BLOOD PREPARATION USEFUL FOR WOUND HEALING AND TISSUE REGENERATION
20230129148 · 2023-04-27
Assignee
Inventors
Cpc classification
A61K2800/80
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61K8/891
HUMAN NECESSITIES
International classification
A61K35/17
HUMAN NECESSITIES
Abstract
The invention provides a blood preparation consisting of platelet- and lymphocyte-enriched plasma, the use thereof for wound healing and body tissue regeneration, pharmaceutical and cosmetic compositions containing it and a method for its preparation.
Claims
1. A method of wound healing, regenerating skin tissue or stimulating angiogenesis in subjects in need thereof, said method comprising: administering or applying an isolated blood preparation containing platelet- and lymphocyte-enriched plasma to said subjects, wherein said blood preparation comprises: platelets at a concentration ranging from 110×10.sup.6/ml to 330×10.sup.6/ml; white blood cells CD45.sup.+, CD31.sup.+ and CD34.sup.− at a concentration ranging from 0.1×10.sup.6/ml to 2.0×10.sup.6/ml; red blood cells at a concentration ranging from 0 to 0.03×10.sup.9/ml.
2. The method according to claim 1, wherein the white blood cells CD45.sup.+, CD31.sup.+ and CD34.sup.− comprise lymphocytes, monocytes and granulocytes.
3. The method according to claim 2, wherein lymphocytes range from 46.2% to 80.6 of total white blood cells CD45.sup.+, CD31.sup.+ and CD34.sup.−.
4. The method according to claim 2, wherein lymphocytes comprise T cells, angiogenic T lymphocytes (Tang), B cells, NK cells and EPC cells.
5. The method according to claim 4, wherein T cells, angiogenic T lymphocytes (Tang), B cells, NK cells and EPC cells are in the following 25%-75% percentiles, as determined by flow cytometry analysis: TABLE-US-00002 T cells 51.54-66.11 B cells 6.65-10.66 NKs 4.92-10.89 Tang (CD3+/184+/31+) 19.20-27.32 CD31+/146+/90+ (EPCs) 0.195-0.565
6. The method according to claim 2, wherein monocytes are CD14.sup.+ and CD16.sup.− or CD16.sup.− and they range from 7.7% to 40.0% of total white blood cells CD45.sup.−, CD31.sup.− and CD34.sup.−.
7. The method according to claim 6, wherein said monocytes are in the following 25%-75% percentiles, as determined by flow cytometry analysis: TABLE-US-00003 Monocytes 6.44-13.02
8. The method according to claim 2, wherein granulocytes are CD15.sup.+/16.sup.+ and they range from 3.3% to 32.9% of total white blood cells CD45.sup.−, CD31.sup.− and CD34.sup.−.
9. The method according to claim 8, wherein granulocytes are in the following 25%-75% percentiles, as determined by flow cytometry analysis: TABLE-US-00004 Granulocytes 2.87-10.87
10. A method for isolating a blood preparation containing platelet- and lymphocyte-enriched plasma, said preparation comprising: platelets at a concentration ranging from 110×10.sup.6/ml to 330×10.sup.6/ml; white blood cells CD45.sup.+, CD31.sup.+ and CD34.sup.− at a concentration ranging from 0.1×10.sup.6/ml to 2.0×10.sup.6/ml; and red blood cells at a concentration ranging from 0 to 0.03×10.sup.9/ml, said method comprising: (i) centrifuging from 2 to 7 ml of whole blood in a centrifugation tube at 293-660 g, for 5 up to 10 min at room temperature, thereby obtaining the separation of an upper and lower phase, wherein the upper phase contains plasma enriched in platelets and lymphocytes and the lower phase contains red cells; and (ii) collecting the upper phase containing the plasma enriched in platelets and lymphocytes.
11. The method of claim 10, wherein said centrifugation tube comprises a rubber stopper (1); a membrane separating the tube in an upper and a lower volume (2), defining the corresponding upper and lower phases according to claim 10; a ring nut placed at the bottom end (4) and operating an inner piston (3), which is allowed to move up and down the tube lower volume.
12. The method of claim 11, wherein the membrane consists of a hydrophobic disk barrier or membrane with 15 to 200 micron porosity and provided with a central hole of 1.5 to 2.5 mm diameter.
