Lich-Gregoir anti-reflux surgery for vesicoureteral reflux -LSB- 7 , 17 -RSB- . The reflux was successfully corrected without morbidity , requiring only a short hospitalisation . Peri-operative ureteral stents were deemed unnecessary . One mild unilateral stenosis did develop later , requiring temporary stenting . Ehrlich et al. described decreased peri - and post-operative pain and improved cosmesis by comparison with open surgery . He suggested that this preliminary report deserved further study . Janetschek et al. on the other hand concluded that the Lich-Gregoir anti-reflux procedure was a complicated one because of the difficult suturing and knot-tying , offering no clear advantage over the conventional procedure . Other teams were very reluctant to join in the efforts to develop this approach for several years to come . The choice for a Lich-Gregoir technique for the first attempts at correction of VUR can be explained by the fact that , at that time , experience with laparoscopy in cavities other than the abdomen was very limited . Five years later Lakshmanan and Fung reported technical modifications to further minimize invasiveness , basically by downsizing ports and instruments and limiting tissue dissection -LSB- 19 -RSB- . A more recent paper by Riquelme et al. again reported excellent outcomes in 15 children , even in cases of bilateral reflux and duplex ureters -LSB- 28 -RSB- . There was no postoperative voiding dysfunction . Laparoscopic ureteral reimplantation with extracorporeal tailoring and stenting of megaureters combined with a Lich-Gregoir type of extravesical reimplantation was recently reported by Ansari et al. in three children -LSB- 1 -RSB- . Although the Cohen procedure was the more widely used in the treatment of VUR , a laparoscopic version thereof was investigated later than the extravesical laparoscopic techniques . The obvious reason is the anticipated difficulties with port placement and the limitations of the intravesical working space . Different approaches were used by Gill et al. and Yeung et al. -LSB- 12 , 33 -RSB- . Gill et al. combined the use of two suprapubic ports with a transurethral resectoscope for unilateral cases whereas Yeung et al. used three suprapubic ports , more closely copying the open Cohen procedure . A recent report by Kutikov et al. on either transvesical laparoscopic cross-trigonal ureteral reimplantation in patients with reflux or a Glenn-Anderson reimplantation in patients with a primary obstructing mega-ureter mentions operative success in 25 of 27 patients with VUR and 4 out of 5 patients with mega-ureters , results that are comparable to the ones obtained in open surgery -LSB- 18 -RSB- . Complications were postoperative urinary leak in four patients and ureteral stricture at the anastomosis in two . The authors noted that most complications occurred in the younger patients with small bladder capacities . For completeness two papers on reimplantations in -LRB- young -RRB- adults can be mentioned . Chung et al. described successful laparoscopic nonrefluxing ureteral reimplantation with a psoas hitch using a submucosal tunnelling technique after submucosal injection of saline under cystoscopy in two adult female patients without postoperative complications -LSB- 5 -RSB- . Also in 2006 , Puntambekar et al. described laparoscopic extravesical ureteroneocystostomy with psoas hitch in five gynaecologic patients , clearly minimizing the procedural morbidity -LSB- 26 -RSB- . Again no intraoperative or postoperative complications occurred . Gradually more relevant series with larger numbers of patients and longer follow-up are being presented . At the 2007 European Society for Paediatric Urology -LRB- ESPU -RRB- annual meeting two groups will present their experience in about 80 patients each , with success rates above 90 % -LRB- http://www.espu.org -RRB- . Robot-assisted techniques Over the last 2 years a few authors reported robot-assisted laparoscopic techniques using the Da VinciR -LRB- Intuitive Surgical , Mountain View , CA -RRB- system for the treatment of VUR , adding yet another approach to this rapidly expanding field -LSB- 3 , 23 , 24 -RSB- . They made good use of the experience gained with conventional laparoscopy , adding the advantages of robotics : enhanced dexterity of the instruments , absence of tremor and 3-D vision . The generally used term of `` robotic surgery '' is to some extent actually misleading because it suggests completely autonomous function of the equipment . In reality it works as a master-slave system , merely transferring the movements of the surgeon 's hands to the tip of the instruments -LRB- Fig. 1 -RRB- . The evolution parallels the one seen in conventional laparoscopy , experience having started with the extravesical approach and later moving to intravesical procedures . The sequence of surgical steps of both techniques will briefly be discussed . As stated , they closely mirror the steps in conventional laparoscopy . Fig. 1Outside view once the draped robotic arms are connected to the laparoscopic ports : the child seems completely `` embraced '' by the machine Extravesical technique To start , a cystoscopic evaluation of the relevant anatomy is carried out . The camera port is then placed in the umbilicus and the two working ports in each lower abdominal quadrant . A small transverse peritoneal incision is made on the laterodorsal side of the bladder where the ureter is retrieved . The ureter is then buried in a trough between the mucosa and detrusor to create the anti-reflux mechanism -LRB- Figs. 2 and 3 -RRB- . The bladder catheter is removed already at the end of the procedure unless a significant perforation needing suturing of the mucosa has been made . Fig. 2Extravesical approach : very gently the detrusor muscle is incised and peeled away until the delicate bladder mucosa starts to bulgeFig . 