Microwave ablation zones are larger than they macroscopically appear - Reevaluation based on NADH vitality staining ex vivo

Abstract

BACKGROUND:

Animal liver is established as an ex vivo model for studies on hepatic microwave ablation (MWA). Macroscopically visible color changes in the ablation zone are used to assess cell destruction and confirm successful ablation ex vivo.

OBJECTIVE:

Macroscopy and histology of MWA zones regarding cell viability in ex vivo porcine livers were compared in this study.

METHODS:

Six MWA were performed in porcine livers post mortem. A 14-G antenna and microwave generator (928 MHz; 9.0 kJ) were used. MWA were cut at the maximum cross section in vertical alignment to the antenna. NADH-diaphorase staining determined cell vitality. Macroscopic and microscopic ablation zones were statistically analyzed.

RESULTS:

Histology showed two distinct ablation zones: central white zone (WZH) with no cell viability and peripheral red zone (RZH) with partial cell viability. However, the macroscopically visible WZM was significantly smaller than the microscopic WZH with an area difference of 43.1% (p < 0.05) and a radius difference of 21.2% (1.6 mm; p < 0.05). Macroscopy and histology showed a very high correlation for the complete lesion area (WZH/M+RZH/M; r = 0.9; p = 0.001).

CONCLUSIONS:

The avital central zone is significantly larger as the macroscopically visible WZ which is commonly used to assess successful ablation in MWA ex vivo studies. Irreversible cell destruction can be underestimated in macroscopic evaluation.

Keywords

Liver animal model microwave ablation (MWA)viability staining NADH-diaphorase

1 Introduction

Liver tumors are among the most common malignant cancers worldwide [1]. Surgical resection is not suitable for the majority of primary and secondary liver tumors [2, 3]. This led to the development of alternative treatment methods such as thermal microwave ablation (MWA) [4 –7]. In MWA, microwave energy is introduced into the target tissue using an antenna with a frequency of 900 to 2500 MHz [8, 9]. The aim of ablation is an irreversible local tissue damage by denaturation. Protein denaturation occurs at temperatures as low as 42–45°C and leads to irreversible cell damage [10]. More precisely, successful ablation ex vivo often is assessed by a visible color change of the liver tissue [11]. Usually, two distinct zones can be differentiated: the “white zone” (WZ) - a beige-grey zone of avital tissue and the “red zone” (RZ) - a rosy-gray zone that is believed to contain vital and avital tissue [12, 13]. As the WZ is assumed to define complete cell death within the tissue it is therefore used to assess successful ablation [13]. In contrast, the RZ is expected to represent partially vital tissue [12, 13]. To improve the safety of MWA as a therapeutic option for liver tumors, animal models are used in research in vivo and ex vivo [9 , 14–22]. Before microwave systems enter clinical application, they are often evaluated in ex vivo studies. Ex vivo tests are essential for a precise understanding of the ablation process in order to ensure the effectiveness of the procedure in a clinical setting [9 , 23]. Many studies are based on macroscopic image data of ex vivo animal models. However, ex vivo ablation zones are usually not validated histologically.

The aim of the present study was to compare the macro-morphological and histological microwave ablation zones in porcine liver ex vivo. We hypothesize that macroscopic MWA zones and microscopic cell viability stains show a high degree of correlation and that macroscopically measured ablation zones can be used to assess successful ablation.

2 Materials and methods

2.1 Liver

Four porcine livers (pig breed: Sus scrofa domesticus) were used within three hours post mortem to minimize false results by autolysis [13]. The samples were transferred immediately after slaughter and cooled to a constant room temperature of 25°C.

2.2 Microwave ablation

A commercially available microwave generator (MWI881, AveCure™, MedWaves Inc., San Diego, California, USA) was used for the experiments. The device generates high frequency energy with a dynamic frequency range between 902 MHz and 928 MHz [9, 24]. Temperature is measured by a sensor located 2 mm behind the active zone of the antenna [25]. All ablations were performed with a 14-gauge microwave probe without external cooling system (AveCure™, MedWaves Inc., San Diego, California, USA) and with a maximum power of 32 W [9].

