Abstract
Purpose: Microwave ablation therapy for secondary splenomegaly and hypersplenism has been shown to be effective from pre-clinical animal models and clinical investigations. This study was performed to determine its effects on the status of peripheral lymphocyte subsets in patients receiving microwave ablation of the spleen.
Materials and methods: Ten patients with secondary splenomegaly and hypersplenism received microwave ablation of the spleen during laparoscopy or percutaneously under ultrasound guidance. The percentage peripheral blood T cells, B lymphocytes and NK cells were measured using flow cytometry before and on days 1, 3 and 7 after therapy, as well as 1 and 3 months afterwards.
Results: Percentages of CD3+ and CD4+ cells increased rapidly 1 month after therapy. There was no significant change in CD8+, CD4+/CD8+ or NK cells of the pre- and post-therapy levels and B lymphocytes increased significantly after therapy. In patients with an ablation volume (AV) less than 20% (group A), T cells increased 1 month after ablation but decreased 3 months after ablation. B lymphocytes increased significantly after surgery. Levels of NK cells were lower than that before therapy on each testing. In patients with 20–40% AV (group B), levels of T cells, B lymphocytes and NK cells showed an increase. Levels of CD4+ cells were significantly higher in group B than in group A, 3 months after therapy.
Conclusions: Microwave ablation therapy for splenomegaly and hypersplenism appears to have a favourable effect on peripheral lymphocyte subsets. A relationship may exist between the ablation volume and the level of peripheral lymphocyte subsets.
Introduction
Hypersplenism is a well-known complication in portal hypertension of hepatic cirrhosis that may increase the incidence of infection and bleeding. It is manifested with symptoms of anaemia, thrombocytopaenia or neutropaenia and may lead to the increased risk of gastrointestinal bleeding. Splenectomy has been widely used for treating hypersplenism in cirrhotic patients Citation[1–3]. However, splenectomy carries a high operative risk, especially in medically compromised patients. It has been found to be associated with overwhelming sepsis from encapsulated micro-organisms Citation[4]. Classic splenectomy is often associated with loss of splenic immune function and high morbidity and mortality rates Citation[5]. Being aware of the importance of the spleen in the immune system, splenic conservative methods have gained prominence in treating benign conditions of this organ Citation[6–8]. These methods aim at preserving part of the splenic anatomic structure to maintain its physiological functions (especially its immune function). Minimally invasive modalities such as transcatheter selective splenic arterial embolization Citation[9], absolute alcohol or ethanolamine oleate intrasplenic injection Citation[10], Citation[11] or radiofrequency ablation for hypersplenism Citation[12] have been investigated clinically. Owing to various complications, however, their clinical application is restricted.
The technique of implanting microwave electrodes into tissues has been used clinically since the late 1970s Citation[13], Citation[14]. Microwave radiation causing tissue coagulation and small vascular occlusion has been used during surgery for haemostasis and tissue resection. In recent years, ultrasound-guided percutaneous microwave coagulation therapy (PMCT) has been adopted for treating hepatocellular carcinoma (HCC) with good clinical results Citation[15–21]. Our team has studied the use of microwaves for 12 years and obtained significant clinical benefits using microwave ablation therapy for HCC Citation[22]. PMCT can produce a controlled volume of necrosis and can be used for haemostasis, so it might be adopted to decrease the splenic parenchyma in treating hypersplenism. It was reported that in patients receiving microwave ablation therapy for HCC, the immune function towards tumour was enhanced Citation[23–27]. However, no studies have been conducted yet studying the effects of microwave ablation therapy for splenomegaly and hypersplenism on parameters associated with the patient's immune system. Variation in the normal percentages of various lymphocyte subsets in the periphery is an important index of immune function. The present study aimed to evaluate the effect of splenic microwave ablation on percentages of peripheral lymphocyte subsets.