3Extravesical approach : the completely freed ureter is hinged into the trough to create an anti-reflux valve mechanism In our experience there were no bladder symptoms post surgery in any of the patients . All cases of reflux resolved , but there was one case of `` de novo '' contralateral low-grade reflux -LSB- 3 -RSB- . Later we successfully performed this operation in an adult male patient after a failed subureteral injection -LRB- unpublished data -RRB- . Interestingly , Elmore et al. recently reported on the use of the open Lich-Gregoir technique as salvage in these patients as well -LSB- 9 -RSB- and already in 2004 one similar laparoscopic patient was reported by Shu et al. -LSB- 29 -RSB- . Both in open and laparoscopic surgery this was a novel approach , meant to avoid the sometimes difficult intravesical dissection due to foreign material after STING . Peters and Borer reported persisting low-grade reflux in 2 of their 24 patients -LSB- 23 -RSB- . Intravesical technique Olsen was the first to experiment with a Cohen cross-trigonal ureter reimplantation by laparoscopic access to the bladder in a pig model using the Da VinciR system -LSB- 22 -RSB- . In all pigs the reflux disappeared after the procedure . The advantage of the robotic equipment seemed to be the better access to submucosal tunnelling of the ureter and the intravesical suturing of the anastomosis . Peters and Woo in 2005 and Callewaert in 2006 reported their experience with robot-assisted Cohen procedures in six and three paediatric patients respectively -LSB- 3 , 24 -RSB- . Initial port placement and closure of the incisions at the end of the procedure were the crucial steps , the rest of the procedure being straightforward . Once inside the bladder the mucosa is circumferentially incised around the ostium using the cautery hook . After both ureters are freed , a submucosal tunnel connecting the most proximal part of the two mucosal incisions is created , using forceps and scissors -LRB- Fig. 4 -RRB- . Creation of the submucosal tunnel and reimplantation of the ureters is remarkably easy because of the three dimensional visualisation and great dexterity inside the very small volume of a child 's bladder . The anatomical detail is such that dissection of the plane between the detrusor and mucosa is achieved with more detail than in open surgery . The bladder catheter is left indwelling for 24 to 48 h. Fig. 4Intravesical approach : creation of the submucosal tunnel connecting the periureteral incisions . -LRB- The jaws of the forceps measure 5 mm in length -RRB- We had one conversion to open surgery out of three cases in our early experience because of port-related problems in a small child -LSB- 3 -RSB- . Kutikov et al. using conventional laparoscopy similarly found that the smaller children were more prone to complications and that these procedures were technically more demanding -LSB- 18 -RSB- . Peters and Woo on the other hand reported no conversions in a series of six children aged between 5 and 15 -LSB- 24 -RSB- . They did however have a case of port-site urinary leakage requiring prolonged bladder drainage . Peters and Callewaert each reported one case of persisting low-grade reflux in their initial experience . Unlike the situation in open surgery , bladder spasms remained completely absent and anticholinergics were unnecessary . This fact is highly suggestive of the minimal invasiveness and limited trauma incurred by the bladder wall . When comparing the robotically assisted intra - and extravesical operations it is our impression that the Lich-Gregoir technique offers some advantages over the intravesical operation : no need for catheters , no haematuria and easier reproducibility . The drawback is that the abdominal cavity needs to be entered . The abdominal cavity even in smaller children is large enough to allow comfortable movement of the instruments , whereas intravesical operations in this patient group can be technically impossible due in part to the relative bulkiness of the robotic instruments . Conclusion Treatment modalities of reflux are evolving rapidly . Conventional or robot-assisted laparoscopic techniques must be considered a possible future alternative to the more traditional ways of treating this condition . There is no proven superiority at this time and experience is limited to a few centres only and relatively small numbers of patients . It is well established that with open surgery very high success rates can be achieved and that morbidity is relatively low and hospitalisation nowadays can be kept short . The first impressions are that morbidity using laparoscopic techniques is lower still and that there is some cosmetic gain , but it is obvious that the most important issue will be whether the long-term success rates are at least comparable . Most surgeons agree that robotics certainly add to the precision and ease of the individual surgical steps when compared to conventional laparoscopy , but the financial costs are very high . The intravesical approach using robotics is feasible , but technical difficulties must be taken into account in smaller children . -LRB- The same holds true for the conventional laparoscopy . -RRB- The extravesical robotic approach clearly seems the more promising , possibly even after failed submucosal injection therapy . Nevertheless we feel that the intravesical approach deserves further pursuing because it may allow surgical correction of other malformations at the level of the bladder neck and ureterovesical junction in a minimally invasive and very precise way . It would be premature to promote laparoscopy as the golden mean between STING and open surgery for a subgroup of reflux patients at this point , as this would imply diverting a large number of patients to a few centres where either the technical laparoscopic expertise or a robotic system is available . However , we remain convinced that in the -LRB- near -RRB- future laparoscopy will find its place in the care for these patients .

