High‐frequency (20 MHz) high‐intensity focused ultrasound: New ablative method for color‐independent tattoo removal in 1‐3 sessions. An open‐label exploratory study

Abstract Background High‐intensity focused ultrasound (HIFU) operating at 20 MHz is new and potentially applicable to ablative tattoo removal. The method was documented safe and rational in preclinical testing. Materials and Methods High‐intensity focused ultrasound was introduced to subjects when lasers and dermatome shaving had failed or caused side effects. Transducers with focal depths between 1.1 mm and 1.7 mm in the skin were used, and settings of 0.4‐1.2 J/shot at pulse durations of 150 ms were applied. Tattoos were covered with synergistic “shoulder‐by‐shoulder” focused ultrasound shots. Effectiveness and side effects were measured. Results Twenty‐two subjects with 67 tattoos were treated. 62% benefitted (19% cleared, 43% partially cleared), and 28% had minor effect. VAS pain was 5‐6 versus 7‐9 with previous lasers removal. Wound healing was longer after HIFU ablation (1‐3 months). 57% of subjects had no scar or minor visible changes of skin surface markings only, while 19% had moderate or major skin thickening. Hypertrophic scar or keloid scars were not observed. Discussion/Conclusion High‐intensity focused ultrasound was effective in removal of difficult tattoos of any color where Nd:YAG lasers had failed. The method only needs 1‐3 sessions. As an ablative method, the wound healing period is longer than with laser removal and needs attention. Focused ultrasound can be used as a first‐line treatment of smaller tattoos independent of color, and second line when Nd:YAG lasers have failed or caused problems. The operator shall be qualified, as with lasers.