Materials and methods
Patients
From March 2005 to April 2006, 10 patients (9 males, 1 female) with secondary splenomegaly and hypersplenism aged 33–67 years (mean 51.8 ± 3.9 years) underwent partial microwave ablation of the spleen during laparoscopy or under percutaneously ultrasound guidance. Two of them had concomitant hepatocellular carcinoma (HCC), and one patient underwent microwave ablation for splenomegaly 2 months after microwave ablation for HCC, while the other had microwave ablation for both HCC and splenomegaly simultaneously. The other eight patients only had secondary splenomegaly and hypersplenism. In all cases, secondary splenomegaly and hypersplenism was a complication of portal hypertension from chronic liver disease. Eight were HbsAg (+), one was anti-HcV (+) and one had drug-induced chronic hepatic disease causing hepatic cirrhosis, portal hypertension and hypersplenism. All of them had hepatic cirrhosis. Before the ablation and on days 1, 3, 7, and 1 and 3 months after ablation, changes in T lymphocyte subsets, B lymphocytes and NK cells were studied. These studies were approved by the institutional review committee, and informed consent documents were obtained from all patients.
Equipment and therapy procedure
A KY-2000 microwave system with an emission of 915 MHz microwave (Kang You Microwave Institute, Nanjing, PRC) was used in this study. It was equipped with 14-gauge needle electrodes (2.0 mm in diameter and 22 cm in length) that were specifically coated and insulated to prevent tissue adhesion and had internally cycling water to cool the pole to prevent burning the skin. The power output ranged from 1 W to 99 W. The spleen was accessed percutaneously via the left lateral subcostal approach under sonographic guidance. During surgery, the electrode was inserted into the splenic parenchyma at the lower pole of the spleen, with the power range at 70–90 W and exposure duration at 600–1200 s. During the laparoscopic procedure, the lower pole was first ablated, then the antenna withdrawn slowly and inserted into the upper pole for further ablation.
Blood specimen preparation and analysis
Venous blood specimens were collected at six time points: 2 days before therapy and on days 1, 3, 7, and 1 and 3 months after therapy, 2 ml of blood were collected into tubes containing EDTA-K2 for detecting T lymphocyte subsets, B lymphocytes and NK cells. Fresh anti-coagulation blood (100 µl) was taken and incubated at room temperature in the dark room with an equal volume of fluorescently labelled monoclonal antibodies: CD45-FITC/CD14-PE, Isotype Control, CD3-FITC/CD19-PE, CD3-FITC/CD4-PE, CD3-FITC/CD8-PE, CD3-FITC/CD16+CD56+-PE (Becton Dickinson) in different tubes. Diluted FACS Lysing Solution (1 ml) was added to each tube and the mixtures were incubated again at room temperature for 8 min, centrifuged at 1200 rpm for 5 min and the supernatant removed. The cells were washed and then resupended in PBS. The samples were studied and analysed with flow cytometry using MultiSET software (Becton Dickinson, San Jose, CA, USA). CD4+ T cells were defined as CD3+ CD4+ lymphocytes, CD8+ T cells as CD3+ CD8+ lymphocytes, B lymphocytes as CD3− CD19+ lymphocytes and NK cells as CD3− CD56/16+ lymphocytes.
Statistical analysis
Statistical analysis was performed using STATA 7.0 software (Stata Corp., College Station, TX, USA). An F-test was used to compare the pre- and post-therapy values of these groups. A t-test was used to determine if there were significant differences of immune cells between the two splenic ablation volume groups before and after therapy. The data were expressed as the mean ± SD.
Results
Changes in T lymphocyte subsets
shows that levels of CD3+ and CD4+ T lymphocytes increased significantly, especially 1 month after microwave ablation therapy (p < 0.05). The ratio of CD4+ : CD8+ cells and the percentage of CD8+ T lymphocytes did not change significantly.
Table I. Changes in T lymphocytes after microwave ablation therapy (mean ± SD).
Changes in B lymphocytes and NK cells
After partial microwave ablation therapy of the spleen, the percentage of B lymphocytes increased significantly compared with pre-therapy levels on each occasion of detection (p < 0.05). The percentage of NK cells did not change ().
Table II. Changes in peripheral blood B lymphocytes and NK cells after microwave ablation therapy (mean ± SD).