O O O O O O O B I O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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ADAMTS13 phenotype in plasma from normal individuals and patients with thrombotic thrombocytopenic purpura The activity of ADAMTS13 , the von Willebrand factor cleaving protease , is deficient in patients with thrombotic thrombocytopenic purpura -LRB- TTP -RRB- . In the present study , the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting . Normal plasma -LRB- n = 20 -RRB- revealed a single band at 190 kD under reducing conditions using a polyclonal antibody , and a single band at 150 kD under non-reducing conditions using a monoclonal antibody . ADAMTS13 was not detected in the plasma from patients with congenital TTP -LRB- n = 5 -RRB- by either antibody , whereas patients with acquired TTP -LRB- n = 2 -RRB- presented the normal phenotype . Following immunoadsorption of immunoglobulins , the ADAMTS13 band was removed from the plasma of the patients with acquired TTP , but not from that of normal individuals . This indicates that ADAMTS13 is complexed with immunoglobulin in these patients . The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis , impaired cellular secretion , or enhanced degradation in the circulation . This study differentiated between normal and TTP plasma , as well as between congenital and acquired TTP . This method may , therefore , be used as a complement in the diagnosis of TTP . Introduction Von Willebrand factor -LRB- VWF -RRB- is a glycoprotein that plays a key role in the primary hemostatic process by inducing platelet adhesion and aggregation at sites of vascular injury under conditions of high shear stress . The main source of circulating VWF is the endothelium , from which it is secreted in the form of ultra-large multimers -LRB- ULVWF -RRB- -LSB- 53 -RSB- . ULVWF multimers are biologically very active -LSB- 2 , 31 -RSB- and , upon release , undergo processing into smaller multimers in normal individuals . This occurs on the surface of endothelial cells -LSB- 10 -RSB- . VWF defects may potentially lead to both bleeding and thrombotic disorders : defective VWF secretion , intravascular clearance , multimer assembly , or increased proteolytic degradation may lead to different types of von Willebrand disease . On the other hand , dysfunctional VWF proteolysis may lead to the thrombotic disorder thrombotic thrombocytopenic purpura -LRB- TTP -RRB- -LSB- 40 -RSB- . TTP is a thrombotic microangiopathy -LRB- TMA -RRB- characterized by microangiopathic hemolytic anemia , thrombocytopenia , fever , neurological and renal manifestations . Chronic recurrent TTP has been associated with the presence of ULVWF in the plasma -LSB- 30 -RSB- . ULVWF multimers are capable of inducing increased platelet retention in children with TTP -LSB- 21 -RSB- . These observations , along with the finding of VWF and platelet-rich -LRB- but fibrin-poor -RRB- thrombi in the microcirculation of the heart , brain , kidneys , liver , spleen , and adrenals in TTP patients -LSB- 3 -RSB- , led to the conclusion that ULVWF multimers are responsible for the disseminated platelet thrombi occurring in TTP and that their degradation to smaller VWF multimers is impaired due to the deficiency of a VWF-cleaving protease -LSB- 15 -RSB- . Recently , the VWF-cleaving protease was purified -LSB- 12 , 13 , 16 , 50 -RSB- and the encoding gene sequenced , linking the protease to the ADAMTS -LRB- a disintegrin-like and metalloprotease with thrombospondin-type-1 motif -RRB- family of metalloproteases -LSB- 27 -RSB- . The protease , named ADAMTS13 , cleaves VWF at the 1605Tyr-1606Met peptide bond in the A2 domain , yielding the 140-kD and 176-kD VWF fragments present in normal plasma -LSB- 13 , 50 -RSB- . Cleavage is made possible by a conformational change in VWF due to shear stress in the circulation , which exposes the cleavage site , making it susceptible to proteolysis -LSB- 55 -RSB- . ADAMTS13 activity is severely deficient -LRB- < 5 % of normal plasma activity -RRB- in TTP patients -LSB- 6 -RSB- , either due to a mutation in the ADAMTS13 gene in the congenital form of TTP or due to auto-antibodies in the acquired form -LSB- 14 , 27 , 52 -RSB- . Autosomal recessive hereditary TTP -LRB- also termed the Upshaw-Schulman syndrome -RRB- typically presents during