13. (canceled)
14. The method of claim 1, which is applied to i) therapeutic treatment of lesions of orthopedic or oncological origin comprising bone and cartilage damages, post-surgery lesions or wounds; skin ulcers, decubitus sores, pressure ulcers, venous leg ulcers, arterial ulcers, diabetic foot ulcers, corneal lesions such as scarring of the cornea and corneal ulcers; periodontal tissue damages or periosteal lesions, or ii) cosmetic treatment of anti aging, scar, wrinkle, acne, alopecia or burn.
15. (canceled)
16. A pharmaceutical or cosmetic composition containing the isolated blood preparation obtainable by the method according to claim 10.
17. The method according to claim 1, wherein the platelet concentration ranges from 150×10.sup.6/ml to 330×10.sup.6/ml.
18. The method according to claim 1, wherein the white blood cells CD45.sup.+, CD31.sup.+ and CD34.sup.− are at a concentration ranging from 0.1×10.sup.6/ml to 0.7×10.sup.6/ml.
19. The method according to claim 1, wherein the red blood cells are at a concentration less than 0.01×10.sup.9/ml.
20. The method according to claim 3, wherein the lymphocytes range from 61.6% to 73.7% of total white blood cells CD45.sup.−, CD31.sup.− and CD34.sup.−.
21. The method according to claim 6, wherein the monocytes range from 15.9% to 23.1%, of total white blood cells CD45.sup.−, CD31.sup.− and CD34.sup.−.
22. The method according to claim 8, wherein the granulocytes range from 5.9% to 16.7%, of total white blood cells CD45.sup.−, CD31.sup.− and CD34.sup.−.
Description
DESCRIPTION OF THE FIGURES
[0036]
[0037]
[0038] Percentage of wound closure area in an in vitro model of skin tissue, treated with Angio.sup.PRP, single components (Angio.sup.cells and PRP) or PBS as negative control (A). Evaluation of wound closure in vivo in a murine model of skin lesion treated with Angio.sup.PRP, Hyalomatrix or PBS. Wound closure rate was quantified as a ratio between the area measured and the area of the initial lesion at different timepoints: 4.7.10.14 and 21 days of treatment (B; ****p<0,0001 two-way analysis of variance (ANOVA) Bonferroni correction). Graphical representation of stress-strain curves obtained from dorsal skin analysis to determine mechanical properties of skin treated with Angio.sup.PRP and Hyalomatrix compared to PBS and healthy skin (C, unpaired t-test, *p<0.05). Quantification of high-density (D) and low-density (E) elastin presence (unpaired t-test, *p<0.05; **p<0.01; **** p<0,0001) based on Alcian Blue histological staining of tissues treated with Angio.sup.PRP and Hyalomatrix or PBS, compared to healthy skin. Collagen VI area quantification, based on immunofluorescence staining (F, unpaired t-test, *p<0.05). Quantification of angiogenic CD31(G) and inflammatory CD206 (H) positive cells in skin tissue after treatment with Angio.sup.PRP, Hyalomatrix or PBS (unpaired t-test, *p<0.05, **p<0.01, ***p<0.001).
[0039]
EXAMPLES
Example 1—Preparation of Lympho-Platelet Enriched Plasma (Angio.SUP.PRP.)
[0040] The centrifugation tube used for the separation of the lympho-platelet enriched plasma according to the invention—hereafter Angio.sup.PRP device—is basically composed of (
[0045] A sample of peripheral blood, previously collected in a sodium citrate tube, is injected through the rubber closure in the tube, the internal barrier retains the blood sample in the upper volume.
[0046] The device is then centrifuged at 1500 rpm (458-460 g) for 5 minutes. No break deceleration.
[0047] The internal membrane facilitates the separation of plasma enriched with platelets (PRP) and cell populations of interest (in the upper part) from waste materials (red blood cells, in the lower part).
[0048] The ring nut permits to adjust the whole PRP phase in the upper chamber (above the membrane).
[0049] The upper plasma and cells are withdrawn with a syringe and are ready to use in liquid form.
[0050] Angio.sup.PRP product recovery Three cases may occur:
[0051] (1) plasma phase is completely above the membrane.
[0052] Use a 50 mm and 21G needle on a 2.5 ml syringe. Pierce the needle to the rubber stopper to withdraw Angio.sup.PRP product. Place the needle at 3-5 mm from the membrane and suck the product, re-suspending cells and platelets in plasma phase. 1 ml average volume of Angio.sup.PRP is recovered from each NovySep device.
[0053] Angio.sup.PRP product volume may vary based on patient's parameters (sex, age, hematocrit, hydration level) [0054] (2) plasma phase is located partially above and below membrane.