2.3 Evaluation of ablation zones

All experiments were performed at room temperature (25°C) with an energy input of 9 kJ. During ablation, liver samples were immersed in 0.9 % sodium chloride solution (B. Braun, Melsungen AG, Melsungen, Germany) within a targeting device to avoid air pockets in liver vessels, which can cause alterations in temperature [26]. After ablation, livers were cut in the middle of the active zone of the antenna, orthogonal to the antenna. A custom-made targeting device ensured a standardized and reproducible cutting process (Fig. 1).

Fig.1

a) Targeting device for exact antenna positioning. Slits on both sides were used to cut the liver orthogonally to the antenna (cross section) with a trimming blade for further analysis. b) Macroscopic cross section of an ablation with white and red zones.

Cross sections were photographed after ablation on a millimeter scale. Tissue blocks were immediately embedded within Tissue-Tek^® O.C.T.™ Compound (Sakura Finetek Germany GmbH, Staufen, Germany). The specimens were snap-frozen with liquid nitrogen and stored at –80°C. Preparations were cut in 8–10 μm thick sections with a cryostat (CryoStar™ NX70 Cryostat, ThermoFischer Scientific, USA) for histological processing. Cryosections were stained with a solution of reduced NADH (nicotinamide adenine dinucleotide) and NBTC (nitro blue tetrazolium chloride) to identify vital and non-vital cells as previously described [27]. Vital cells with an intact histological structure were stained blue, whereas non-vital cells remained pale. Stained cryosections were scanned with a flatbed scanner (CanoScan, 9000F, Mark II, Canon Europa N.V., Netherlands) alongside the millimeter scale. Macroscopic and histologic images were aligned along anatomic landmarks, such as vessels and connective tissue (Fig. 2). The microwave antenna was defined as the ablation center. The distance between the center point and the ablation border was measured every 45 degrees in all macroscopic and microscopic images. The ablation areas for the WZ and RZ were measured with the scientific image software ImageJ (National Institute of Health, Bethesda, USA). Lesion shapes were assessed by a regularity index $(RI = \frac{Rmin}{Rmax})$ [28, 29]. An RI close to 1.0 indicated a circular round and a ratio till 0.8 an oval shaped ablation [29]. The qualitative analysis of histological MWA zones was based on the following cellular characteristics: loss of cell structure and loss of NADH-diaphorase stain in the WZ. The WZ is surrounded by the RZ.

Fig.2

Cross section of a microwave ablation (MWA) lesion with outlined borders of the white zone (WZ). a) Macroscopic image with an outlined white zone (WZM). b) Histological image with NADH-diaphorase staining. Vital cells appear blue. The homogenous red zone (RZ) surrounds the microscopically visible white zone (WZH) and is clearly demarcated from the native liver tissue. c) Comparison of the macroscopic (WZM) and microscopic (WZH) white zones. The ablation area and the radius of ablations were measured every 45 degrees. The antenna (A) was the center point of the ablation. Images were aligned along anatomic landmarks (LM), such as vessels.

2.4 Statistical analysis

Statistical analysis was performed with statistics software (SPSS, version 24, SPSS Inc., IBM Corp, Armonk, NY, USA). Data are expressed as median (minimum – maximum). The Wilcoxon signed-rank test was used to assess differences between histology and macroscopic findings. The Pearson coefficient was used to assess the correlation of histological and macroscopic area differences. The level of significance was 0.05 (two sided) for each statistical testing.

3 Results

Six ablations with an ablation temperature of 110°C (91–120°C) were performed. Ablation time was 6:21 minutes (05:56–07:02).

3.1 Qualitative analysis

The macroscopic ablation area showed zones of different colors directly around the antenna: a brown carbonization zone was observed in close proximity to the antenna. This zone was surrounded by a pale white-gray coagulation zone, known as the WZM. The microscopic WZH showed a complete loss of cell structure and a loss of color in the NADH-diaphorase stain. The macroscopic and microscopic WZ was enclosed by one transition zone, called the RZ. This RZ is characterized by its gradual progression into the native tissue. Predominantly, the microscopic RZH showed partial cell destruction with remaining cell borders and exhibited partly damaged and partly vital cells.