the neonatal period or early childhood -LRB- < 10 years of age -RRB- , but may also manifest during adolescence and adulthood . Recurrent TTP episodes may occur as often as every third week . TTP recurrences are associated with cerebral vascular accidents in approximately 30 % of cases , and these episodes may lead to neurological complications . Renal manifestations may be mild or may result in acute renal failure due to hemoglobinuria and TMA . About 20 % of patients progress to end-stage renal failure -LSB- 28 -RSB- . Hemolytic uremic syndrome -LRB- HUS -RRB- is a similar microangiopathic disorder characterized by microangiopathic hemolytic anemia , thrombocytopenia , and acute renal failure -LSB- 5 -RSB- . Two forms of HUS have been described : D + or typical -LRB- diarrhea-associated -RRB- HUS and D - or atypical -LRB- non-diarrhea-associated -RRB- HUS . D + HUS occurs after infection with Shiga-like toxin producing bacteria , typically , enterohemorrhagic Escherichia coli . The patients are usually children presenting with abrupt onset of diarrhea , followed by the development of HUS 2 -- 10 days later . A prothrombotic state precedes the acute renal failure -LSB- 8 -RSB- , but the pathogenetic mechanism is , as yet , unclear . It is assumed that bacterial virulence factors gain access to the circulation , circulate on blood cells , activate platelets , and reach the kidney , where the endothelium is injured -LSB- 36 , 47 -RSB- . D - HUS is associated with mutations in certain complement regulatory factors , such as factor H , factor I , and membrane co-factor protein -LRB- CD46 -RRB- . The mutations lead to activation of the complement system on host endothelial cells -LSB- 29 , 58 -RSB- . The resulting vascular damage may lead to the formation of thrombotic lesions in the kidneys . Although HUS patients are , typically , young children with a history of diarrhea and acute renal failure , the clinical manifestations of HUS and TTP often overlap , making differentiation between the two syndromes based solely on clinical presentation difficult . ADAMTS13 antigen levels can differentiate between HUS and TTP , as they are severely deficient in patients with congenital TTP and normal to moderately reduced in HUS -LSB- 51 -RSB- . Assays for ADAMTS13 activity can , therefore , differentiate between TTP -LRB- congenital and acquired -RRB- and HUS -LSB- 26 , 48 -RSB- . Several ADAMTS13 assays are available today based on antigen detection and activity -LSB- 15 , 17 , 25 , 38 -RSB- , showing the presence of the protease -LRB- by enzyme-linked immunosorbent assay , ELISA -RRB- and its bioactivity in normal plasma and the lack of protease and activity in the plasma from patients with congenital TTP . These assays have also shown that patients with acquired TTP have auto-antibodies that neutralize the activity of ADAMTS13 . The present study utilized a different method , immunoblotting , and two anti-ADAMTS13 antibodies against specific domains , to investigate the presence of ADAMTS13 antigen in normal plasma , TTP plasma -LRB- congenital and acquired -RRB- , and in heterozygous carriers of ADAMTS13 mutations , demonstrating the presence of ADAMTS13 and its size in normal plasma , the lack thereof in congenital TTP , and auto-antibody-bound protease in acquired TTP . Materials and methods Subjects Citrated plasma was available from patients with congenital -LRB- n = 5 -RRB- and acquired -LRB- n = 2 -RRB- TTP . The patient data are presented in Table 1 . The ADAMTS13 activity level was assayed as previously described -LSB- 15 , 17 -RSB- . Table 1Clinical and laboratory data regarding thrombotic thrombocytopenic purpura -LRB- TTP -RRB- patientsPatient no.SexAge at debut Age at sampling -LRB- years -RRB- Current age -LRB- years -RRB- Symptoms during episodesNo . of episodesADAMTS13 activity levelADAMTS13 mutationADAMTS13 inhibitor Reference1aM2 d1619Jaundice , hemolytic anemia , thrombocytopenia , macroscopic hematuria , pathological urinalysis , fever , neurological symptoms , elevated serum creatinine > 5 < 5 % 4143insAbcNone -LSB- 4 , 20 , 43 -RSB- 2aM5 .3 y1718Jaundice , hemolytic anemia , thrombocytopenia , fever , neurological symptoms , pathological urinalysis > 5 < 5 % 4143insAbcNone -LSB- 4 , 20 , 43 -RSB- 3F20 m1523Hemolytic anemia , thrombocytopenia , hematuria , epileptic attacks , slightly elevated serum creatinine > 5 < 5 % P353Ld , P457LeNone -LSB- 4 -RSB- 4M3 y79Hemolytic anemia , thrombocytopenia , purpura , pathological urinalysis4 < 5 % P671Lf , 4143insANone -LSB- 43 -RSB- 5M2 d3939Jaundice , hemolytic anemia , petechiae , thrombocytopenia , transitory neurological deficits and aphasia , elevated creatinine > 5 < 5 % 4143insAbNone -- 6F54 y7075Recurrent hemolytic anemia and thrombocytopenia , reduced consciousness , pathological urinalysis > 5 < 5 % NA0 .5 U/ml -- 7F25 y4244Thrombocytopenia , hemolytic anemia , elevated creatinine during viral infection and pregnancy2 < 5 % NA0 .2 U/ml -- aPatients 1 and 2 are siblingsbPatients 1 , 2 , and 5 are homozygous for the ADAMTS13 mutationc4143insA leads to a mutation in the second CUB domaindP353L is a mutation in the disintegrin-like domaineP457L is a mutation in the cysteine-rich domainfP671L is a mutation in the spacer domainNA : not assayed The study also included the parents of patients 1 -- 4 . The parents of patients 1 and 2 are both heterozygous for the 4143insA mutation , and have protease activity levels of 20 % -LRB- mother -RRB- and 50 % -LRB- father -RRB- , as assayed by the VWF multimeric structure analysis -LSB- 15 -RSB- . The parents of patient 3 are heterozygous for the P353L -LRB- mother -RRB- and P457L -LRB- father -RRB- mutations , and both have 50 % ADAMTS13 activity . The parents of patient 4 are heterozygous for P671L -LRB- mother -RRB- and 4143insA -LRB- father -RRB- , and have 50 % ADAMTS13 activity . All parents are clinically unaffected . Plasma samples from 20 healthy adult volunteers were used as controls . The study was conducted with the approval of the ethics committee of Lund University and the plasma samples were collected with the informed consent of the patients , their parents , and the controls . Plasma samples At the time of sampling , the patients were treated regularly with fresh frozen plasma or Octaplas -LRB- Octapharma , Stockholm , Sweden ; patients 1 -- 5 -RRB- . Patient 6 was treated with plasma infusions every sixth week . Patient 7 did not receive any plasma treatment at sampling . All blood samples were obtained at least three weeks -LRB- patients 1 -- 4 and 6 -RRB- or one week -LRB- patient 5 -RRB- after the last treatment . Venous blood from patients and controls was collected , and the plasma obtained as previously described -LSB- 20 -RSB- . Anti-ADAMTS13 antibodies A polyclonal anti-peptide antibody was raised in New Zealand white rabbits against a unique sequence in the second CUB domain -LRB- AA1413-1427 -RRB- and affinity-purified against the peptide . Antibody specificity was tested by ELISA -LRB- plates coated with the peptide -RRB- and by immunoblotting with purified plasma ADAMTS13 -LSB- 50 -RSB- under reducing -LRB- SDS-PAGE -RRB- and non-reducing -LRB- dot blot -RRB- conditions . The monoclonal antibody A10 -LSB- 56 -RSB- , directed against the disintegrin-like domain , was used to confirm the results of the polyclonal antibody . The polyclonal antibody reacted with ADAMTS13 under reducing -LRB- reduced by the addition of 2-mercaptoethanol to disrupt disulfide bonds in the protease -RRB- and non-reducing conditions , whereas the monoclonal antibody reacted with ADAMTS13 only under non-reducing conditions . Immunoblot analysis for the detection of ADAMTS13 in plasma The plasma samples -LRB- 1:20 -RRB- were subject to SDS-PAGE under reducing -LRB- for blotting with the polyclonal antibody -RRB- and non-reducing conditions -LRB- for blotting with the monoclonal antibody -RRB- -LSB- 19 , 58 -RSB- . Purified plasma ADAMTS13 -LRB- 1:100 -RRB- was used as the control for the polyclonal antibody . Immunoblotting was performed with rabbit anti-ADAMTS13 IgG 1.6 μg/ml followed by goat anti-rabbit IgG HRP -LRB- DakoCytomation , Carpinteria , CA -RRB- 1:2000 , or with mouse anti-ADAMTS13 IgG 0.6 μg/ml followed by goat anti-mouse IgG HRP -LRB- DakoCytomation , Carpinteria , CA -RRB- 1:2000 . The signal was detected by chemiluminescence . The specificity of the signal obtained with the polyclonal antibody was tested by preincubation with a 50-fold molar surplus of blocking peptide followed by immunoblotting with the blocked antibody . The specificity of the secondary antibodies was tested by omission of the primary antibodies . Immunoblotting with the polyclonal antibody revealed , in addition to ADAMTS13 , two unspecific bands at 130 kD and 170 kD , which were identified as C3 and alpha-2-macroglobulin . These proteins were removed by incubating the plasma samples with protein A-sepharose coupled rabbit anti-C3 IgG and rabbit anti-alpha-2-macroglobulin IgG . The results obtained using the polyclonal antibody show samples from which these proteins have been removed . In order to investigate the presence of ADAMTS13-autoantibody complexes in the plasma from patients with acquired TTP , samples were passed onto protein G-sepharose -LRB- Amersham Biosciences , Buckinghamshire , UK -RRB- prior to immunoblotting . Normal plasma -LRB- n = 2 -RRB- was used for comparison . Results Detection of ADAMTS13 in plasma samples from normal individuals Normal plasma under reducing conditions revealed a single immunoreactive band at 190 kD when blotted against the polyclonal antibody -LRB- Fig. 1a -RRB- . Purified plasma ADAMTS13 showed a similar band under the same conditions -LRB- Fig. 1a -RRB- . The monoclonal antibody detected an immunoreactive band at 150 kD in normal plasma under non-reducing conditions -LRB- Fig. 1b -RRB- . Preincubation of the polyclonal antibody with the blocking peptide abolished the ADAMTS13 band in normal plasma -LRB- data not shown -RRB- . Immunoblots in which the primary antibodies had been omitted showed no bands -LRB- data not shown -RRB- . Fig. 1a -- gDetection of ADAMTS13 in normal and thrombotic thrombocytopenic purpura -LRB- TTP -RRB- plasma . a The polyclonal antibody detected a single band at 190 kD in normal plasma -LRB- NP -RRB- under reducing conditions . Purified plasma ADAMTS13 in the right lane showed a similar band . b Normal plasma under non-reducing conditions revealed an immunoreactive band at 150 kD when blotted against the monoclonal antibody . c Immunoblotting with the polyclonal antibody revealed that patients 1 -- 5 with congenital TTP all lacked the ADAMTS13 band , whereas patients 6 and 7 with acquired TTP presented a normal expression pattern . Normal plasma -LRB- NP -RRB- was run on the same gel for comparison . d These results were confirmed by the monoclonal anti-ADAMTS13 antibody . e Immunoblot using the polyclonal antibody . Immunoadsorption of immunoglobulins from the plasma samples of patients 6 and 7 with acquired TTP lead to the simultaneous removal of the ADAMTS13 band , indicating that ADAMTS13 is complexed with the anti-ADAMTS13 auto-antibodies . In contrast , the ADAMTS13 band remained visible in normal plasma -LRB- NP -RRB- treated similarly . f A schematic presentation of the mechanism by which the removal of immunoglobulins leads to the removal of the ADAMTS13 antigen from the plasma of patients with acquired TTP . The plasma sample contains ADAMTS13 -LRB- filled circles -RRB- , auto-antibodies to ADAMTS13 -LRB- ⤙ -RRB- , and various other plasma proteins -LRB- open circles -RRB- . Immunoblotting of the plasma sample prior to the removal of immunoglobulins detects the presence of ADAMTS13 antigen . Passage of the plasma sample through a protein G-sepharose column leads to the binding and removal of all immunoglobulins from the sample . Since ADAMTS13 is bound to the anti-ADAMTS13 auto-antibodies , it is removed along with them . Immunoblotting of the flow-through shows no ADAMTS13 band . g Immunoblot with the monoclonal antibody showing a normal ADAMTS13 band in the plasma of the parents , which are all heterozygous for one mutated allele and are clinically unaffected . Lane 1 : the mother of patients 1 and 2 , 4143insA ; lane 2 : the mother of patient 3 , P353L ; lane 3 : the father of patient 3 , P457L ; lane 4 : the mother of patient 4 , P671L ; lane 5 : the father of patient 4 , 4143insA . Normal plasma -LRB- NP -RRB- was run on the same gel for comparison Detection of ADAMTS13 in plasma samples from TTP patients The plasma samples from all patients with congenital TTP , regardless of the mutation , lacked the ADAMTS13 band -LRB- Fig. 1c -RRB- . Patients 6 and 7 with acquired TTP revealed the same ADAMTS13 protein expression pattern as the controls -LRB- Fig. 1c -RRB- . These results were obtained using both the polyclonal -LRB- Fig. 1c -RRB- and the monoclonal antibody -LRB- Fig. 1d -RRB- . When immunoadsorption of plasma immunoglobulins