those who regretted. Medical practitioners used surgical excision as wells as dermabrasion and salabrasion, however with limitations due to size, anatomical site and sequelae observed particularly as scars.
Today, nanosecond Q-switched lasers, for example, Nd:YAG, alexandrite, and ruby lasers, are used as the methods of choice, recently followed by the launch of picosecond lasers. [3][4][5] Lasers are of different wavelength and therefore have color-and pigment-dependent energy absorption, which is an advantage and a limitation at the same time. Some colors, particularly yellow, green, and blue, have low energy absorption to available laser wavelengths and are therefore difficult to remove. White titanium pigment used as pure pigment or in toned ink may turn dark after high-energy laser exposure. Lasers may furthermore produce photochemical effects on the tattoo pigment with formation of breakdown substances that are allergenic, carcinogenic, or otherwise toxic. 6,7 Lasers produce a focused ultra-short thermal burn with temperatures reaching 300-400°C in the pigment. These high temperatures are conducted to nearby skin structures, which are then damaged. For this reason, laser treatment often is very painful, and the thermal insult is followed by a wheal and flare reaction. The long-term sequelae are consequently scarring and dyspigmentation with hypoor hypermelanosis. 8 Lasers are, despite these known limitations, positioned as the gold standard treatment of today. Ablative CO 2 lasers are used only exceptionally due to the high risk of scars. Lasers shall not be applied to allergic tattoo reactions, since the reaction can be boosted through neoformation of allergenic breakdown products. 9 Allergic reactions are better treated with dermatome shaving. 10 Caustic, strongly acidic, or strongly oxidant products for tattoo removal are marketed as cosmetic products, and on free sale despite the obscure dose-effect control and the very high risk of scars. 11,12 Laser removal requiring 8-12 treatment sessions, and given with a minimum interval of 6 weeks, is expensive and cumbersome to fulfill, and there is therefore a need for new or supplementary methods.
The new method for tattoo removal presented in this article is innovative and based on high-intensity focused ultrasound (HIFU).
HIFU systems operating at frequencies from 500 kHz to approximately 3 MHz were since decennia established for non-invasive treatment of a variety of medical indications exemplified by internal cancers of major organs and cerebral pathologies, with the HIFU focal point located deep within the body or the brain. [13][14][15][16][17][18][19][20][21] Selective HIFU treatment of the thalamus has revolutionized the treatment of invalidating essential tremor. HIFU used at even lower frequencies can be used to treat kidney stones. In all such treatments, accurate positioning of the focal point at targets within organs is guided by magnetic resonance imaging or ultrasound imaging. Thus, the principle of targeted and surveyed treatment using HIFU is verified and internationally accepted.
The size of the focal zone generated by a HIFU transducer is inversely dependent on the operating frequency, that is, the higher the frequency the smaller the focal zone. For HIFU treatments to be relevant within the field of dermatology, the focal zone must be small to match the 1-2 mm total thickness of the human skin. This requires an operating frequency of at least 15 MHz as confirmed by theoretical considerations and preclinical testing. 22,23 Commercially available HIFU systems for aesthetic wrinkle reduction and body contouring operate at about 3-10 MHz. Such frequencies create focal zones that exceed the total thickness of the skin, and are therefore not feasible for dermatology indications addressing the dermal end epidermal layers only. 24,25 The ultrasonic device introduced for tattoo removal in this article is based on 20 MHz high-intensity focused ultrasound (HIFU). 26 The HIFU device uses a focused acoustic transducer with a concave surface that concentrates ultrasound beams over pulse durations of typically 100-200 ms in a very narrow focal zone. In the focal zone, where the convergent beam concentrates, the energy density is greatly intensified with a resultant rapid heating of the tissue temperature up to approximately 65°C, which induces a highly localized acute necrosis. It shall be emphasized that the dept control of the thermal lesion in a medium is optimized; that is, the delineation in depth is reproducible and sharp under a given setting of the instrument.
The method is "color blind" and "content-neutral," as the thermal lesion has no special preference in the compartment or layer of the skin exposed to HIFU treatment. Different transducers can be selected to accurately position the lesion as preferred at any vertical depth of the skin. Thus, the method can be adapted for both individual differences in skin thickness and the vertical position of a lesion. With the focal point positioned in the outer skin, the method is ablative. With the focal point in the deep dermis, at the interface to subcutis or directly in the subcutis the method is non-invasive and non-ablative, see Figure 1.
F I G U R E 1 Schematic illustration of the high-intensity focused ultrasound (HIFU) principle. The transducer is a focused piezoelectric element constructed to deliver non-invasive acoustic energy of high intensity into a confined focal zone under the skin surface. The zone of concentrated heating to about 65°C is in the focal point, and the adjacent part of the medium above the focal point. The energy attenuates abruptly below the focal point. Exposed to 65°C, cells die and the vitality of the exposed skin is lost Laboratory study and preclinical study in Göttingen pigs have demonstrated reproducibility and a linear energy-dependent dose-effect curve. Preclinical testing indicated a realistic range of operation within a dose range of 0.6-1.5 J/dose in the clinic. 23,26 The method produced the intended thermic insults in the dermis and was confirmed as ablative or non-ablative depending on setting and choice of transducer. In the pig model, scarring was not a problem, albeit histology revealed subclinical fibrous change in cases exposed to high-energy multiple doses positioned in close proximity to each other.
Such horizontal synergy between ultrasound lesions applied nearby each other, that is, "shoulder by shoulder" mode, was compared to single dose, the latter clearly having lower effect. 23,27 Thus, the dose delivered to the skin, as determined by instrument setting, had an additional operator-dependent dimension, namely the number and closeness of applied treatment doses.
This horizontal synergy is important in the practical use of the method for clinical applications. Synergy can also be produced in the vertical plane by "double pass" mode of use, that is, administering double dosing over the same site within a minimum time interval, thereby deliberately increasing the tissue temperature and affected volume. A third option is use of two different probes with different depths of the focal point on the same site, thus creating a "sandwich" mode of use.
The 20 MHz HIFU system used in the present study has recently been successfully introduced to actinic keratosis and selected cases of basal cell carcinoma and Kaposi sarcoma. 28 The HIFU treatment, as found in the study of pigs, was often followed by a wheal and flare response that peaked after 5-10 minutes and faded over the subsequent 10-30 minutes. After a few days a superficial dry wound and inflammatory reaction in the treated area was observed, followed by the necrotic material being expelled from the skin leaving an excavated inflamed wound that healed gradually.
Based on these observations from the animal study, a hypothetical mode of action of HIFU applied to tattoo removal was deducted according to the process shown in Figure 2.
The study presented below is the very first study of the novel 20 MHz HIFU method applied to tattoo removal according to this mode of action. A clinical material of challenging cases of tattoo removal that mostly had been unsuccessfully treated with lasers F I G U R E 2 Schematic illustration of ablative effects of 20 MHz high-intensity focused ultrasound (HIFU) treatment dosed "shoulder by shoulder" according to preclinical study of pigs, extrapolated to treatment of tattoos. 27 The illustration shows the hypothetical mode of action and sequence of events of tattoo removal using 20 MHz HIFU. A, cross-sectional illustration of the skin with black tattoo pigment concentrated in the outer dermis, which is a typical location. B, HIFU dosed "shoulder by shoulder" and positioned to target the outer dermis. C, immediate wheal and flare reaction due to the thermal ultrasound trauma causing histamine release; edematous center and perilesional axonal flare. D, HIFU-induced thermal necrosis of the outer dermis, holding the pigment in the necrotic mass. E, scab and loosened necrotic material. F, the necrotic material, with tattoo pigment, is expelled from the skin leaving an excavated inflamed wound that will heal gradually. Inflammation of the wound and the border of the wound are not illustrated. While the wound is open, with broken barrier and secretion, tattoo pigment particles still can migrate out of the skin into the bandage. Thus, clearing of pigment has two phases, that is, a first via the necrotic crust and later via the post-necrotic open wound until the epidermis and the barrier are restored, and an additional mode of metabolic breakdown of pigment affected by inflammation of the wound bed extending into the border of the wound before is presented including illustrative case reports. Thus, this open-label exploratory study primarily addressed difficult-to-remove tattoos.