3.2 Quantitative analysis

All ablations were oval and confluent with a regularity index of 0.81 (macroscopically) and 0.83 (histologically). The area of the WZ was 102.3 mm² (macroscopically) and 176.0 mm² (histologically, Table 1). The comparison of both WZ showed a significant area difference of 43.1% (p = 0.03). The total lesion area (L, area of WZ + RZ) was not significantly different (Fig. 3). A very high correlation (r = 0.97; p = 0.001) was determined for histologically and macroscopically measured areas of the lesion. The WZ radius showed a significant difference of 21.2% (1.6 mm, p≤0.001) between the macroscopic and histological measurements (Table 2). Based on the median difference, we propose the following correction factor for the contouring of the ablation zone: r(x) + 21.2 % (Fig. 3). As a proof of concept, we compared the corrected macroscopic ablation zone with the microscopic zone and found no significant difference (p = 0.615).

Table 1
Median values (minimum-maximum) of macroscopic and histological ablation areas of White Zone (WZ) and complete lesion (WZ + RZ). ^* p < 0.05

Area Macroscopy Histology Difference Macro/Histo

(mm²) (mm²) (mm²) % p

White Zone (WZ) 102.3 (93.0–136.0) 176.0 (156.0–296.0) 77.5^*(56.0–160.0) 43.1(35.9–54.1) 0.028

Lesion (WZ+RZ) 264.0 (216.0–328.0) 300.5 (246.0–395.0) 36.0 (13.0–86.0) 11.5 (3.2–33.4) 0.075

Area	Macroscopy	Histology	Difference Macro/Histo
	(mm²)	(mm²)	(mm²)	%	p
White Zone (WZ)	102.3 (93.0–136.0)	176.0 (156.0–296.0)	77.5^*(56.0–160.0)	43.1(35.9–54.1)	0.028
Lesion (WZ+RZ)	264.0 (216.0–328.0)	300.5 (246.0–395.0)	36.0 (13.0–86.0)	11.5 (3.2–33.4)	0.075

Fig.3

Comparison of the macroscopic WZM and histologic WZH and the total lesion area (L). The median radius difference between histological radius (rH) and the macroscopic radius (rM) was 1.6 mm. As a result, we propose that the macroscopic WZ (WZM) should be outlined with an added distance of 1.6 mm (dotted black line, see inlay). A: Antenna.

Table 2

Median values (minimum-maximum) of macroscopic and histological ablation radius and lesion circumference. ^* p < 0.05

Radius and circumference	Macroscopy	Histology	Difference Macro/Histo
	(mm)	(mm)	(mm)	%	p
White Zone (WZ)	6.0 (5.2–9.1)	7.5 (6.0–11.9)	1.6^* (–1.0–5.7)	21.2 (–24.1–48.3)	0.000
Lesion circumference	62.6 (57.1–76.0)	66.1 (59.8–78.2)	3.2 (1.9–11.2)	4.9 (2.8–18.0)	0.027

4 Discussion

This study demonstrated a higher extent of histological non-vital tissue exceeding the borders of the white zone defining cell death in macroscopic evaluation.

Macroscopic tissue cuts are commonly used to visually assess microwave ablation in experimental studies ex vivo. Ablation success is judged by the macroscopically visible white zone (WZ), which is a distinct color change in liver tissue and contains non-vital cells. Therefore, it is essential that the macroscopically seen white zone correlates to lethally destructed tissue. This is the first study that examines the correlation between microscopic and macro-morphological results of MWA ex vivo. Our hypothesis was that the macroscopic and the microscopic assessment of the WZ show a high correlation. However, the non-vital zone significantly exceeds the visible white zone. We calculated a radius correction factor of r(x) + 21.2 % between estimated macroscopy and actual histological detection. One reason for this difference could be a graduation between cell death and tissue appearance in MWA-zone. Especially at the border to the native liver tissue, low temperatures are reached which lead to incomplete cell damage. However, these damages are not yet completely visible in macroscopy. Tissue necrosis by immediate cell death occurs from a temperature of 60°C [30, 31] and visible carbonization from temperatures of 105°C [32].