Changes in T lymphocyte subsets according to the ablation volume
Using the splenic ablation volume, as calculated by three-dimensional CT, patients were divided into two groups (group A and group B), each of five patients. In both groups, the percentage of T cells did not change significantly pre- and post-therapy. Levels of CD3+ and CD4+ T lymphocytes of group B patients began to increase after therapy. The percentage of CD3+ and CD4+ T lymphocytes of group A patients increased 1 month after ablation but decreased 3 months after therapy. Three months after therapy, the level of CD4+ T cells in group A was significantly lower than in group B (p < 0.05; ).
Table III. Changes in T lymphocytes subsets grouped by the ablation volume (mean ± SD).
Changes in B lymphocytes and NK cells according to ablation volume
In , we can see that the percentage of B lymphocytes increased after therapy in both ablation groups and was significantly higher than the pre-therapy levels (p < 0.05). On each testing, no significant difference was seen in the percentage of B lymphocytes between the two ablation groups. After ablation, levels of NK cells in group A were lower than pre-therapy levels while levels of NK cells in group B were higher than pre-therapy levels. However, these differences were not significant.
Table IV. Changes in B lymphocytes and NK cells groups by ablation volume (mean ± SD).
Discussion
Hypersplenism is a well-known complication in portal hypertension of hepatic cirrhosis that may favour infection and bleeding. Patients with cirrhosis, splenomegaly and hypersplenism often show complications contraindicating splenectomy. Being aware of the spleen's function in the immune system Citation[28], Citation[29] and preserving as much of the splenic tissue as possible is recommended when treating benign conditions of the spleen Citation[5–7]. Moreover, the immune function of cirrhotic patients with secondary splenomegaly and hypersplenism is low. Patients easily contract infections and HCC Citation[30], Citation[31]. Splenic immune function is important and removal of the spleen is not to be taken lightly. New strategies for conserving as much of the spleen as possible are needed. Based on our clinical experience accumulated over 12 years, we determined that microwave ablation for secondary splenomegaly and hypersplenism is safe and effective. Previous studies have shown that immune function was enhanced in HCC patients receiving microwave ablation therapy for tumours Citation[23–27]. However, changes in immune status of patients receiving microwave ablation for the spleen remained unknown. Peripheral lymphocyte subsets are important indices of immune function; thus we investigated the changes in peripheral lymphocytes in the human body after microwave ablation therapy for secondary splenomegaly and hypersplenism.
T lymphocytes mainly control cellular immunity. Microwave thermal ablation therapy for tumours can enhance the body's cellular immunity Citation[32]. Previous studies using experimental animals or peripheral blood of patients have shown that patients receiving microwave ablation therapy for HCC have increased levels of T lymphocytes Citation[25–27]. In our study, the percentage of CD3+ and CD4+ T lymphocytes increased significantly 1 month after therapy. Although the levels decreased over the next 2 months, they remained higher than the pre-therapy levels. Although the CD8+ T lymphocyte level showed a decreasing trend between 1 and 3 months after therapy, the ratio of CD4+ : CD8+ T cells increased during this time. These results suggested that microwave ablation therapy for secondary splenomegaly and hypersplenism may increase patient's peripheral T cell levels.
B lymphocytes mainly keep charge of humoral immunity. They can produce immunoglobulin. Thermal ablation therapy increases body humoral immunity by increasing production of immunoglobulin Citation[33]. According to Wu et al.'s research, in 89 cases of lung cancer patients receiving radiofrequency ablation for tumours, the level of B lymphocytes in peripheral blood increased Citation[34]. In our study, the level of B lymphocytes in patients on microwave ablation therapy of the spleen increased significantly on each testing compared with pre-therapy level. Our results were in agreement with data in the literature.
Our results demonstrate that T and B lymphocyte levels returned to normal in some patients after therapy, but not in all. However, all lymphocyte levels increased compared with pre-therapy levels. We conclude, first, that hyperthermia can enhance body immunity, as manifested by an increase in the peripheral lymphocyte subsets; second, that after splenic therapy, peripheral blood constituents, such as white blood cells and platelets, as well as blood flow to the liver through the hepatic artery increase. Increased hepatic arterial blood flow can supply increased oxygen to the liver and improve the capacity of the hepatic oxygen exchange Citation[35]. By improving hepatic function, the overall patient condition is improved, allowing an increase in the levels of peripheral lymphocyte subsets. In one patient undergoing microwave ablation therapy for both HCC and splenomegaly, the increase of peripheral lymphocyte subsets might have been influenced by the HCC ablation therapy. This patient was the only one undergoing ablation therapy for splenomegaly and HCC simultaneously. The peripheral lymphocyte subsets increased in all other patients receiving ablation therapy exclusively for the spleen. This should not affect the results of our therapy trial.
