Cryotherapy using liquid nitrogen (LN) is the most common form of treatment of actinic keratosis [
Sixteen patients were selected (
Patients received the treatment of cryopeeling using the PS for one forearm (a) and using LN for the other forearm (b), randomly. The cryopeeling was performed by applying the freezing substance with brush movements along the extension of the forearm until the area was frozen. After this, actinic keratoses lesions were individually treated.
A 4 mm skin biopsy punch was performed in all patients on each forearm, at the lateral region, before and 60 days after cryopeeling. Actinic keratoses lesions were intentionally avoided and the second sample was taken in the same region but 1 cm apart from the first biopsy in order to avoid the scaring area. Random codes were established for all cases in order to allow for blind analysis of data in all histological evaluations regarding the time of biopsy (before or after treatment) and which method was used (LN or PS). At the end of the study each patient had performed four skin biopsies. The specimens were fixed in 10% formalin and submitted to routine histological processing. Microscopic sections were made for hematoxylin and eosin (H&E), Fontana Masson, and Weigert stains. Several parameters were analyzed as described below, by the same observer at the same day.
We analyzed the thickness of the epidermis and Grenz zone (area of loose papillary dermis spared by elastosis) using the Image-Pro software program version 4.5.123. The thickness of the epidermis was calculated by using the three smallest measurements found (Figure
Evaluation of epidermal thickness using the average of consecutive measurements of the three thinnest areas found.
The Grenz zone thickness measurement was obtained by the average of the three thickest measurements. The largest was discarded when it was more than twice compared to the second largest measure and replaced by a fourth measure.
Blindly, a dermatopathologist analyzed the pairs of slides before and after procedure from each patient. Taking into account a comprehensive global review of the quality of the skin (appearance of the stratum corneum, epidermal thickness, presence of solar lentigines, and quality/quantity of elastosis) it was aimed to establish which slide corresponded to the pretreatment and posttreatment, or if this was not possible to define. A second analysis was performed in the same way, but considering only the analysis of the stratum corneum.
We analyzed the distribution of melanin pigment in the basal layer using a weighted average as follows: the total length of the epidermis was divided into 5 segments, each of which corresponded to 20% of the total. Each segment was classified according to the amount of pigment: weak, moderate, or intense corresponding to weights 1, 2, and 3, respectively. Thus, each specimen had a score for pigment distribution. For example: 20% with weak pigment, 20% with moderate pigment, and 60% with intense pigment; (0.2 × 1 + 0.2 × 2 + 0.6 × 3)/6 = score of 0.4.
We analyzed the density of solar elastosis in paired slides before and after procedures for each patient, blindly. The aim was to define which slide had a higher density of elastic fibers or if there was no difference between them.
The student's
We analyzed a total of 16 patients and 31 skin biopsies from 16 forearms treated with LN (
Histological findings are summarized. Epidermal and Grenz zone thicknesses values were obtained by average of three measurements. Pigment intensity score was based on a weighted average considering intensity and distribution of pigment. The intensity of pigment decreased after PS treatment (
Before treatment | After treatment |
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Epidermal thickness average ( | ||||
LN | 39.34 (SD 7.83) | 43.37 (SD 9.40) | 16 | 0.09 |
PS | 38.26 (SD 6.52) | 42.28 (SD 8.24) | 15 | 0.13 |
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Grenz zone thickness average ( | ||||
LN | 61.41 (SD 29.50) | 64.37 (SD 18.07) | 16 | 0.71 |
PS | 71.08 (SD 25.98) | 80.58 (SD 37.04) | 15 | 0.36 |
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Pigment intensity score | ||||
LN | 75.42 (DP 13.27) | 71.88 (DP 17.68) | 16 | 0.54 |
PS | 69.55 (DP 11.04) | 58.89 (DP 12.39) | 15 |
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LN: liquid nitrogen. PS: portable system. SD: standard deviation.
Considering the analysis of the epidermal thickness, in 54% of the cases, an increased thickness was demonstrated after treatment. Regarding the Grenz zone thickness, in 52% of the cases, there was an increased thickness after treatment, although for both epidermal and Grenz thicknesses no significant difference was demonstrated after treatments (
Quantification of melanic pigment, considering the weighted average intensity of pigment correlated with the extent of the area analyzed, showed that in 61% of the cases the pigment decreased, in 32% increased, and in 7% remained unchanged. There was a tendency of decrease of pigment intensity with both treatments but this difference was significant only in the group of the PS (
The density of elastic fibers was higher after the procedure in 52% of the cases analyzed after both treatments, but no statistically significant difference was found (
In the analysis of H&E slides, both global analysis and specific analysis of stratum corneum did not show significant differences. Trying to presume which specimen was submitted to treatment, the percentage of correct answers was 48% in global analysis and 61.3% in specific analysis of stratum corneum with no statistically significant difference for correct answers (Figure
Blind assessment of pre- and postprocedure specimens in order to presume which one underwent treatment. Results are demonstrated by percentage of correct answers, considering a global analysis (stratum corneum, epidermal thickness, presence of lentigines, and elastosis) and a second analysis considering only the aspect of stratum corneum.
Although cryotherapy is a method widely used in clinical practice, few studies using cryopeeling technique have been conducted so far [
Histological changes due to photoaging are highly debated in the literature. It is believed that there is a decreased cell turnover resulting in reduced turnover rate of the stratum corneum, epidermal atrophy, increased healing time, and less effective scaling. There is an accumulation of corneocytes making the skin surface rougher. Within the dermis, there is a reduction of about 20% of its thickness, with disorganization of collagen fibers and accumulation of abnormal elastin-containing material [
In photoaging, there is no consensus regarding the epidermis behavior: some studies show thinning of the epidermis and others thickening [
Although there was a decrease of the pigment after both treatments, this difference was only significant with the PS. Cryotherapy is a method that is well known to induce hypopigmentation. During the use of the PS for actinic keratoses treatment an unexpected increase of the frozen area was often observed. This fact deserves special attention since it may lead to a high risk of an uncontrolled frozen area which might have contributed to this finding.
Three cases showed dermal fibrosis: one before the procedure, one after LN treatment, and one after PS treatment. Thus one may not attribute this fibrosis to the procedures and a random finding should be considered.
The medium peel with trichloroacetic acid is able to induce an increase of elastic fibers in the papillary dermis after the procedure, establishing what was called Grenz zone [
Cryopeeling technique is well tolerated and effective in the treatment of actinic keratoses, with clinical benefit evidence in improving the surface appearance of the skin [
In blind assessment of pre- and postprocedure slides in order to presume which one underwent treatment, considering the global analysis of the quality of the skin (stratum corneum, epidermis, lentigines and solar elastosis), it was not being able to identify the cases submitted to treatment accurately (
Furthermore previous studies have demonstrated a hyperplastic response of epidermis after several peeling sections, although only one session with this treatment was not able to induce the same response [
The conclusion of this study was that cryopeeling technique was not able to induce measurable histological medium term changes and has doubtable benefit in the treatment of photodamage despite of the improvement of the superficial aspect of the skin. Besides it should be taken into consideration the possible risk of hypopigmentation using the method.
The PS was provided freely by the company neo-Latin, but without any financial assistance for the study. There is therefore a conflict of interests among researchers.