MWA zones were morphologically consistent with previous studies [8 , 33]. Because of the lack of blood circulation only two zones were histologically visible [12]. In vivo, the RZ usually is divided into the inner and the outer RZ [13]. Siriwardana et al. found similar zone characteristics [12]. Typical signs of bleeding and edema were missing as described in an in vivo study by Gemeinhardt et al. [13]. The biological variance by vessels and connective tissue structures did not affect the shape of the lesion, mainly because of missing perfusion.

A limitation in our study might be the ex situ setting for vitality assessment. However, livers in our study were only used for up to three hours after extraction to avoid autolysis [13]. The rate of autolysis also depends the temperature, which is why livers were cooled down to room temperature. The 37°C warm liver samples were not stored on ice in order to avoid tissue alteration caused by reheating the samples to room temperature and the time needed torestore a physiologic temperature. One study reported that liver tissue showed only minor autolytic modifications up to 24 h post mortem compared to normal histological aspects [34].

We used NADH for vitality staining. NADH is a co-enzyme of the mitochondria and is involved in metabolic cascades of energy supply. Hyperthermia destroys the protein structure of NADH and inhibits enzyme activity. A lack of enzyme function and the destruction of intact mitochondrial DNA can be regarded as destruction of the cell Gemeinhardt et al. [13] showed, that cryosections of MWA with vital staining (NADH) were possible up to three hours post mortem.

Percutaneous ablation of liver malignancies has established in clinical routine and is anchored in national and international guidelines [6]. Ablations are rarely removed and histologically processed. However, understanding the histological zone distribution of MWAs may be helpful for the treatment of patients with imaging.

5 Conclusion

In an ex-vivo-setting the macroscopically visible white zone underestimates complete cell destruction extent in hepatic microwave ablation. In our experiments the total extent of avital tissue was 43.1% larger than the macroscopic white zone. Based on the results of this study, the validation of the macroscopic measurement with the radius calculation (r(x) + 21.2 %) of ex vivo image data for MWA can be improved.

Statement of ethics

All authors disclose any financial and personal relationships with other people or organizations that could inappropriately influence their work.

Role of the founding source

This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Ref.-No. LE 1343/2-1.

Author contributions

B. Geyer: conceptualization; data curation; formal analysis; investigation; methodology; project administration; supervision; writing – original draft

F. G. M. Poch: conceptualization; formal analysis; writing – review and editing

O. Gemeinhardt: conceptualization; formal analysis; writing – review and editing.

C. A. Neizert: supervision; writing – review and editing.

S. Niehues: supervision; writing – review and editing.

J. Vahldiek: supervision; writing – review and editing.

R. Klopfleisch: supervision; writing – review and editing.

K. S. Lehmann: conceptualization; funding acquisition; methodology; project administration; supervision; writing – review and editing.

References

Ferlay

, Soerjomataram

, Dikshit

, Eser

, Mathers

, Rebelo

, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer. 2015;136(5):E359–86.

Solbiati

, Ahmed

, Cova

, Ierace

, Brioschi

, Goldberg

. Small liver colorectal metastases treated with percutaneous radiofrequency ablation: Local response rate and long-term survival with up to 10-year follow-up. Radiology. 2012;265(3):958–68.

Forner

, Llovet

, Bruix

. Hepatocellular carcinoma. Lancet (London, England). 2012;379(9822):1245–55.

Gruber-Rouh

, Marko

, Thalhammer

, Nour-Eldin

, Langenbach

, Beeres

, et al. Current strategies in interventional oncology of colorectal liver metastases. The British Journal of Radiology. 2016:20151060.

Pathak

, Jones

, Tang

, Parmar

, Fenwick

, Malik

, et al. Ablative therapies for colorectal liver metastases: A systematic review. Colorectal disease: The Official Journal of the Association of Coloproctology of Great Britain and Ireland. 2011;13(9):e252–65.