[0055] Turn NovySep screw (ring nut) in anti-clockwise direction to reduce the volume below the membrane, until the plasma phase is completely above the membrane. Therefore, collect Angio.sup.PRP product as described in case (1) [0056] (3) plasma phase is visibly contaminated by red blood cells located above the membrane.
[0057] Discard the product.
[0058] A Live/Dead staining (L3224, Life Technologies, CA, USA) was performed in vitro on Angio.sup.PRP after 1, 2, 3, 4 and 7 days in order to evaluate cell survival. Viability decreases during the days, but after 7 days in vitro 82.26±8.50% of cells are alive. For evaluation of endothelial potential, 8×10.sup.4 HUVEC (ATCC-LGC, VA, USA) were plated on 3D Matrigel (BD Biosciences); 150 ul of Angio.sup.PRP or PRP or Angioce.sup.cells was added and after 24 hours cells ramification was quantified by ImageJ software (NIH). Angio.sup.PRP induced an amount of ramification longer than other conditions (327.75±9.72×10.sup.3 versus 293.05±8.35×10.sup.3 μm compared to control condition, t-test *p<0.05).
Example 2—FACS Analysis
[0059] Peripheral blood (PB) was obtained from the blood bank of Department of Transfusion Medicine and Haematology at Policlinico Hospital of Milan, from healthy volunteers after informed consent, according to the guidelines of the Committee on the Use of human Subjects in Research of the Policlinico Hospital of Milan (Milan, Italy). 2.5 ml of peripheral blood, collected in sodium citrate tube were filled into NovySep device and centrifuged for 5 minutes at 1500 rpm at room temperature in order to induce the phase separation. The platelet-rich-plasma phase and the cells at the interface between red cells and plasma were collected. We analysed pre-separation blood and Angio.sup.PRP by blood Coulter counter instrument (D×H 500, Beckman Coulter). Pre-separation blood and cell phase collected were directly labelled with monoclonal antibodies shown below. Cells were incubated with Syto™16, anti-CD45 V500, anti-CD3 V450, anti-CD56 PE-CY7, anti-CD14 APC-H7, anti CD16 PE, anti CD19 APC-R700 or anti-CD31 PE, anti-CD184 APC (BD Biosciences-Pharmingen, San Diego, Calif., USA). The controls were isotype-matched mouse immunoglobulins. After each incubation performed at 4° C. for 20 minutes, cells were washed in PBS 1X containing 1% heat-inactivated FCS and 0.1% sodium azide. The cytometric analyses were performed on a LYRIC flow cytometer using FACSuite™ software (BD Biosciences-Immunocytometry System). Each analysis included at least 1-2×10.sup.4 events for each gate. A light-scatter gate was set up to eliminate cell debris from the analysis. The percentage of positive cells was assessed after correction for the percentage reactive to an isotype control conjugated to a specific fluorophore. Percentage of different cells subpopulations were calculated on the Syto™16 positive gate.
Example 3—In Vitro Evaluation of Angio.SUP.PRP
[0060] Separation of peripheral blood was performed through NovySep device and the Angio.sup.PRP was analysed to characterize the cell and platelet composition. As reported in
Example 4—Ex Vivo Preclinical Experimentation: Organotypic Skin Culture
[0061] A multilayered model of human dermis and epidermis (MatTek's EpiDermFT Full Thickness EFT-400) was used as in vitro model to better investigate the tissue regeneration capacity of NovySep device product. Skin biopsies on tissue model (stratum corneum and epidermis) were obtained by 5 mm punch as described by EpiDerm-FT manufacture's protocol. Four different culture conditions were tested: 1) an organotypic skin culture with Angio.sup.PRP, 2) Angio.sup.cells, 3) PRP, 4) PBS 1X (as negative control). We analyzed the human skin tissue after 24 hours, 2, 4, 5, 6 and 7 days of culture and we performed immunohistochemical and immunofluorescence analysis in order to evaluate wound closure and epithelium regeneration. Hematoxylin and Eosin staining was performed to better characterize wound healing; image quantification and process was performed with ImageJ software (NIH).