Stimulation of NK cells needs no antigen and NK cells are considered to play a vital role in the first line of defence against bacterial infections or tumours Citation[36]. Previous studies have shown that levels of NK cells were enhanced after PMCT Citation[26]. In our study, although NK cells levels increased 1 month after therapy, the increase was not statistically significant. To determine better the influence of ablation therapy on NK cells, we should increase the number of patients in the study. This work is currently under investigation.
In the research of PSE for secondary splenomegaly and hypersplenism, it was reported that the larger the splenic embolization volume, the better the symptoms caused by splenomegaly and hypersplenism are relieved and the longer the efficacy lasts Citation[37]. In our study, we found that the spleen ablation volume may influence the level of peripheral lymphocyte subsets. The maintenance of recovery levels of peripheral lymphocyte subsets in group B patients was better than in group A. We think that the symptoms from splenomegaly and hypersplenism could be relieved better in a larger ablation volume, so the level recovery of peripheral lymphocyte subsets was better. Sangro et al. Citation[38] thought that the spleen embolization volume should be at least 50% to achieve good long-term results in PSE. In our study, the largest ablation volume was 40%. Being a preliminary study, we did an ablation volume of ≤40% to be safe. The ablation procedure was well tolerated in all without any severe complications. Moreover, the minimally invasive microwave ablation therapy for secondary splenomegaly and hyper-splenism can be repeated. To maintain better recovery results and patient tolerance, sequential ablations may be employed.
However, our study was a preliminary one. The sample size was small; more significant changes may be seen enlarging sample size. The follow-up period was rather short and the indices that we used for evaluation need to be more detailed. There may also be changes in absolute numbers of lymphocyte subsets and cytokines, etc. Studies with larger sample size, longer follow-up period and more detailed indices (information on absolute values of peripheral lymphocyte subsets and cytokines, etc.) are needed to confirm further the efficacy of microwave ablation therapy for secondary splenomegaly and hypersplenism, to answer questions as to how long the peripheral lymphocyte subsets increased level lasts and results of having an ablation volume >50%. Prospective future randomized control studies may also be useful to compare the efficacy of microwave therapy with other therapeutic approaches such as splenectomy or PSE for secondary splenomegaly and hypersplenism.
In conclusion, microwave ablation for secondary splenomegaly and hypersplenism is a new, minimally invasive method that means local partial splenectomy. Our results suggested that partial ablation of the spleen can improve the levels of peripheral lymphocyte subsets, results of which may be related to ablation volume. Basing on our research, splenic ablation volume between 20% and 40% may help to enhance and maintain the level of the peripheral lymphocyte subsets. Further study is warranted to observe the therapeutic effect.
Acknowledgement
We thank Professor Ye Hui Fang for her help in the English writing of the manuscript.
