- To evaluate the mid-term effects of haemostatic sealant application during tubeless percutaneous nephrolithotomy (PCNL) on renal drainage and histology in an in vivo porcine study.
Since 1976 when Fernstorm and Johannson  introduced percutaneous renal stone management, the urological community has focused its efforts on improving percutaneous nephrolithotomy (PCNL) techniques in terms of morbidity. To this end, several alternative approaches have been established, including tubeless PCNL [2, 3]. The concept behind tubeless PCNL is to spare patients the postoperative percutaneous nephrostomy tube drainage that has been associated with substantial postoperative pain and morbidity [4-6].
While several studies in humans have shown that the use of absorbable haemostatic sealants may be safe, feasible and a useful adjunct for a safer tubeless PCNL, the precise effect of these haemostatic agents on renal histology is still not clear [7-10]. Haemostatic sealant use during PCNL remains an ‘off-label’ use of haemostatics and so no guidelines exist to elucidate which sealant is indicated and which is not. Currently, almost the entire range of haemostatic agents has been used in the clinical setting. Nevertheless, concerns regarding a potential negative impact on renal tissue stem from the fact that such haemostatic agents may have varying degrees of bio-absorbability, antigenicity, anaphylactic/hypersensitivity reactions and risk of inducing adverse reactions such as fever, granuloma, abscess or foreign body reaction.
In an attempt to address this issue we examined the mid-term effects of various common haemostatic sealants in the drainage and histology of porcine renal units, using an in vivo totally tubeless PCNL model.
For the purposes of this study 14 female domestic pigs weighing 30–35 kg were used. After protocol approval from the Animal Care Committee of our institution, the pigs were supplied from a local vendor and were housed in the animal care unit of the University Hospital of Patras, following all regulations for the good care and use of laboratory animals.
The pigs were allowed a minimum period of 72 h before the procedure to recover from the stress of transportation. Food was restricted for 12 h before anaesthesia. Induction to anaesthesia was achieved by i.m. administration of a combination of ketamine, xylazine and atropine sulphate. The pigs were then endotracheally intubated and ventilated, and i.v. propofol 5% was used to maintain anaesthesia for the duration of the procedure. Prophylactic, peri-operative antibiotics were administered to all pigs.
With the pig in the supine position, a cystoscopy was performed and, under fluoroscopic guidance, a hydrophilic 0.89 mm (0.035-inch) guidewire was advanced up to the renal pelvis of each kidney. Then, a hydrophilic 5-F ureteric catheter was inserted over each wire and secured with stitches at its entrance to the urethra. The guidewires were removed and the pig was placed in the prone position. Fluoroscopic opacification of the collecting system was then performed by retrograde infusion of contrast medium and a fluorosocopy-guided percutaneous nephrostomy access was established using the Amplatz Renal Dilator Set (Cook Urological, Spencer, IN, USA). All nephrostomy tracts were dilated up to 30 F. The sheath of the last dilator remained in place for 15 min before haemostatic sealant application. Haemostatic sealants were then applied to the nephrostomy tract through the nephrostomy sheath. At the end of the procedure all drains, including the dilator and embedded ureteric catheter, were removed and skin incision was sutured.
The haemostatic sealants investigated in this study were the Helisorb® particles (Eucare Pharmaceuticals, Chennai, India), a type 1 absorbable fish origin collagen powder, the Tachosil® (Nycomed Austria GmbH, Linz, Austria), a human fibrinogen- and thrombin-coated sponge and the Floseal® (Baxter, Deerfield, IL, USA), a cross-linked gelatin granule/topical thrombin matrix. Each haemostatic agent was placed either only in the tract of the nephrostomy (from renal capsule up to the skin), or to both tract and ipsilateral kidney (from the renal parenchyma up to the skin). To avoid direct injection of haemostatic agents into the calyces, the technique described by Nagele et al.  was followed. A nephroscope was used to identify the entrance to the collecting system and direct withdrawing of the nephrostomy sheath either outside the pelvicalyceal system or the renal capsule (in the case of renal instillation or tract-only application, respectively). Then the nephroscope was exchanged with the haemostatic sealants applicator. Both the haemostatic sealant applicator and nephrostomy sheath were slowly pooled together outside the nephrostomy tract as haemostatic agent injection was carried out. In total, each sealant was placed in eight nephrostomies (four cases of both tract and renal unit and four cases of tract only). Four nephrostomies were left intact and served as controls. The study design is shown in Table 1.
