World J. Surg. 25, 882-885. 2001 DOh 10.1007/s00268-001-0045-0 WORLD

Journal of SURGERY r 2001 by the Sock"tv;

[nternadonale de Cbirurgie'

Evaluation of Risk of Splenosis during Laparoscopic Splenectomy in Rat Model

Joan Josep Espert , M.D., 1 Edua rd M. Targarona, M.D., Ph.D., L2 Ernest Bombuy, M.D., t Josep Setoain, M.D., 3 Josep Visa, M.D., Ph.D., 1 Manuel Trias, M.D., Ph.D. z

~Service of General and Digestive Surgery, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain "-Service of General and Digestive Surgery, Hospital de Sant Pau, Avenida P. Claret 165, 08025 Barcelona, Spain ~Service of Nuclear Medicine, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain

Abstract. Laparoseopic splenectomy (LS) is an alternative to open sur- gery. However, there is a theoretic risk of splenosis and abdominal cavity dissemination of splenic cells if the splenic capsule is broken, as seen by experimental evidence of tumoral cell mobilization by the pneumoperito- neum. We evaluated the features of splenosis after splenectomy operated via an open approach or under laparoseopic control in an experimental model in the rat. A total of 65 Sprague-Dawley rats were distributed in seven groups that included the open approach, CO2 pneumoperitoneum LS, or wall lift LS with or Without a splenic graft. Splenic function was evaluated 90 day later through (1) scintigraphy with Tc-labeled heat- damaged erythrocytes; (2) determination of circulating "pitted" cells; and (3) analysis of the distribution of splenic pulp in the peritoneal cavity. Scintigraphy did not show viable residual tissue in any group after spleneetomy; splenic activity in the splenic fossa was observed in 40% of the animals with grafts. Splenectomy increased the "pit" cell count, but it was reduced to normal values with a splenic graft. Necropsy showed normal splenic tissue in the splenic fossa in 100% of animals with a graft. Abdominal implants were observed significantly more frequently after CO, LS than after the open surgery or a wall lift LS (80% vs. 20% vs. 30%; p < 0.05). In addition, trocar site implants were observed with CO 2 LS (n = 3) or wall lift LS (n = 2), whereas there were no implants in the wound in the open group. We conclude that in an e~xperimental rat model the pueumoperitoneum may facilitate abdominal splenosis after LS if the splenic capsule is ruptured or if splenic tissue spills compared with surgery without gas (open or laparoseopic).

Laparoscopic splenectomy (I_N) has become accepted as an alterna- tive to open surgery [1-3], and some authors have suggested that it should be the technique of choice for benign hematologic diseases and normal-size spleens [4, 5]. However, splenectomy is not fre- quently performed in surgical units, and LS is a technically demand- ing procedure. These facts have precluded the realization of random- ized trials to evaluate the potential advantages of this new alternative.

Splenic tissue is frail; and if spilled in the abdomen it favors the

This article was presented as an original scientific report during the 7th International Congress of the European Association for Endoscopic Sur- gery, Linz, Austria, June 1999. It was also accepted for poster presentation at the Society of American Gastrointestinal Endoscopic Surgeons Scien- tific Meeting, Atlanta, GA, USA, March 2001).

Correspondence to: E.M. Targarona, M.D., Ph.D., Sen'ice of General and Digestive Surgery., Hospital de la Santa Creu i Sant Pau, Sant Antoni M. Claret 167, 08025 Barcelona, Spain. e-mail: 7666@hsp.santpau.es

development of splenosis, a severe complication that can induce the relapse of hematologic diseases, mainly autoimmune plaquetopenias (low platelet count). Several authors have found evidence of residual intraabdominal tissue after LS [6, 7]; and in recent years the poten- tially adverse effect of pneumoperitoneum on the dissemination of intraabdominal malignancies has been well evaluated [8, 9]. The aim of this study was to evaluate the existence and features of postoper- ative splenosis after splenectomy operated on through an open ap- proach or under laparoscopic control.

M a t e r i a l s a n d M e t h o d s

Sixty-five 325 g Sprague-Dawley rats (Pantab, Barcelona, Spain) were distributed in seven groups: group I, control (n = 5); group II, open splenectomy (n = 10); group [II, open splenectomy plus splenic graft (n = t0); group IV, laparoscopic splenectomy (n = 10); group V, laparoscopic splenectomy plus splenic gr~fft (n = t0); group VI, laparoscopic splenectomy with abdominal wall lift (n = 10); group VII, laparoscopic splenectomy with abdominal wall lift plus splenic graft (n = t0). In the groups with splenic graft, the whole spleen was morcellated, the pulp localized in the splenic tbssa, and the pneu- moperitoneum maintained for 20 minutes.