References
- Yang Z, Qiu F. Pericardial devascularization with splenectomy for the treatment of portal hypertension. Zhonghua Wai Ke Za Zhi 2000; 38: 645–648
- Sugawara Y, Yamamoto J, Shimada K, Yamasaki S, Kosuge T, Takayama T, Makuuchi M. Splenectomy in patients with hepatocellular carcinoma and hypersplenism. J Am Coll Surg 2000; 190: 446–450
- Tomikawa M, Hashizume M, Akahoshi T, Shimabukuro R, Gotoh N, Ohta M, Sugimachi K. Effects of splenectomy on liver volume and prognosis of cirrhosis in patients with esophageal varices. J Gastroenterol Hepatol 2002; 17: 77–80
- Hansen K, Singer DB. Asplenic-hyposplenic overwhelming sepsis: Postsplenectomy sepsis revisited. Pediatr Dev Pathol 2001; 4: 105–121
- Kyaw MH, Holmes EM, Toolis F, Wayne B, Chalmers J, Jones IG, Campbell H. Evaluation of severe infection and survival after splenectomy. Am J Med 2006; 119: 276e1–7
- Trobs RB, Bennek J. Partial splenectomy, transposition of the spleen to the abdominal wall or splenohepatoplasty in portal hypertensive rats. Effects on portal venous pressure and homeostasis–microscopical appearance of the transposed spleen. Eur J Pediatr Surg 1998; 8: 155–162
- Jiang HC, Sun B, Qiao HQ, Xu J, Piao DX, Yin H. Clinical application of serial operations with preserving spleen. World J Gastroenterol 2001; 7: 876–879
- Zhang H, Chen J, Kaiser GM, Mapudengo O, Zhang J, Exton MS, Song E. The value of partial splenic autotransplantation in patients with portal hypertension: A prospective randomized study. Arch Surg 2002; 137: 89–93
- Obatake M, Muraji T, Kanegawa K, Satoh S, Nishijima E, Tsugawa C. A new volumetric evaluation of partial splenic embolization for hypersplenism in biliary astresia. J Pediatr Surg 2001; 36: 1615–1616
- Liu FQ, Chu GW, Tong JM, Zhang XJ, Qin DJ, Liu YL, Chen GH. Studies of treatment of hypersplenism with injection of absolute alcohol into spleens. Shijie Huaren Xiaohua Za Zhi 2000; 8: 1381–1384
- Shina S, Aoyama H, Shiratori Y, Mutoh H, Kurita M, Ota S, Terano A, Sugimoto T. Ultrasound-guided percutaneous injection of ethanolamine oleate for hypersplenism. An experimental study in dogs. Invest Radiol 1990; 25: 651–657
- Liu QD, Ma KS, He ZP, Ding J, Huang XQ, Dong JH. Experimental study on the feasibility and safety of radiofrequency ablation for secondary splenomagely and hyper-splenism. World J Gastroenterol 2003; 9: 813–817
- Tabuse K, Katsumi M, Kobayashi Y, Noguchi H, Egawa H, Aoyama O, Kim H, Nagai Y, Yamaue H, Mori K, Azoma Y, Tsuji T. Microwave surgery: Hepatectomy using a microwave tissue coagulator. World J Surg 1985; 9: 136–143
- Hornback NB, Shupe R, Shidnia H, Joe BT, Sayoc E, George R, Marshal C. Radiation and microwave therapy in the treatment of advanced cancer. Radiology 1979; 130: 459–464
- Ohmoto K, Miyake I, Tsuduki M, Shibata N, Takesue M, Kunieda T, Ohno S, Kuboki M, Yamamoto S. Percutaneous microwave coagulation therapy for unresectable hepatocellular carcinoma. Hepatogastroenterology 1999; 46: 2894–2900
- Mikami Y, Shimizu M, Manabe T. Tumorous necrotic nodule in the liver: Unexpected effect of the microwave tissue coagulator. J Clin Pathol 1997; 50: 703–705
- Seki T, Kubota Y, Wakabayashi M, Kunieda K, Nakatani S, Shiro T, Inoue K. Percutaneous transhepatic microwave coagulation therapy for hepatocellular carcinoma proliferating in the bile duct. Dig Dis Sci 1994; 39: 663–666
- Sato M, Watanabe Y, Kashu Y, Nakata T, Hamada Y, Kawachi K. Sequential percutaneous microwave coagulation therapy for liver cancer. Am J Surg 1998; 175: 322–324
- Matsukawa T, Yamashita Y, Arakawa A, Nishiharu T, Urata J, Murakami R, Takahashi M, Yoshimatsu S. Percutaneous microwave coagulation therapy in liver tumors. A 3-year experience. Acta Radiol 1997; 38: 410–415
