2.1. Study design
In this prospective and open label clinical study, the participants were selected according to the inclusion criteria that included healthy individuals, with adipose tissue deposits in the abdomen greater than 2.0 mm as measured by the Lafayette Adipometer and a body mass index between 21.0 and 33.6 in men and 22.9 and 45.8 kg/m in women. A total of 20 participants were divided by computer-generated simple randomization into two groups, A and B, with each group consisting of 10 participants (5 men and 5 women). Body fat mass was measured and blood samples were collected at time zero (baseline), time one (2 days), time two (30 days), time three (60 days), and time four (90 days) after the procedure.
2.2. Cryolipolysis Procedure and Body Fat Assesment
Cryolipolysis technique was applied to the lower and upper abdomen. During the 60-minute experiment, the temperature was -5ºC for group A and -10ºC for group B. Applications were performed in the upper abdomen (above the navel) and lower abdomen (below the navel) in a single application. The Dermasul company supplied the Adoxy Medical, Asgard VC10 model equipment to carry out the procedure. Two types of handles, 360º conventional “shielded” models were used. Before being exposed to low temperatures, the skin was protected with an anti-freeze membrane. Diuretic drugs were not taken for at least 24 hours before tests, food and beverages were not consumed was avoided up to 4 hours before tests, and patient was kept at rest. Body fat mass was measured using the Bioelectrical Impedance Analysis (BIA), following of the Brazilian Medical Association (AMB) and the Federal Council of Medicine (CFM) guidelines (2009).
2.3. Cryolipolysis procedure steps
2.3.1. 1. Installation of equipment before the activities.
2.3.2. Acclimatize room at 17°C before starting the application.
2.3.3. Setup the equipment.
2.3.4. Skin protection of participants with anti-freeze membrane.
2.3.5. Start application, reduce the vacuum to 10% after 5 minutes and proceed for 60 minutes.
2.3.6. Remove the handles from the applied area.
2.3.7. Remove and discard the used antifreeze membrane.
2.4. Hematological and inflammatory parameters
Each study participant had 3 mL of venous blood drawn into an EDTA tube via venin puncture. A blood liquate was extracted from the whole blood sample for hematological analysis. Hematological parameters such as white blood cells (WBC) and red blood cells (RBC), hematimetric indices and platelets, were determined using an automated hematology analyzer, the ADVIA 120 (Siemens Healthcare, Berlim, Germany), a hematology analyzer that performs a complete blood count and provides results in printouts. An experienced Senior Laboratory Technologist who had special training and was certified on the automated hematology analyzer performed hematological analysis in accordance with the standard protocol and manufacturer instructions of the hematology analyzer machine and the Clinical and Laboratory Standards Institute (CLSI) guideline. Inflammatory biomarkers such as mean platelet volume (MPV), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR) and the immune inflammation index (SII) were calculated as described previously [9–11].
2.5. Statistical analysis
Quantitative variables were expressed as mean ± standard deviation. Gaussian distribution (i.e., normality) were analyzed by the Shapiro-Wilk test. The levels in biomarkers and percentage of body fat mass between groups were performed using analysis of variance (ANOVA) followed by Holm-Sidak post hoc test or Friedman´s test followed by Dunn´s pos hoc test for multiple comparisons. All tests were two-tailed, with a value of p<0.05 considered statistically significant. All statistical analyzes were performed using GraphPad Prism 9.2.0 software (GraphPad Software, Inc., La Jolla, California, USA).