Surgical Techniques Used for Splenectomy in the Rat

All procedures were performed using a clean technique with sterile material. Anesthesia was induced with ether and main- tained with intramuscular ketamine (2.5 ml/kg).

Open splenectomy was performed through a midline incision. After mobilizing the stomach the spleen was eviscerated, and the splenic hilum was severed between ligatures. The abdominal wall was closed with a running suture of nonabsorbabte material.

Laparoscopic splenectonn' was performed following a previously described technique [i0]. Briefly, the rat lies in supine decubitus and 6 mmHg pneumoperitoneum is induced at a rate of 0.8 to 1.2 L/min after introducing a 5 mm disposable sheath through a 5 mm skin incision in the umbilicus. Then a 3 or 5 mm 0-degree scope is inserted. Two additional trocars are used: a 2 mm Swan-Ganz catheter sheath in the right iliac fossa and a 5 mm trocar (Tyco Auto Suture, Norwalk, CT, USA) in the left iliac fossa. A 2 mm

Espert et al.: Splenosis and Laparoscopic Spleneetomy 883

arthroscopy forceps (Storz, Tuttlingen, Germany) is inserted through the left port, and a 5 mm endoclip applier (Tyco Auto Suture) is inserted through the right port. Gentle dissection re- veals the rat's spleen, which is loosely attached to the stomach by a filmy epiploon. The splenic hilum and tail of the pancreas are identified, and two or three windows are created between the vessels. Then, while holding back the spleen with the left instru- ment, the vessels are ligated with a multiclip applier. A 2- or 5-mm pair of scissors (Tyco Auto Suture) replaces the endoclip, after which the splenic hilum is severed. The spleen is extracted through the right incision.

Laparoscopic splenectomy without pneumoperitoneum was per- formed using a Foley balloon catheter as a wall lifter. The surgical technique was similar to that used for LS with pneumoperitoneum.

Induction of SpIenosis

A preliminary study using a 30% morcellated spleen implant showed minimal residual tissue after 90 days without uptake of the scintigraphic isotope. We therefore used a 100% morcellated spleen implant. Prior to closure of the abdomen or exhaustion of the pneumoperitoneum, the whole spleen was morcellated in situ and left in place, and the pneumoperitoneum was maintained for 20 minutes. Great care was taken to avoid dropping the splenic tissue from laparoscopic instruments into the abdominal cavity once the spleen was morcellated.

Evaluating the Existence of SpIenosis

Ninety days later the animals were evaluated for the existence of splenosis through (1) determination of "pitted" cells; (2) scintig- raphy with technetium (Tc)-labeled heat-damaged erythrocytes; and (3) anatomic and histologic analysis of the distribution of splenic pulp in the peritoneal cavity.

"Pitted" Cell Measurement. For the pitted cell measurement [11] A drop of fresh venous blood was mixed with 0,5 ml 3% glutar- aldehyde. Around I000 red blood cells were examined in a wet preparation (magnification • 1000) with direct interference con- trast microscopy with a "Nomarsky" optic. The percentage of cells with one membrane abnormality or more, visible as pits under interference microscopy, was calculated.

Scintigraphic Analysis. Technetium 99m sodium pertechnetate heat-damaged red blood celt scintigraphy was performed using 1.5 ml of autologous blood. After blood sample retrieval, estanium pyrophosphate 2 p.g/ml was added, and the sample was incubated for 10 minutes. 99mTc 2 mCi was added, and the red blood cells were damaged by immersing the container in water heated to 49.5~ Images were obtained 60 minutes after injecting the iso- tope in an LFOV 609 gamma camera high-resolution collimator (Elscint, Haifa, Israel)

Anatomic and Histologic Analysis. Al~ter scintigraphy, the animals were killed with an anesthesia overdose. A systematized laparot- omy was performed to evaluate the number and site of the splenic nodules: (1) splenic fossa; (2) another site in the abdominal cavity; and (3) trocar sites or laparotomy wound. All the samples ob- tained were studied histologically (hematoxylin-eosin).

Table 1. Comparative duration of splenectomy according to the technique used.

Duration of splenectomy (minutes)

Open splenectomy CO 2 LS Wall lift LS Condition (n = 10) (n = 10) (n = 10)

No graft 15.4 ~ 2.8 33.7 +- 4* 36.7 +- 5.8* 100% Graft 17.3 • 2.3 33 • 9.5 :~ 33.0 -- 3.0*

LS: taparoscopic splenectomy. *p < .001 vs. open procedure.