| Patient recruitment
The study was open-label and integrated in the ongoing clinical treatment practiced at the Department of Dermatology of Bispebjerg University Hospital, Denmark. The department has a specialized Tattoo Clinic treating tattoo complications, and a laser section which on special indications performs Nd:YAG-laser removal of tattoos. [29][30][31][32] The surgical unit has special experience in removal of tattoos with allergic reactions to red azo pigment by dermatome shaving. 10  was an open-label production control assessment. The study was conducted from November 2018 to February 2020. Patients were informed before treatments and gave their consent. The principles of the Helsinki Declaration II were followed.

| Equipment
Treatments of this study were performed using the novel System ONE from TOOsonix A/S, Denmark. 22,26 The system operates at 20 MHz ± 5%. The HIFU equipment was safety tested and approved and registered by the Dept. of Medical Engineering of the Hospital. The equipment fulfills the general requirements for basic safety and essential performance according to IEC 60601-1:2006 including its collateral standards.
The system, shown in Figure 3, consists of an ultrasound power unit responsible for generation and regulation of ultrasound signals, handpieces with a range of ultrasound transducers, and a software to manage treatment settings. HIFU doses, or "shots," are activated manually by a footswitch. High-resolution real-time monitoring of the treated area is integrated in the system using a digital video camera that operates as a dermoscope. Different handpieces of the system are characterized by their −6 dB focal zone depth, that is, the maximum extend of the zone where acoustic intensity is within 25% of the maximum intensity in the center of the zone. Handpieces with focal depths ranging from 1.1 to 2.7 mm were available and selected depending on the tattoo under treatment. The treatment for tattoo removal was primarily intended to be ablative, and handpieces were therefore chosen accordingly.

| Procedure of tattoo removal
Prior to treatment, the transducer chamber was filled with nongaseous distilled water and closed with a thin polyethylene film. A standard ultrasound coupling gel was used between the skin surface and the probe.
The duration of each ultrasound shot was chosen at 150 ms in all treatments. This has previously been found adequate for sufficient energy transfer, and at the same time minimize influence of movement of the handpiece during HIFU transmission. The acoustic peak energy was preferably 0.6, 0.9 or 1.2 J/shot depending on the thickness of the tattooed skin and the anatomical site.
Guided by a red pointer on the screen and observing the skin surface with the integrated dermoscope camera, the HIFU shot was positioned precisely over the target in the tattoo. The system was activated with the footswitch, and a shot was fired. Whitening or contraction of the treated skin was displayed directly on the screen in real-time and surveyed to control that an effective dose was taken up by the tattoo (Figure 4).
A full treatment consisted of consecutive shots administered shoulder-by-shoulder, that is, placed with approximately 1-2 mm Treatments included 1-2 shots in the surrounding skin immediately outside the tattoo to enforce treatment of the demarcation of the tattoo, for example, the "lining." The tattoo contour or lining, most often made in black, is normally made by the tattooist with a thin liner needle that installs the pigment rather deep in the dermis. Thin lines in tattoos, such as texts, were deliberately treated in the surrounding skin as well to benefit from the synergy of "shoulder by shoulder" applications.