was carried out prior to immunoblotting , the ADAMTS13 band remained visible in normal plasma , but was completely removed from the samples of the patients with acquired TTP -LRB- Fig. 1e -RRB- , probably due to its association with the immunoglobulin inhibitor . Detection of ADAMTS13 in the parents of the TTP patients The parents of the TTP patients are all carriers of one ADAMTS13 mutation and one normal allele . All parents presented a normal ADAMTS13 phenotype using both the polyclonal -LRB- data not shown -RRB- and the monoclonal antibody -LRB- Fig. 1g -RRB- . Discussion In the present study , we detected ADAMTS13 in plasma using a polyclonal and a monoclonal antibody . This assay was capable of distinguishing TTP patients from normal individuals , as well as differentiating between congenital and acquired TTP . Plasma from the patients with congenital TTP lacked the ADAMTS13 antigen . In contrast , the plasma of patients with acquired TTP expressed a normal ADAMTS13 phenotype . Previous studies describing the ADAMTS13 phenotype in normal plasma by immunoblotting with other specific anti-ADAMTS13 antibodies have shown immunoreactive bands of the same molecular weight using similar conditions -LSB- 33 , 46 -RSB- . The ADAMTS13 antigen in patients with congenital and acquired TTP has recently been shown by ELISA , demonstrating low to undetectable ADAMTS13 levels in patients with congenital TTP -LSB- 11 , 38 -RSB- and decreased , but mostly detectable , levels in patients with acquired TTP -LSB- 11 , 38 , 45 -RSB- . In the present study , the ADAMTS13 phenotype in TTP patients is described by immunoblotting , confirming the lack of ADAMTS13 antigen in the plasma of patients with congenital TTP and the presence of circulating complexes in acquired TTP . Furthermore , we showed that heterozygous carriers of the ADAMTS13-related mutations who , thus , have reduced ADAMTS13 bioactivity have a normal phenotype . The plasma of the patients with congenital TTP did not present the ADAMTS13 band . This may be due to altered synthesis , secretion or antigenicity , or due to increased breakdown of the protease in plasma . The fact that two antibodies directed to two different domains in ADAMTS13 were unable to detect the protease band makes altered antigenicity less likely to be the cause for the lack of the ADAMTS13 band in these patients . Previous studies have shown impaired secretion of the 4143insA -LRB- patients 1 , 2 , 4 , and 5 -RRB- -LSB- 35 , 42 , 44 -RSB- and P353L -LRB- patient 3 -RRB- -LSB- 42 -RSB- mutants from cells , thus , indicating that the protease may accumulate intracellularly , at least in some patients with congenital TTP . This may be due to a missing cell sorting signal , as in the case of 4143insA -LSB- 44 -RSB- or due to conformational changes in the protein , impairing its secretion . Similar findings regarding two other ADAMTS13 mutations , V88M and G239V , have been recently reported -LSB- 34 -RSB- . A total lack of ADAMTS13 activity in the plasma is thought to be incompatible with life -LSB- 27 -RSB- , thus , the patients may have very low amounts of ADAMTS13 activity in their plasma , which we were unable to detect with this method . The patients with acquired TTP presented with a normal ADAMTS13 band , which is consistent with the fact that ADAMTS13 protease genotype and expression are normal , but their activity is lower , due auto-antibodies -LSB- 11 , 14 , 41 , 45 , 52 -RSB- . The finding that the immunoadsorption of immunoglobulins from the plasma of the patients with acquired TTP also led to the removal of the ADAMTS13 antigen from their samples indicates that the protease is complexed with the auto-antibodies in the circulation of these patients , and that this occurs even during clinical remission . The majority of the ADAMTS13 assays available today detect ADAMTS13 activity levels in plasma -LRB- Table 2 -RRB- . This is performed either by detecting the VWF products resulting from ADAMTS13 cleavage -LRB- assays 1 -- 5 -RRB- or by measuring the residual VWF activity -LRB- assays 6 -- 7 -RRB- . The VWF substrate utilized in these assays may be high molecular weight VWF -LRB- plasma-derived or recombinant ; assays 1 -- 4 -RRB- or VWF domains or short synthetic peptides -LRB- assay 5 -RRB- . Two multicenter studies evaluating methods 