Statistical Analysis. Statistical analysis was performed with Stu- dent's t-test for quantitative variables with normal distribution. Differences between proportions were analyzed using Fisher's exact test and Bonferroni's correction.

Results

Scintigraphy showed splenic actMty in all control rats. The pitted cell percentage in control groups was 4.0% - 1.1%. The duration of the procedure for laparoscopic splenectomy (CO, or wall lift) was significantly longer than that for open splenectomy (Table 1).

Scintigraphic study and necropsy of groups with total splenectomy performed by any surgical technique did not show residual splenic tissue. The percentage of pitted cells in these groups was significantly higher than in the control group (p < 0.05 vs. control) (open sple- nectomy 25.7% • 10.5%, laparoscopic splenectomy 12.7% • 5.0%, wall lift splenectomy: 19.9% -+ 7.0%) (Tables 2, 3, 4).

The open splenectomy plus implant group showed a pitted cell count of 7.5% • 4.5%. (p < 0.005 vs. group II). Scintigraphy showed splenic activity in 30% of rats, but necropsy and histolo~c analysis revealed viable splenic tissue located in the splenic fossa in 100% of animals. Abdominal implants were observed in two animals without implants at the abdominal wound. (Tables 2, 3, 4).

The laparoscopic splenectomy group with implants had a mean pitted cell count of 5.9% • 4.5% (p < 0.005 vs. group IV). Five animals had positive scintigraphy, and the images were located in the left hypochondrium in all cases. However, in the necropsic study, all 10 animals had implants in the splenic fossa, and in 8 of the 10 (80%) implants were also located in the abdomen. Three cases showed trocar site implants. Histologic analysis showed normal splenic tissue in all cases (Tables 1, 2, 3, 4).

Walt lift laparoscopic splenectomy plus implants showed a mean pitted cell rate of 7.6% • 3.0% (p < 0.005 vs. group II). Four animals showed isotope uptake, but all 10 had residual tissue in the splenic fossa. In three cases additional splenic tissue was observed in the abdominal cavity, and two trocar site implants were observed (Tables 2, 3, 4).

Discussion

In recent years, laparoscopic splenectomy has become accepted as an alternative to the open approach, as it presents all the advan- tages of laparoscopic surgery, especially for an excisional proce- dure [1-3]. However, after the demonstration of port-site recur- rences during laparoscopic surgery for malignant conditions, concern has arisen regarding the risk of splenic tissue dissemina- tion if the splenic capsule is broken and cell spillage occurs. Another controversial question is the difficulty of identifying ac- cessory spleens during LS.

884 World J. Surg. Vol. 25, No. 7, July 2001

Table 2. Percentage of "pitted" cells observed in peripheral blood cell smears after sptenectomy.

% Pit cells after splenectomy

Condition Control Open (n = 10) CO 2 LS (n = 10) Wall lift LS (n = I0)

No graft - - 26 +- t4 i3 + 6 ..... 20 _+ 8 100% Graft 4 --+ 1 7.5 + 6* 4 + 3*'*** 7.6 .-2 4*

*p < 0.001 vs. no-graft groups; **p < 0.05 vs. open and wall lift LS without graft; ***p < 0.05 vs. open and wall lift LS with graft.

Table 3. Scintigraphic exploration with Tc-labeled erythrocytes

Scintigraphic findings during splenectomy

Open CO 2 LS Wall lift LS Condition (n = t0) (n = 10) (n = 10)

No graft Negative Negative Negative 100% Graft

Splenic fossa 3 5 4 Abdomen 0 0 0 Trocar site 0 0 0

Table 4. Histologic analysis of the distribution of splenic pulp in the peritoneal cavity.

Splenic pulp in peritoneal cavity after splenectomy (%)

Open CO~ LS Wall lift LS Condition (n = 10) (n = 10) (n = 10)

No graft - - - - - - 100% Graft

Splenic fossa 100 100 100 Abdomen 20 80* 30 Trocar site 0 30 20

*p < 0.05 vs. open or wall lift LS.