|Right-side nephrostomy||Left-side nephrostomy|
|Nephrostomy tract||Renal unit||Nephrostomy tract||Renal unit|
The haemostatic agent application technique of differed from agent to agent, owing to the different characteristics of each product. Helisorb® application was considered the most problematic because of its powdered nature. To apply the sealant in the deep aspect of the nephrostomy tract, the applicator tube provided by the company was used. The former was visible during fluoroscopy. Nevertheless, we were unable to estimate the exact amount of powder that was actually reaching the open tip of applicator. Two packs of Helisorb® powder were used in each nephrostomy. Tachosil® application was achieved by folding the sponge into a tubular shape. The sponge was then inserted from the open tip of the nephrostomy sheath and was advanced to the indicated place under flurosocopic guidance (the Tachosil® sponge itself was radiolucent, but the folded sponge was visible during fluoroscopy as a result of contrast medium transposition as the sealant was inserted inside the nephrostomy sheath). Floseal® application was considered the most convenient of all. Using the metal applicator provided by the company, the agent was injected to the indicated place under flurosocopic guidance (Fig. 1).
The variables studied were assessment of renal drainage and identification of urinoma formation during the observational period as well as histological lesions observed in the nephrostomy tract and the penetrated renal unit at the end of the follow-up.
Preoperative assessment of individual collecting systems and ureteric anatomy was performed by CT urography in all the pigs, with the pigs under deep sedation. Postoperative urinary drainage and urinoma formation was evaluated by CT urography on postoperative days 1, 15, 30 and at the termination of the experiment on postoperative day 40, immediately before the pigs were killed.
The pigs were killed using high doses of anaesthesia and both kidneys and nephrostomy tracts of each pig were harvested and sent for pathological examination. A representative area of each specimen was processed for haematoxylin and eosin staining and reviewed by a single pathologist.
Nephrostomies were established bilaterally in all pigs without significant bleeding or other complications. All animals tolerated the procedure well and survived throughout the whole follow-up period.
On CT urography on postoperative days 1, 15, 30 and 40 normal drainage was observed in all control nephrostomy tracts (left sides of pigs 1–4). No evidence of postoperative urinoma or haematoma formation was noted during the follow-up. Histological evaluation of control kidneys showed no significant microscopic lesions. In the perinephric tissue, the presence of hemosiderin as well as small thrombi in the lumen of several blood vessels were observed. Focal lesions of haemorrhagic infarct were observed in the rest of the tract.
In two cases of Helisorb® application (right side of pigs 1 and 8) the presence of a foreign body (most likely a clot) inside the renal pelvis and upper ureter was identified on the first postoperative day (Fig. 2). Both cases had the sealant placed inside the renal parenchyma which indicates that a part of the sealant may have entered the collecting system leading to thrombus formation. The latter formation did not occlude normal drainage in either case and resolved spontaneously on postoperative day 15.
Histological evaluation of the renal parenchyma injected with Helisorb® (right sides of pigs 1, 8, 12, 13) showed several areas of chronic tubulointerstitial nephritis. Hystiocytes, lymphocytes and plasma cells formed the bulk of the inflammatory infiltrate. Vascular dilatation and hyperplasia was also recognized, mainly involving the interstitium near the collecting system. As far as the urothelium was concerned, foci of chronic inflammation with the presence of granulation tissue underlying sites of simple urothelial hyperplasia were also observed (Fig. 3). In the tissue around the tract area, non-specific lesions, such as focal fibrosis and sites of chronic inflammation, were seen. In the pigs where Helisorb® had been injected only in the tract region (right sides of pigs 5, 6, 9 and 10), the main histological findings were localized in the urothelium of the renal pelvis where sites of focal urothelial hyperplasia were observed. In these renal units, the renal parenchyma showed no significant histopathological changes.
Postoperative CT urography on day 1 showed the presence of hydronephrosis and a significant urinoma in one pig (left side of pig 13) in which Tachosil® had been introduced inside the renal parenchyma. In this particular case, urinary drainage towards the bladder was absent until postoperative day 15. On postoperative days 30 and 40, normal drainage was evident with shrinkage of ipsilateral hydronephrosis and urinoma. The urinoma was finally organized into a relatively large perinephric mass (Fig. 4). The postoperative period was uneventful for the rest of renal units in the Tachosil® group, all demonstrating normal urinary drainage throughout the follow-up.
Histological evaluation of the single renal unit where drainage occlusion and urinoma formation had been observed, showed a dense inflammatory infiltrate in the renal parenchyma with the presence of inflammatory cells, both in the interstitium and within the tubular lumina, accompanied by focal tubular damage. Fibrosis of the kidney parenchyma was observed, as well as rare foci of glomerulal sclerosis. Many areas of vascular thrombosis were identified, mainly near the renal capsule while areas of necrosis and abscess formation were also present outside the renal capsule, accompanied by a foreign body reaction. The urothelium appeared focally hyperplastic (simple urothelial hyperplasia) with lesions similar to those seen in cystitis glandularis. The most notable histological finding observed in the tract area of this nephrostomy were sites of inflammation as well as severe vascular damage with multiple calcifications (Fig. 5).