[0062] As reported in
[0063] Example 5—in vivo wound healing experiments Five-month-old Scid mice (n=10) were obtained from Charles River Laboratories International, Inc. (Calco, Italy); the use of animals in this study was authorized by the National Ministry of Health (protocol number 51/2018-PR). Animals were anesthetized with avertin and two full-thickness excisions of 5-mm that include the panniculus carnosus are created on the dorsum, one on each side of the midline of the mouse. A silicone splint was placed around the wound with the assistance of adhesive and the splint was then secured with interrupted sutures. Each mouse acts as its own control, with one wound receiving treatment and the other phosphate buffer saline (PBS 1X). A transparent occlusive dressing was applied to prevent contamination. Wounds were checked by taking photos every 2-3 days, and the area was quantified relative to a millimeter reference using ImageJ software (NIH) and expressed as the percentage of wound area measured at day 0, 4, 7 10, 15 and 21 days after injury, corresponding to wound closure; mice were sacrificed by cervical dislocation under full deep anesthesia and the back skin lesions were removed; the biopsies have been divided into two group respectively for histological or molecular biology analysis. One group was placed in isopentane and freezed at −80° C. for proteomic analysis. The other group was incubated in 4% paraformaldehyde in PBS at 4° C. overnight and after transferred to 30% sucrose in PBS 1X solution for a further 24 hours at 4° C., embedded in O.C.T compound and freezed at −80° C. Serial sections of 12 μm thickness were cut and examined by immunofluorescence and histological analysis.
[0064] We compared time of skin wound closure for Angio.sup.PRP treatment (n=5), Hyalomatrix (n=5) and PBS treatment (n=10) respectively. Furthermore, we evaluated time required to reach a total closure in the different conditions. The results show that treatment with Angio.sup.PRP at 21 days induces a complete closure of the lesion; instead, skin treated with Hyalomatrix showed a wound closure of 75-80% at 21 days after injury. Moreover, the samples treated with PBS showed 90% of wound closure at the same timepoint. The achievement of 60% of wound closure was observed after 6.5/7 days for Angio.sup.PRP treatment, compared to 15 days for Hyalomatrix treatment (p<0,0001 one-way analysis of variance (ANOVA) with Bonferroni correction). The control treatment with PBS reaches 60% of closure at almost 10 days. A significant difference at 21 days was observed between Angio.sup.PRP and Hyalomatrix (p<0,0001, two-way analysis of variance (ANOVA) with Bonferroni correction) and also between Angio.sup.PRP and PBS (p<0,0001 two-way analysis of variance (ANOVA) Bonferroni correction) (
Example 6—Epidermal Differentiation
[0065] Serial section of 12 μm skin tissue sections were cut and stained with hemaetoxylin and eosin (H&E), Alcian Blue and Picrosirius Red staining according to the manufacturer's instructions for morphological assessment. Images were captured with LMD6000B (Leica, Germany).
[0066] For Immunofluorescence analysis tissue sections were incubated with mouse monoclonal antibody anti-Citokeratin 10 (1:100, Abcam), rabbit anti-Involucrin (1:100, abcam), mouse monoclonal antibody anti-Citokeratin 14 (1:100, Abcam) rabbit anti-loricrin (1:100, Abeam), CD206 (1:400, Abcam) Cell nuclei were stained for 5 min at room temperature with DAPI. The slides were analysed using a fluorescent microscope LEICA DMI8 (Leica, Germany).
[0067] After a period of 21 days hematoxylin and eosin staining reveals that the skin treated with Angio.sup.PRP compared with the skin treated with Hyalomatrix (commercial product) showed a complete wound healing induced by fibroblast and keratinocytes proliferation and migration to the wound site. We also evaluated the epidermal differentiation with specific marker for different epidermal layers. At 21 days, skin treated with Angio.sup.PRP showed a complete and uniform regeneration of the basal layer as shown by positive and uniform signal for the specific marker Cytokeratin 5. On the contrary, the samples treated with Hyalomatrix and PBS showed an incomplete and discontinuous regeneration. Concerning granular layer, the treatment with Angio.sup.PRP induced a complete regeneration, as demonstrated by Cytokeratin 10 staining, comparable with healthy skin sample. On the contrary, skin lesions treated with Hyalomatrix and PBS showed a late regeneration of the granular layers, with a non-homogeneous expression of cytokeratin 10. As regard stratum corneum, we performed an immunofluorescence staining for Loricrin in order to visualize the most external layer; we observed a complete re-epithelization only in samples treated with Angio.sup.PRP, while in skin treated with Hyalomatrix and PBS we observed an incomplete epithelium at day 21.