We have shown evidence of the existence of residual splenic tissue after LS [6]. Gigot et al., in a prospective series of 18 LSs, also demonstrated the existence of residual splenic tissue by de- naturated red blood cell scintigraphy (DRBCS) and single photon emission computed tomography (SPECT) reconstruction in up to 50% of cases [7]. Seeding of spilled splenic cells in a high-pressure pneumoperitoneum during the slightly longer operation is a new problem and one that is specific to LS. In our series of 122 LS cases, 45% were performed for treatment of idiopathic thrombo- cytopenic purpura (ITP); in one case that required conversion because of splenic bed oozing, the plaquetopenia relapsed and computed tomography (CT), scintigraphy, and the pitted cell count showed splenic residual function [6]. Gigot et al. [7] found splenic seeding in three of four cases in which the splenic capsule was turned out. A more worrisome finding was the presence of positive isotopic scans in patients without AS or capsule rupture.

Splenic function can be assessed by several means. Imaging studies may identify amounts of tissue up to 1 cm (CT scan, ultrasonography). DRBCS shows the existence of tissue able to take up isotope. Correct functioning of the splenic architecture is evaluated by the rate of pitted cells or Howell-Jolly bodies in peripheral blood smears [11]. Residual splenic tissue should be enough in quantity as well as in adequate architecture to restore splenic function and potentially to induce relapse of the hemato-

logic diseases. A lesson learned from the treatment of residual accessory spleens after splenectomy for ITP is that the disease is cured after accessory splenectomy in only half of the cases [12].

We designed an experimental model to assess features of splenosis during LS. We used a whole morcellated spleen model to ensure that there was enough viable splenic tissue, because previous preliminary analysis with a 30% or 50% morcetlated spleen model was not followed by viable implants. This model is not comparable to the clinical setting, but it is well known that even a small quantity of splenic tissue may induce relapse of ITP [11]. Despite absolute anatomic differences between the rat spleen and the human spleen, it is clear that when LS is performed and the splenic capsule is maintained intact there is no risk of cell spillage. However, we obtained evidence of intraabdominal diffuse splenosis and port-site implants following application of a pneumoperitoneum in a model of spleen morcetlated implants in the splenic fossa after LS using a CO2 pneumoperitoneum. Splenic implants after open splenectomy were always localized in the splenic fossa; and after wall lift splenectomy we also observed a reduced incidence of intraabdominal implants not localized in the splenic fossa. This means that, in much the same way as with models of intraabdominal dissemination of malignant tumors, the pneumoperitoneum is able to mobilize splenic tissue if the spleen capsule does not protect it. This possNility is related to the amount of splenic tissue implanted. Possible explanations of the dissemina- tion of the tissue are diverse. The key factor may be the closed cavity and a tension pneumoperitoneum, with continuous gas exchange, which can mobilize splenic tissue during morcellation. Support for this option is provided by observation of a larger number of implants in the pneumoperitoneum ~oup than in the wall lift LS ~oup. Another reason could be the transport of cells by contaminated instruments during splenic manipulation, but the role of the pneu- moperitoneum is also highlighted because implants were more fre- quent in groups with pneumoperitoneum than in laparoscopic groups without gas.

A striking finding is that after laparoscopic splenectomy with CO2 pneumoperitoneum we observed an increase in the pitted celt rate, but it was significantly smaller than with the other two types of splenectomy without pneumoperitoneum. In addition, pitted cell counts in laparoscopic groups with implants were sig- nificantly lower than after total sptenectomy, and the reduction in the CO 2 LS group was larger than in the other groups. These results are difficult to interpret, but a link with CO. is suggested.

The results of this study have clear clinical implications. The spleen has a capsule; and if it is maintained intact, there is no reason for cell spillage during LS. Current LS technique with a lateral or semitateral approach makes it possible to open the lesser sac, search for accessory spleens, and clip the ~u'tery with minimal handling of the spleen. Mobilization of the posterior face of the spleen and stapling of the hilum can be done without damage to the capsule

Espert et al.: Splenosis and Laparoscopic Splenectumy 885

thanks to the wide mobility of the spleen that is obtained. Careful exposure with probes, rather than gasping the spleen or adjacent tissue, may avoid capsule tears. Furthermore, methods tbat can spread splenic tissue (e.g., argon beam coagulators) should be avoided when trying to control surface splenic bleeding.

In conclusion, great technical care is required during LS to avoid rupture of the splenic capsule, which may induce splenic spilling facilitated by the pneumoperitoneum and secondary fail- ure of the hematologic disease.