| Clinical assessment and rating scales
Immediate wheal and flare reaction directly after treatment was rated as follows: 0 no reaction, 1 + redness only, 2 + redness and edema, 3 + perilesional redness in the surrounding skin additional to redness and edema. 1 + and 2 + are histamine mediated; the flare of 3 + is an axonal reflex not accompanied by edema.
High-intensity focused ultrasound treatment causes instant pain directly when a shot is fired. Pain was measured on a visual analogue scale, ranging from 0 to 10 with 10 marking unbearable pain.
The effectiveness of HIFU on tattoo color reduction was rated as follows: "cleared" (no remnant pigment), "partly cleared" (removal or diminishment of up to half of the pigment or the color of the tattoo), "minor effect" (removal or diminishment of less than half of the pigment or the color of the tattoo), and "no effect" meaning no visual effect on color or pigment.
Scar at follow-up was rated as follows: no scar, 1 + visible change of skin surface markings only, 2 + slightly increased skin thickness with slightly increased skin tension, 3 + moderate skin thickening with definitely increased tension, 4 + major thickening and tight skin, 5 + hypertrophic scar or keloid. Rating of scar was not conducted at start of study, and outcome rating of scar is thus the accumulated sum of scarring resulting from tattoo needle trauma, previous laser or dermatome shaving and, finally, HIFU used as rescue intervention.

| RE SULTS
The study included 22 subjects with 67 tattoos treated with 20 MHz HIFU. Most tattoos were black and made in a tattoo parlor, but also red and green tattoos had been included. One had gun powder tattoos in the face, and one subject had X-ray field markings made by a hospital.
Most tattoos were recalcitrant and had been treated before with Q-switched Nd-YAG lasers or dermatome shaving, the latter applied as first-line treatment of red tattoos with allergic reactions.
Subjects in the study were treated with 1-3 HIFU sessions with an interval of no less than 6 weeks to observe the immediate outcome. While different probes were used for special cases, the preferred probes had focal depths of 1.1 and 1.3 mm to obtain an ablative treatment mode. The preferred power settings were 0.9 and 1.2 J/shot. Follow-up ranged from 3 to 12 months.
The results of the study are summarized in Table 1, and clinical findings are discussed below. The process of tattoo removal by HIFU is furthermore exemplified in selected case reports.

| Wheal and flare reactions immediately after HIFU -dose titration
All subjects developed wheal and flare directly after treatment, most subjects grade 1 + and 2+, and a few with grade 3+. However, it was intended that 1 + and 2 + wheal and flare was used as an indicator of clinically relevant dose setting. In the first phase of the study, 12 subjects were assessed by dose titration, where 3-4 doses of 0.4, 0.6, 0.9, and 1.2 J/shot were given to separate test sites in the tattoo or in the normal skin. The dose of best benefit/ adverse effect ratio was chosen for further treatment and subsequently applied to the entire tattoo. The same dose was given in the next session (session number 2) after about 6 weeks when the course of healing had been observed. A green tattoo treated with a test dose titration is shown in Figure 5. The preferred dose also could be decided immediately from reading of the wheal and flare reaction in normal skin. Pre-treatment dose titration was therefore deemed valuable.

| Pain of treatment
All patients who had tried laser removal before HIFU rated painless by HIFU compared to lasers according to VAS (data not F I G U R E 4 Close-up screen photograph of the dermoscopy picture of a black tattoo treated with "shoulder by shoulder" shots. The red cross at the center is the marker of position being treated when an ultrasound shot is fired shown). Typical VAS pain scores of HIFU were between 5 and 6, compared to scores between 7 and 9 reported by the subjects who had experienced pain of laser treatment. Pain from HIFU was reported variable in the treated field with sporadic points of higher sensitivity as compared to HIFU treatment shots in other parts of the field. This was attributed to pain sensors that may be TA B L E 1 Overview of results in 22 subjects with 67 tattoos treated with high-intensity focused ultrasound aiming at tattoo pigment removal A single subject self-administered an anesthetic topical (EMLA ® , AstraZeneca) on the targeted tattoo prior to treatment. In this case, pain score was very low (VAS 0.5), while treatment response and post-treatment healing remained within the range observed in the subjects.