[0068] Example 7—Skin elastic properties Following sacrifice the skins for mechanical testing were placed in metal screw clamps with rubber pieces covering the clamped ends. Clamps were placed into a Bose Electroforce 3100 instrument. Applying an initial traction of 0.15 N. The traction measured in MPa was increased by 0.2% per second up to the breaking point. Force (N) and displacement (mm) were measured on a xy plotter and these points were subsequently recorded as stress (σ=force per cross-sectional area) and strain (ε=change in length/initial length) and re-plotted in Excel (Ashley W. Seifert et. Al; 2012).
[0069] To assess skin weakness, we compare mechanical properties of skin treated with Angio.sup.PRP, PBS, Hyalomatrix (as negative control) and healthy skin (as positive control). We derived stress-strain curves from dorsal skin to determine the mean tensile strength. Skin treated with Angio.sup.PRP showed a higher resistance to traction compared with skin treated with Hyalomatrix or with PBS (3 MPa±0.7, 0.3 MPa±0.3 and 1.8 MPa±0.70) (
[0070] In addition to epidermal characterization, we performed an analysis of dermal composition. Alcian Blue staining was used to evidence the presence of collagen fibers in dermal area. Thanks to the intensity of this staining, it was possible to distinguish between high- and low-density elastic fibers in the skin samples and quantify the surface occupied by these two density types in the different treatments; as reported in
Example 8—Proteomic and Biochemical Analysis
[0071] Skin samples were extracted for proteomic analysis, centrifuged at 17,000×g for 10 min at 4° C. and supernatants were subjected to ultracentrifugation at 200,000×g for 1 h at 4° C. Pellets, re-suspended in 0.1 M ammonium bicarbonate, pH 7.9, were trypsinized according procedures previously reported (Bari, Perteghella et al. 2018), using 50±0.5 μg of proteins from each sample. One microliter of trypsin-digested mixtures was analyzed by nano-chromatography equipped with cHiPLC-nanoflex system (Eksigent, AB SCIEX, USA) coupled to Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA), through a 65 min gradient of 5-45% of eluent B (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile), at a flow-rate of 300 nL/min. Full mass spectra were recorded in positive ion mode over a 400-1600 m/z range at 70000 FWHM resolution, followed by 10 MS/MS spectra, at resolution of 17,500 FWHM, generated in data dependent manner on the most abundant ions. All data generated were searched using Proteome Discoverer 2.1 platform (Thermoscientific) based on SEQUEST search engine and human protein database (70726 entries, downloaded on January 2017 from UNIPROT website, www.uniprot.gov). The obtained protein lists were aligned, normalized (Sereni, Castiello et al. 2018) and then processed by means of Linear Discriminant Analysis (LDA) (Hilario and Kalousis 2008). For assigning each subject to specific group it was calculated the α-value parameter according the extracted marker proteins (Brambilla, Lavatelli et al. 2012).
[0072] In order to understand the protein profile of different wound treatments, we performed a nLC-MS/MS analysis; single PBS treatments were used as internal control for the relative group (NovySep or Hyalomatrix), while healthy skin was analyzed to identify a normal control profile of the skin from the same animal (internal control). Total number of proteins identified was 1514, with a high technical and group reproducibility. Proteomic profiles from Hyalomatrix wound tissue are markedly different compared to profiles of control skin samples from the same animal. The principal component analysis of the samples shows that control samples form a similar cluster from samples collected from Angio.sup.PRP treated wounds. In fact, samples treated with Hyalomatrix show a correspondence with their internal control, while single Angio.sup.PRP samples correlates with their single relative internal control, suggesting a systemic effect of the treatment. Healthy skin samples clusterize and correlate more with Angio.sup.PRP than Hyalomatrix group. After Bonferroni correction, 179 proteins were differentially expressed at least between 2 groups, although up- and down-regulated proteins were similar in the same comparison or condition evaluated. Analyzing the single treatment and its specific control, half number of differential proteins is changed for Angio.sup.PRP then for Hyalomatrix treatment. The same behavior is reported if the treated conditions are compared to healthy skin. This suggests a trend for Angio.sup.PRP closer to healthy skin than the Hyalomatrix one. The Proteome Recovery index (PRi) (Roffia, De Palma et al. 2018, Sereni, Castiello et al. 2018) was then applied to compare three different conditions, as the algorithm generates and compares healthy