Rdsum6

La splrnectomie par laparoscopie (SL) est une alternative h la chirurgie ouverte. Cependant, 6tant donn6e l'rvidence exp&imentale de dissdmination tumorale en rapport avec le pneumoprritoine, il existe un risque throrique de spldnose et de diss6mination de cellules splrniques dans la cavit6 abdominate si la capsule splrnique est rompue. Nous avons 6valur, darts un modrle exprrimental chez le rat, les caractrristiques de splrnose apr~s spldnectomie effecturc soit par chirurgie ouverte soit par laparoscopie (SL). On a divis6 65 rats Sprague Dawley en sept groupes comprenant une approche ouverte, une spl6nectomie laparoscopique avec pneumopdritoine ou par ,~wall lift,,, avec ou sans greffe spldnique. On a 6vain6 la fonction spldnique 90 jours apr~s l'intereention par : 1) la scintigraphie aux 4rythrocytes traitrs par ta chaleur et marqurs au Tc; 2) la drtermination des cellules circulantes contenant des corps de Howell-Joly, et 3) l'analyse de la distribution de la pulpe splrnique darts la cavit6 abdominale. La scintigraphie n'a montr6 de cellules viables rdsidnelles darts aucun des ~oupes apr~:s splrnectomie. Une activit6 splrnique a 6t4 observre dans I'hypochondre droit chez 40% des arfimaux greff4s. Le tatLx de globules rouges avec corps de Howell-Joly a augment6 aprhs spldnectomie, mais est rest6 dans les limites de la normale aprhs geffe. L'autopsie a montrdu tissu sptrnique normal dans l'hypochondre droit chez 100% des animaux avec greffe. Les implants abdominaux ont 6t6 observds significativement plus frdquemment apr~s pneumoprritoine au CO2 qu'apr& splrnectomie par laparotomie ou avec ,~all lift,> (80 vs 20 vs 30%, p < 0,05). De plus, les implants au niveau des sites de trocart ont 6t6 observ6s apr& SL au CO 2 (n:3) ou rdtracteur pari6tal (n:2), mais pas apr6s spl6nectomie par laparotomie. Nous concluons que dans ce mod61e expdrimental, chez le rat, le pneumopdritoine pourrait faciliter la spldnose abdominale apr~s SL si la capsule est rompue ou si on perd du ti~u spl6nique.

Resumen

La esplenectomia laparosc6pica ( IS) constituye una alternativa a [a cirugia abierta. Sin embargo, al menos desde el punto de vista te6rico y basados en el hecho de que el neumoperitoneo facilita la movilizacidn y diseminacidn de las crtulas tumorales, existe el riesgo, sl la cfipsula del bazo se rompe, de producir una esplenosis, con diseminacidn de las c61ulas esplrnicas pot la cavidad abdominal. Tratamos de evaluar la posibilidad de esplenosis en un modelo experimental en ratas tras esplenectomia abierta y laparoscdpica. 65 ratas Sprague Dawley se distribuyeron en 7 grupos que incluyeron: cirugia abierta, cirugia laparoscdpica con neumoperitoneo de CO2, cirugia laparosc6pica con elevacidn de la pared abdominal, con o sin injerto espKnico. La funcidn espldnico ka funci6n espldnica se valor6 a los 90 dias de la intervencidn mediante: 1 ~ Escintigrafia con

Tc-marcado con eritrocitos lesionados mediante calor. 2 ~ Determination de c61ulas "pitted" circulantes. 3 ~ AnAlisis de la distribucidn de ta pulpa esptdnica en ta cavidad abdominal. Los estudios escintigr~ificos no mostraron tejido residual viable, tras esplenectomia, en ninguno de los grupos; constatamos actividad esplrnica en rosa espldnica en el 40% de los animales que fueron sometidos a un injerto de bazo. La esplenectomia incremental el nfimero de crlulas "pit" (con superficie deprimida) circutantes, que se redujeron a valores normales tras el injerto espldnico. En la autopsia se observ6 tejido esplrnico normal en rosa esplrnica en el 100% de los animales con injerto de bazo. Los implantes espldnicos abdominales fueron mils frecuentes tras esplenectomia laparosc6pica con neumoperitoneo con CO a, que tras druNa abierta o laparosc6pica con elevacidn de la pared abdominal (80% vs. 20% vs. 30%; p < 0.05). Se observaron implantes en las puertas de los trdcares en 3 casos de esplenectomia laparoscdpica con CO2 y en 2 con esplenectomia laparoscdpica con elevacidn de la pared abdominal. No se observ6 implante alguno en los esplenectomizados con cirugia abierta (convencionat). Conclusidn: En el modelo experimental en ratas, el neumoperitoneo propicia la esplenosis abdominal tras esplenectomia laparosc6pica, si se rompe la c@sula o se vierte tejido esplrnico, 1o que no ocurre tras cirugia sin utilizaci6n de neumoperitoneo (abierta o laparoscrpica).

Acknowledgment

This study was supported by grant 97/970 of FIS.

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