| Phases of wound, crustation, and healing of HIFU-treated sites
Instantly on HIFU application, an epidermal reaction was obligatory, as intended. The epidermis became whitened, loose, and chapped.
Occasionally, vesicles were seen.
Within a few days, a superficial crust formed, followed by denser and drier crustation. After 1-2 weeks, a wound fully covered by a necrotic debris was observed. The necrotic tissue was extruded

| Efficacy of treatment
The tattoo was deemed cleared in 4 subjects (19%) and deemed partly cleared with some remnant pigment in 9 (43%) subjects (one subject lost for follow-up). Thus, in total 13 subjects, that is, 62%, benefitted from the treatment with respect to full or partial clinically relevant tattoo pigment removal. Six subjects (28%) only had minor effect of HIFU. Two subjects (10%) had no effect, including the case with gun powder tattoo.

| Side effects (scar)
Twelve (57%) had no scar, 5 (24%) had 1 + scar, and 4 (19%) had 2 + scar. Scar degree 3 + to 5 + was not observed. Effect versus scar was a scattered plot with no clear relationship. The two subjects with no effect had no scar and were considered non-responders.
One subject with hypopigmentation without a scar was registered.

| C A S E REP ORT 1: B L ACK TAT TOO ACQU IRED TO SUPP ORT RE S E ARCH OF FIR S T APPLIC ATI ON OF 2 0 MHz HIFU TO TAT TO O R E M OVA L
Subject (TB) was a 48-year-old male volunteer, who had a squared black tattoo made on his right buttock for the purpose of testing.
The tattoo was performed by a professional tattooist. The case is shown in Figure 6.

| C A S E REP ORT 2: TAT TOO REG RE T, TE S T TRE ATMENT OF A S ELEC TED B L ACK TAT TOO WHEN PI COS ECOND L A S ER IN THE PA S T HAD C AUS ED S E VERE LO C AL RE AC TI ON , UNB E AR AB LE PAIN AND G ENER AL MAL AIS E
Subject (SK) was a 33-year-old woman, Fitzpatrick Type 3, with ethnic predisposition to pigmentation. The subject had various tattoos with three on the arm causing social problems. Picosecond laser removal of a tattoo was attempted but given up, despite a new try with F I G U R E 6 First treatment using 20 MHz high-intensity focused ultrasound (HIFU) for tattoo removal, performed in a 48-year-old volunteer (TB), illustrating treatment effects and phases of healing. A, before HIFU treatment. The upper squared field marked red was treated with a probe with focus depth 1.7 mm, and the lower squared field marked blue was treated with a probe with focus depth 2.7 mm.
Half of the tattoo served as untreated reference. B, immediately after HIFU treatments. Loosening and chapping of both treated fields are observed, with associated perilesional edema and disturbance of the untreated reference. Lacking edema in the treated fields contrasting untreated tattoo indicates damage to the vascular supply of the epidermis and outer dermis. C, 2 days post-HIFU treatment. A confluent necrotic crust has formed. D, 10 days post-HIFU treatment. The necrotic crust is expelled. The wound bed is flat and at a lower level in the field treated with the 2.7 mm probe (blue field) in comparison with the field treated with the 1.7 mm probe marked red. Reepithelization is active in the wound margin. There are major inflammation and swelling of the surrounding skin, with edema extending into the untreated reference site. E, 4 weeks post-HIFU treatment. The epidermis is healed without restitution of skin markings, and the wound is closed. Ongoing inflammation in the dermis is causing redness. F, 20 weeks post-HIFU treatment. Healing without scar is completed, with slight edema remaining. One-session HIFU treatment has completely removed the tattoo, using the shallow probe (red field), while few discrete spots of pigment are remaining in the field treated with the deep probe (blue field). There is no sign of dyschromia, neither hypo-or hypermelanosis. Case supports to a high degree the hypothetic mode of action and course of healing introduced in Figure 2 (A) (B) (C) a low dose. The problem was unbearable pain and extraordinary swelling of the laser-treated tattoo affecting her general condition and requiring analgesics and oral prednisone treatment.
A small black tattoo on the forearm, a 1.5 cm star, was selected for test treatment with HIFU, performed in one session, probe depth 1.1 mm, settings 150 ms, and 0.9 J/shot, with 20 shots applied. A pre-test in normal skin with reading of wheal and flare had been performed. Figure 7 illustrates the tattoo directly after treatment, after approximately 10 days and after 7 months. This tattoo was completely removed after one HIFU session leaving a mild scar rated 1+, however, with some upcoming skin markings. There was a narrow rim of postinflammatory hyperpigmentation in the surrounding skin, maybe with her ethnic predisposition as a background. It is estimated that the esthetic outcome will normalize over time. HIFU-induced pain was rated 3 (1-7), much less than experienced with the laser. High-intensity focused ultrasound was given in one session with approximately 50 shots and a 1.1 mm focal depth probe, settings 0.9 J/shot, and pulse duration 150 ms. Figure 8 illustrates the red pigmentation before HIFU treatment and at a follow-up visit after 10 months. The remnant red pigmentation has been completely removed, and subject had not experienced any allergic reaction during the healing phase.