and disease profiles in order to identify the proteins involved in the homeostasis recovery. We identified 6 proteins that are absent in wounded tissue and recovered after Angio.sup.PRP treatment, and 12 proteins present in the untreated lesion and not in Angio.sup.PRP samples (PBS samples were used as negative controls). Levels of 97 proteins are recovered after Angio.sup.PRP treatment (57 up-regulated and 40 down regulated in damaged tissue), while only 23 were detected after Hyalomatrix treatment (14 in common with Angio.sup.PRPgroup). Clusterization of normalized data confirmed the results, with Hyalomatrix and relative controls in the same macro group, and healthy skin and Angio.sup.PRP single samples correlating with their relative negative controls. The differentially expressed proteins were analyzed using Cytoscape platform to evaluate the metabolic. Network analysis underlined the variation of different metabolic clusters both for wound induction and two relative treatments. In particular, clusters relative to serpins, proteins involved in defense response, cytoskeleton organization, complement and coagulation cascades, extracellular matrix (ECM) and ribosome are overexpressed in the Hyalomatrix treatment and its control condition, while in Angio.sup.PRP and relative control are normalized, as in healthy samples. On the opposite, clusters of keratins and proteins involved in lipid metabolism are more expressed in Angio.sup.PRP conditions than in Hyalomatrix ones. Inside these eight target groups, we focused our attention on 17 wound-related proteins expressed in a different way after the two treatments and compared to the healthy tissue: Caveolin-1, EGFR, Fibronectin, Decorin, Plectin, SERPINB2, Discoidin domain receptor-2, Prothrombin, Heme Oxygenase 1, Fibrinogen alpha- beta- and gamma-chains, Tenascin, Plastin-2, Alpha-Tropomyosin, Cytokeratin-6A and Annexin Al. The expression of these proteins displays an opposite trend for Hyalomatrix and Angio.sup.PRP treatments: for example, Caveolin 1 and Decorin are up regulated after Angio.sup.PRP treatment, but down-regulated in Hyalomatrix samples. On the opposite, factors involved in coagulation, such as fibrinogen, SERPINB2 and Prothrombin are more expressed after Hyalomatrix then Angio.sup.PRP treatment.
Example 9—Network and Statistical Analysis
[0073] Starting from the list of proteins selected by differentially and LDA procedures, the corresponding Homo sapiens Protein-Protein Interaction (PPI) network was extracted, and by means of Cytoscape plug-in, STRING 8 database (Pawlowski, Muszewska et al. 2010), known interactions were retrieved from several databases such as Prolink, DIP, KEGG and BIND. In addition, PPI were examined using Cytoscape 3.5 (Shannon, Markiel et al. 2003) and only experimentally verified interaction with >0.15 score were retained. Finally, Bingo 2.44 (Maere, Heymans et al. 2005) was used to emphasize modules based on functionally organized GO terms.
[0074] Criteria for identification of peptide sequences and related proteins were: trypsin as enzyme; three missed cleavages permitted per peptide; mass tolerances of 10 ppm for precursor ions and +0.6 Da for fragment ions were applied. Percolator node was used with target-decoy strategy to give final false discovery rates (FDR) at Peptide Spectrum Match (PSM) level of 0.01 (strict) based on q-values, considering maximum deltaCN of 0.05 (Kall, Canterbury et al. 2007); minimum peptide length of six amino acids, and rank 1 were considered, and protein grouping and strict parsimony principle were applied.
[0075] Differences of categorical variables were evaluated using X-squared test. Kruskal Wallis test was applied for comparing quantitative variables. Analyses were performed using R (3.5.2) statistical analysis software; p-value<0.05 was considered statistically significant.
[0076] SpCs were evaluated by t-test, too (Zhang, VerBerkmoes et al. 2006), proteins selected by both LDA and MAProMA were evaluated by hierarchical clustering (Zhao and Karypis 2005) applying Ward's method and Euclidean distance metric. LDA. using common covariance matrix for all stratified groups and Mahalanobis distance (Jain A K 1999) and hierarchical clustering were performed by JMP 5.1 software. To select proteins discriminating the stratified children groups, we considered those with largest F ratio (≥5) and smallest p-value (≤0.05). Based on a direct correlation between the spectral counts (SpC, also called PSM) and relative abundance of identified proteins, DAve (Differential Average) and DCI (Differential Coefficient Index) indices of MAProMa (Multidimensional Algorithm Protein Map) software (Mauri, Riccio et al. 2014) were used to process average (aSpC) corresponding to each analyzed children group. The threshold values imposed were DAve>|0.2| and DCI>|100|.