Histological evaluation of the remaining renal units in the Tachosil® group were found to have significantly less histological deterioration than the one described in the above case of obstruction. From these units, in all kidneys that had the sealant placed inside the renal parenchyma (right side of pigs 2 and 14 as well as left side of pig 7) moderate chronic inflammatory infiltrates and focal fibrosis of the renal cortex and medulla were observed. In the renal pelvis, lymph follicle formation and the presence of hemosiderin were the main histological findings, accompanied by several foci of fibrosis and the presence of fibrin in some blood vessels (Fig. 6). Moreover, sites of severe thrombosis and lesions of haemorrhagic infarct were identified in the perinephric tissue, outside the renal capsule. In the tract area, a foreign body inflammatory reaction and focal haemorrhagic lesions were seen. In the pigs where Tachosil® had been injected only in the tract region, no significant histopathological changes were detected in the renal parenchyma.
Normal drainage was preserved in all renal units in the Floseal® group, throughout the follow-up period. Histological evaluation of units that had the sealant placed inside the kidney (the right side of pigs 3, 4, 14 and left side of pig 12), showed chronic inflammation in the renal parenchyma with distinctly focal fibrosis as well as several sites of vascular thrombosis and dilatation. Additionally, the urothelium was focally inflamed. Foci of vasculitis and vascular damage with accompanying necrosis were identified in the perinephric tissue. The tract tissue showed foci of inflammation, vascular dilatation and multiple calcifications (Fig. 7). In one case (right side of pig 3), the formation of a fibrino-adipose mass outside the renal capsule accompanied by a foreign body reaction was identified. As this formation had the same histological characteristics as the one observed in the Tachosil® group, the latter was attributed to organization of a mild urinoma (not initially identified by the CT urography) and not to a specific reaction against the applied haemostatic. In the case of renal units where the sealant was placed in the tract only, no significant histological lesions were identified in the renal parenchyma.
The lack of postoperative drainage is considered to be a disadvantage of the tubeless PCNL technique, mainly with regard to potential delayed bleeding. Application of haemostatic agents in the nephrostomy tract after the procedure has been proposed as a useful adjunct to tubeless PCNL, inducing immediate haemostasis and thus eliminating the need for postoperative drainage. Fibrin glue, gelatin matrix and oxidized cellulose application are haemostatic sealants that have already been evaluated in several clinical studies [8-14]. Nevertheless, the exact effect of these haemostatic agents on the renal drainage and histology is still not clear.
Several experimental studies question the safety of this haemostatic sealant application, mainly focusing on the potential of such materials to occlude urinary drainage. Uribe et al. , in an in vitro experimental setting evaluated the mixing of several haemostatic agents with urine. Fibrin glue and oxidized regenerated cellulose maintained a solid form when initially mixed with urine and then assumed to a semisolid gelatinous state, which remained in the same form during the 5 days of observation. Polyethylene glycol formed a solid clot, that did not dissolve after 5 days. Only the gelatin matrix formed a fine particulate suspension. In addition, Kim et al.  examined the effect of direct injection of various haemostatic agents in the porcine collecting system through a nephrostomy tube. A risk of obstruction in >50% of applied cases that did not resolve over a 5-day period was reported. In the present study, drainage occlusion was observed in only 5% of cases where a haemostatic sealant was used (1 out of 20 renal units). The difference between the two studies can be attributed to the fact that, in the present experimental setting, haemostatic agents were not purposely injected inside the collecting system. Yet, the single case of obstruction, which was observed in one case of Tachosil® application, as well as the presence of clot inside the renal pelvis in two cases in the Helisorb® group, show that the danger of occlusion existed for all renal units but was avoided in the majority of cases. Consequently, the present study verifies that haemostatic agent application during tubeless PCNL presents a risk for urinary drainage obstruction and, therefore, measures to avoid the insertion of such materials to the collecting system (e.g. sealing the collecting system with a balloon catheter) are recommended.