- Shibata T, Niinobu T, Ogata N, Takami M. Microwave coagulation therapy for multiple hepatic metastases from colorectal carcinoma. Cancer 2000; 89: 276–284
- Murakami T, Shibata T, Ishida T, Niinobu T, Satoh T, Takamura M, Shibata N, Takami M, Nakamura H. Percutaneous microwave hepatic tumor coagulation with segmental hepatic blood flow occlusion in seven patients. AJR Am J Roentgenol 1999; 172: 637–640
- Liang P, Dong BW, Yu XL, Yu DJ, Wang Y, Feng L, Xiao QJ. Prognostic factors for survival in patients with hepatocellular carcinoma after percutaneous microwave ablation. Radiology 2005; 235: 299–307
- Dong BW, Zhang J, Liang P, Yu XL, Su L, Yu DJ, Ji XL, Yu G. Sequential pathological and immunologic analysis of percutaneous microwave coagulation therapy of hepatocellular carcinoma. Int J Hyperthermia 2003; 19: 119–133
- Zhang J, Dong BW, Liang P, Yu XL, Su L, Yu DJ, Ji XL, Yu G. Significance of changes in local immunity in patients with hepatocellular after percutaneous microwave coagulation therapy. Chin Med J 2002; 115: 1367–1371
- Nakayama J, Kokuba H, Kobayahshi J, Yoshida Y, Hori Y. Experimental approaches for the treatment of murine B16 melanomas of various sizes. II: Injection of ethanol with combinations of beta-interferon and microwaval hyperthermia for B16 melanomas with a size of greater than 10 mm in diameter. J Dermatol Sci 1997; 15: 82–88
- Szmigielski S, Sobczynski J, Sokolska G, Stawarz B, Zielinski H, Petrovich Z. Effects of local prostatic hyperthermia on human NK and T cell function. Int J Hyperthermia 1991; 7: 869–880
- Hong X, Baowei D, Xingshi L. Changes of T-lymphocyte subsets in patients with liver cancer treated by ultrasound-guide percutaneous microwave coagulation. Chin J Phys Ther 2000; 1: 15–17
- Siemion IZ, Kluczyk A. Tuftsin. On the 30-year anniversary of Victor Najjar's discovery. Peptides 1999; 20: 645–674
- Vliet E, Melis M, Ewijk W. Marginal zone Macrophages in the mouse spleen identified by a monoclonal antibody. Anatomical correlation with a B cell subpopulation. J Histochem Cytochem 1985; 33: 40–44
- Fan WC, King KL, Loong CC, Wu CW. Hepatocellular carcinoma after renal transplantation: The long-term impact of cirrhosis on chronic B virus infection. Transplant Proc 2006; 38: 2080–2083
- Joseph FP, Gregory LA, Leigh AF, Yvan JFH, Beth PB. The contribution of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol 2006; 45: 529–538
- Meyer JL. Hyperthermia as an anticancer modality: A historical perspective. Front Radiat Ther Oncol 1984; 18: 1–22
- Santini R, Hosni M, Douss T, Deschaux P, Pacheco H. Increasing antigenecity of B16 melanoma cell fraction with microwave hyperthermia. J Microw Power Electromagn Energy 1986; 21: 41–44
- Wu Y, Jiang NY, Cheng QS, Li XF, Wang XP, Liu K. Assessment of immune cells from patients with lung cancer before and after radiofrequency ablation. J Fourth Mil Med Univ 2004; 25: 671–672
- Troisi R, Hoste E, Langenhove PV, Decruyenaere J, Voet D, Hesse UJ, Cuomo O, Hemptinne BD. Modulation of liver graft hemodynamics by partial ablation of the splenic circuit: A way to increase hepatic artery flow?. Transplant Proc 2001; 33: 1445–1446
- Kim S, Lizuka K, Aguila HL, Weissman IL, Yokoyama WM. In vivo natural killer cell activities revealed by natural killer cell-deficient mice. Proc Natl Acad Sci USA 2000; 97: 2731–2736
- Zhu KS, Shan H, Li ZR, Shen XY, Meng XC, Guan SH, Jiang ZB, Huang MS. Long-term efficacy of partial splenic embolization for hypersplenism in cirrhosis. Chin J Radiol 2004; 38: 732–736
- Sangro B, Bilbao I, Herrero I, Corella C, Longo J, Beloqui O, Ruiz J, Zozaya JM, Quiroga J, Prieto J. Partial splenic embolization for the treatment of hypersplenism in cirrhosis. Hepatology 1993; 18: 309–314