1 , 3 -- 5 , and 6 -- 7 showed that all assays were able to detect severely ADAMTS13-deficient plasma samples and indicated that methods 1 , 6 , and 7 were the most consistent and reliable methods -LSB- 48 , 49 -RSB- . A recent smaller study evaluating the FRETS-VWF73 method -LRB- assay 5 -RRB- showed that this is a reliable assay which provides results in good accordance with other methods -LSB- 26 -RSB- . Table 2ADAMTS13 assaysASSAYPRINCIPLEREFERENCEDetection of ADAMTS13 activityDetection of VWF cleavage products 1 . VWF multimer structure analysisDetection of the breakdown of high-molecular-weight VWF -LSB- 15 , 23 -RSB- 2 . Immunoblotting of VWF Detection of cleavage products of native VWF or recombinant VWF domains -LSB- 37 , 50 -RSB- 3 . IRMADetection of VWF cleavage products -LSB- 32 -RSB- 4 . Flow assayDetection of the breakdown of ULVWF-platelet strings attached to endothelial cells -LSB- 1 -RSB- 5 . Various methods using VWF domains or short synthetic VWF peptides as the substrate , such as the FRETS-VWF73 assayDetection of cleavage products of the VWF domains or VWF peptides -LSB- 9 , 18 , 22 , 25 , 59 -- 61 -RSB- Detection of VWF residual activity 6 . Collagen bindingDetection of VWF binding to collagen ; binding correlates to VWF multimer size -LSB- 17 -RSB- 7 . Ristocetin cofactor activityDetection of platelet aggregates ; VWF ability to induce platelet aggregates in the presence of ristocetin correlates to multimer size -LSB- 7 -RSB- Detection of ADAMTS13 antigen and auto-antibodies 8 . ELISADetection of ADAMTS13 antigen or anti-ADAMTS13 auto-antibodies -LSB- 11 , 38 , 39 , 41 , 54 -RSB- 9 . ImmunoblottingDetection of anti-ADAMTS13 auto-antibodies -LSB- 57 -RSB- 10 . Present assay -LRB- immunoblotting -RRB- Detection of ADAMTS13 antigen and size and -LRB- indirectly -RRB- of auto-antibodies -- Mutation analysis 11 . PCRDetection of mutations in the ADAMTS13 gene -LSB- 27 -RSB- IRMA : immunoradiometric assay ; FRET : fluorescence resonance energy transfer ; FRETS-VWF73 : a 73-amino-acid-long synthetic peptide which provides a minimal substrate for ADAMTS13 -LSB- 24 -RSB- that has been made fluorogenic ; PCR : polymerase chain reaction Few recent studies have shown that ADAMTS13 antigen was detectable in plasma by ELISA -LRB- assay 8 in Table 2 -RRB- . ELISA assays are a valuable complement to the ADAMTS13 activity assays , and offer a fast analysis of the ADAMTS13 antigen levels in plasma samples using antibodies directed to ADAMTS13 . These assays are able to distinguish ADAMTS13-deficient TTP plasma from normal plasma samples . Other methods are capable of detecting anti-ADAMTS13 auto-antibodies -LRB- Table 2 , assays 8 -- 9 -RRB- . Mutational analysis can be carried out by polymerase chain reaction -LRB- PCR -RRB- in order to detect mutations in the ADAMTS13 gene -LRB- assay 11 in Table 2 -RRB- . In the present study , we developed a qualitative , semi-quantitative assay capable of detecting ADAMTS13 antigen and anti-ADAMTS13 auto-antibodies in plasma and , thus , distinguishing between TTP and normal plasma , as well as distinguishing between congenital and acquired TTP . All patients with congenital TTP , regardless of their ADAMTS13 mutations , presented with undetectable levels of ADAMTS13 antigen , whereas patients with acquired TTP presented a normal phenotype . Heterozygous carriers of ADAMTS13 mutations also revealed a normal ADAMTS13 antigen band . Although not a quantitative method like the above-mentioned ELISA assays , the present assay offers the advantage of not only demonstrating the presence or absence of ADAMTS13 antigen , but also the molecular size of ADAMTS13 present in plasma samples . Furthermore , this assay was able to show the presence of anti-ADAMTS13 antibodies indirectly by performing immunoadsorption of plasma immunoglobulins prior to immunoblotting . In conclusion , this assay is able to distinguish between normal and TTP plasma , and also between congenital and acquired TTP . It is easy to perform and can be used at any hospital laboratory routinely using SDS-PAGE electrophoresis and immunoblotting , and offers a complement to existing ADAMTS13 methods in the diagnosis of TTP .

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

adamts13
von willebrand factor
thrombotic thrombocytopenic purpura
immunoblotting
plasma