Bruenn

, Beyer

, Haimerl

, Pregler

, Stroszczynski

, Jung

, et al. Comparison of computed tomography (CT) and contrast-enhanced ultrasound (CEUS) for the quantitative evaluation of an ablation defect following radiofrequency ablation of malignant liver lesions. Clinical Hemorheology and Microcirculation. 2017;67(3-4):445–51.

da Silva

NPB

, Beyer

, Hottenrott

, Hackl

, Schlitt

, Stroszczynski

, et al. Efficiency of contrast enhanced ultrasound for immediate assessment of ablation status after intraoperative radiofrequency ablation of hepatic malignancies. Clinical Hemorheology and Microcirculation. 2017;66(4):357–68.

Brace

. Microwave ablation technology: What every user should know. Current Problems in Diagnostic Radiology. 2009;38(2):61–7.

Hoffmann

, Rempp

, Erhard

, Blumenstock

, Pereira

, Claussen

, et al. Comparison of four microwave ablation devices: An experimental study in ex vivo bovine liver. Radiology. 2013;268(1):89–97.

10.

Vanagas

, Gulbinas

, Pundzius

, Barauskas

. Radiofrequency ablation of liver tumors (I): Biological background. Medicina (Kaunas, Lithuania). 2010;46(1):13–7.

11.

Ahmed

. Image-guided tumor ablation: Standardization of terminology and reporting criteria–a 10-year update: Supplement to the consensus document. Journal of Vascular and Interventional Radiology: JVIR. 2014;25(11):1706–8.

12.

Siriwardana

, Singh

, Johnston

, Watkins

, Bandula

, Illing

, et al. Effect of Hepatic Perfusion on Microwave Ablation Zones in an Ex Vivo Porcine Liver Model. Journal of Vascular and Interventional Radiology: JVIR. 2017;28(5):732–9.

13.

Gemeinhardt

, Poch

, Hiebl

, Kunz-Zurbuchen

, Corte

, Thieme

, et al. Comparison of bipolar radiofrequency ablation zones in an in vivo porcine model: Correlation of histology and gross pathological findings. Clinical Hemorheology and Microcirculation. 2016;64(3):491–9.

14.

Rieder

, Poch

, Ballhausen

, Lehmann

, Preusser

. Software Tool for the Analysis of the Coagulation Zone from Multipolar Radiofrequency Ablation 2012.

15.

Farina

, Weiss

, Nissenbaum

, Cavagnaro

, Lopresto

, Pinto

, et al. Characterisation of tissue shrinkage during microwave thermal ablation. International Journal of Hyperthermia. 2014;30(7):419–28.

16.

Amabile

, Farina

, Lopresto

, Pinto

, Cassarino

, Tosoratti

, et al. Tissue shrinkage in microwave ablation of liver: An ex vivo predictive model. International Journal of Hyperthermia: The official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group. 2017;33(1):101–9.

17.

Poch

, Rieder

, Ballhausen

, Knappe

, Ritz

, Gemeinhardt

, et al. The vascular cooling effect in hepatic multipolar radiofrequency ablation leads to incomplete ablation ex vivo. International Journal of Hyperthermia: The official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group. 2016;32(7):749–56.

18.

Singh

, Siriwardana

, Johnston

, Watkins

, Bandula

, Illing

, et al. Perivascular extension of Microwave ablation zone - demonstrated using an ex-vivo porcine perfusion liver model. International journal of hyperthermia: The Official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group. 2017:1–25.

19.

Cavagnaro

, Amabile

, Cassarino

, Tosoratti

, Pinto

, Lopresto

. Influence of the target tissue size on the shape of ex vivo microwave ablation zones. International Journal of Hyperthermia: The official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group. 2015;31(1):48–57.

20.

Vahldiek

, Erxleben

, Bressem

, Gemeinhardt

, Poch

, Hiebl

, et al. Multipolar RFA of the liver: Influence of intrahepatic vessels on ablation zones and appropriateness of CECT in detecting ablation dimensions - results of an in-vivo porcine liver model. Clinical Hemorheology and Microcirculation. 2018.