| C A S E REP ORT 4: TAT TOO C AUS ING SO CIAL IN COMPE TEN CE , WITH A S PECIAL NEED OF FA S T REMOVAL
Subject was a younger man, who carried two tattooed droplets under the eye, with strong negative social impact and risks. He also had various larger tattoos on the head. Previous laser treatments had not reduced the color significantly. He was treated with HIFU, 1.1 mm probe and 0.9 J/shot, but the effect was barely visible. A second treatment was given with a 1.3 mm probe and 1.2 J/shot.
A satisfactory result with no sequelae was noted after 4 months ( Figure 9).

| D ISCUSS I ON
In this introduction of 20 MHz HIFU for tattoo removal, the effectiveness of HIFU was confirmed with 62% benefitting from HIFU.
Nevertheless, many subjects were only partly cleared from pigment or had minor effect, and only a few were completely cleared.
However, HIFU was put on a real acid test, where no method was likely to produce ideal results. HIFU was used as a rescue when The presumptive pros and cons of lasers and HIFU are presented in

F I G U R E 9
Droplets under eye as a marker of serious event or danger, of high social impact. A, before high-intensity focused ultrasound (HIFU). B, directly after the first HIFU treatment; chapping of treated field after ultrasound shots, and wheal and flare reaction. C, status after first treatment after healing. D, a second treatment with HIFU was given; removal is completed with no significant remnant pigment and no scar. The need for swift treatment was high priority and had been met. The tattoos were made with a liner needle; thus, the pigment was installed deeply in the skin Another first-or second-line potential indication is difficult tattoos, where the outcome of laser removal is a failure or suboptimal due to poor match of the wavelength of the laser source and the absorbance of the pigment. There is presently no experience with HIFU applied to large tattoos, and the size of the tattoo is a logic limitation of HIFU because of the longer healing phase of the ablation wound.
Twenty megahertz HIFU was recently documented effective in the treatment of actinic keratosis, with a cure rate of 97%. 28 HIFU was found to be well-suited for field eradication and has the potential to replace or supplement photodynamic therapy. HIFU was also applied to skin cancers. HIFU is being studied for other indications: venous spiders, venous lakes, hemangiomas, lentigines, and benign skin tumors, thus common concerns in the elderly population.
In conclusion, 20 MHz HIFU has a significant potential for future use in dermatological clinics, particularly in the laser clinics as a new and specialized member of the family of advanced machines. There is also a hitherto little studied potential in the beauty industry.
High-intensity focused ultrasound is an advanced piece of equipment, and skills and experience of the operator are certainly needed as it is the case with lasers. Thus, this new method shall be met respectfully, and first-time users shall invest proper time for study, learning, and practical training before clients or patients are treated.

CO N FLI C T O F I NTE R E S T
This study has been funded by TOOsonix A/S, Denmark.