Histological data from the present study show that haemostatic agent application after percutaneous nephrostomy is not harmless for the kidney. All three examined agents induced an inflammatory reaction in the renal units (Table 2). It is worth mentioning that renal histological lesions were identified not only around the nephrostomy tract but, to a lesser degree, to parenchymal areas located away (>1 cm) from the site of penetration as well. It remains to be investigated whether a possible renal inflammatory reaction was responsible for the increased pain (resolved 1 month postoperatively) observed after haemostatic sealant application, as reported in a prospective randomized study comparing tubeless Floseal® closure with fascial stitch or Cope loop nephrostomy tube placement after PCNL .
|Site of application (no. of renal units)||Main renal histopathological findings||Comments|
|Control||None (4)||No significant lesions|
|Helisorb®||Tract and kidney (4)||Chronic tubulointerstitial nephritis, chronic urothelial inflammation, urothelial hyperplasia.||Clot inside renal pelvis and upper ureter in two cases without drainage occlusion.|
|Tract only (4)||Mild urothelial hyperplasia in the renal pelvis.|
|Tachosil®||Tract and kidney (4)||Moderated chronic inflammatory infiltrates and focal fibrosis of the renal cortex and medulla.||One case of drainage occlusion/significant histological deterioration in the particular case.|
|Tract only (4)||No significant lesions.|
|Floseal®||Tract and kidney (4)||Moderate chronic inflammatory infiltrates and focal fibrosis of the renal cortex and medulla.|
|Tract only (4)||No significant lesions.|
Owing to the limited number of animals, reliable statistical analysis of the histological data was not possible and thus, the present study failed to demonstrate any superiority of one haemostatic sealant over the others in terms of induced histological reaction. Nevertheless, it was evident that the solid form of the Tachosil® sponge and the gel form of Floseal® were superior to the powdered form of Helisorb®. In the case of Helisorb®, renal reaction was observed even in cases where the agent was not applied inside the renal parenchyma, but was injected in the s.c. part of the nephrostomy tract only. In addition, clot formation inside the renal pelvis in two cases of parenchymal application was an indication that some amount of substance may have slipped inside the collecting system. By contrast, Tachosil® and Floseal® placement was more accurate and signs of relocation from tract to kidney were not observed. Finally, tubeless PCNL without sealant application in this experimental series was associated with no morbidity (urinoma or clot formation) and the fewest histopathological changes. Based on the latter, it might be safer to perform tubeless PCNL without the use of any sealants in properly selected cases.
A limitation of the present study is that differences between porcine and human kidney reaction to the examined haemostatic sealants might exist. Moreover, potential functional deterioration of the examined renal units was not evaluated (porcine renal scintigraphy was not available and biochemical markers are unreliable for examining unilateral functional deterioration). Thus, further studies in other experimental models are needed to reinforce the conclusions of this work. In addition, the rationale of extending the follow-up period to 40 days after the procedure, was based on the maximum period of housing allowed by our animal care unit. Nevertheless, inflammatory reactions seen in the post mortem specimens could potentially subside after a longer period, which is quite possible given that particular haemostatic sealants have been traced in the nephrostomy tract even after 30 days postoperatively . Consequently, given that the time for absorption of fish origin collagen, human fibrinogen and thrombin as well as bovine-derived gelatin matrix components of the applied haemostatic sealants is unknown for the porcine organism, the relatively short duration of this trial should also be considered to be a limitation.
Furthermore, particular aspects of our model, including suturing of the nephrostomy incision and overdilatation of the nephrostomy tract could induce a tissue reaction which, in turn, could complicate the interpretation of our histological data. Suturing of the nephrostomy site itself could induce local inflammation. Nevertheless, given that the particular part of the procedure was identical for all groups, and histological analysis did not reveal significant inflammatory reaction at tissues located near to the suturing site we consider the impact on our results of suturing to be small. Additionally, nephrostomy tract dilatation by up to 30 F in the relatively small kidney of porcine experimental subjects can be considered an overdilatation which might be a contributing factor to the histological findings of the study. Such dilatation was required to imitate exactly our clinical practice in humans, allow nephroscope insertion and ease the Tachosil® folded sponge application which was relatively thick. Nevertheless, the effect of overdilatation was equal in all nephrostomy tracts and consequently its impact on the observed histological differences between study groups should be considered minimal. Finally, owing to the lack of adequate statistical power of the study, further investigation is required so as to define which haemostatic sealant is superior to the others in terms of inducing the least histological reaction to the injected renal units.
In conclusion, data from the present study indicate that haemostatic agent application in the nephrostomy tract is associated with a risk of renal drainage occlusion and measures to prevent the injection of these agents into the collecting system should be recommended. Moreover, taking into consideration all limitations related to an experimental study, it was revealed that haemostatic agents induce an inflammatory reaction to the injected renal units. While none of the studied haemostatics was proven superior to the others, solid and gel sealants were more accurately placed in the nephrostomy tract and showed no signs of relocation. Further investigation into the effects of each haemostatic sealant in the kidney is recommended before this tool can be regularly adopted for use during tubeless PCNL in humans.