21.

Bressem

, Vahldiek

, Erxleben

, Poch

, Hiebl

, Lehmann

, et al. Instant Outcome Evaluation of Microwave Ablation With Subtraction CT in an in vivo Porcine Model. Investigative Radiology. 2019.

22.

Guo

, Li

, Wang

, Li

, Liu

, Chen

, et al. Stiffness distribution in the ablated zone after radiofrequency ablation for liver: An ex-vivo study with a tissue elastometer. Clinical Hemorheology and Microcirculation. 2019.

23.

Lehmann

, Ritz

, Valdeig

, Knappe

, Schenk

, Weihusen

, et al. Ex situ quantification of the cooling effect of liver vessels on radiofrequency ablation. Langenbecks Arch Surg. 2009;394(3):475–81.

24.

Sommer

, Arnegger

, Koch

, Pap

, Holzschuh

, Bellemann

, et al. Microwave Ablation of Porcine Kidneys in vivo: Effect of two Different Ablation Modes (“Temperature Control” and “Power Control”) on Procedural Outcome. Cardiovascular and Interventional Radiology. 2012;35(3):653–60.

25.

Hoffmann

, Kessler

, Weiss

, Clasen

, Pereira

, Nikolaou

, et al. Preclinical evaluation of an MR-compatible microwave ablation system and comparison with a standard microwave ablation system in an ex vivo bovine liver model. International Journal of Hyperthermia: The Official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group. 2017;33(6):617–23.

26.

Andreano

, Brace

. A comparison of direct heating during radiofrequency and microwave ablation in ex vivo liver. Cardiovascular and Interventional Radiology. 2013;36(2):505–11.

27.

Neumann

, Knobler

, Pieczkowski

, Gebhart

. Enzyme histochemical analysis of cell viability after argon laser-induced coagulation necrosis of the skin. Journal of the American Academy of Dermatology. 1991;25(6 Pt 1):991–8.

28.

Mulier

, Ni

, Miao

, Rosiere

, Khoury

, Marchal

, et al. Size and geometry of hepatic radiofrequency lesions. European Journal of Surgical Oncology: The Journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology. 2003;29(10):867–78.

29.

Mulier

, Ni

, Frich

, Burdio

, Denys

, De Wispelaere

J-F

, et al. Experimental and Clinical Radiofrequency Ablation: Proposal for Standardized Description of Coagulation Size and Geometry. Annals of Surgical Oncology. 2007;14(4):1381.

30.

Habash

, Bansal

, Krewski

, Alhafid

. Thermal Therapy, Part III: Ablation Techniques. Crit Rev Biomed Eng. 2007;35(1-2):37–121.

31.

Meloni

, Chiang

, Laeseke

, Dietrich

, Sannino

, Solbiati

, et al. Microwave ablation in primary and secondary liver tumours: Technical and clinical approaches. International Journal of Hyperthermia: The Official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group. 2017;33(1):15–24.

32.

Vogl

, Nour-Eldin

, Hammerstingl

, Panahi

, Naguib

NNN

. Microwave Ablation (MWA): Basics, Technique and Results in Primary and Metastatic Liver Neoplasms - Review Article. RoFo: Fortschritte auf dem Gebiete der Rontgenstrahlen und der Nuklearmedizin. 2017.

33.

Chiang

, Willey

, Del Rio

, Hinshaw

, Lee

, Brace

. Predictors of thrombosis in hepatic vasculature during microwave tumor ablation of an in vivo porcine model. Journal of Vascular and Interventional Radiology: JVIR. 2014;25(12):1965–71.e2.

34.

Cocariu

, Mageriu

, Staniceanu

, Bastian

, Socoliuc

, Zurac

. Correlations Between the Autolytic Changes and Postmortem Interval in Refrigerated Cadavers. Romanian Journal of Internal medicine=Revue Roumaine de Medecine Interne. 2016;54(2):105–12.