Sampling the skin surface chemistry for diagnosis and prognosis

Abstract Skin and wound blotting are non‐invasive techniques used to sample the skin and wound surface chemistry, whereby a nitrocellulose membrane is applied to an intact or broken cutaneous surface to detect biomarkers. However, there has been no comprehensive review of the evidence for the techniques used and data obtained to date. The primary aim of this study was to review the utilities of surface blotting for the diagnosis and prognosis of physiological, pre‐disease, and pathological states. The secondary aim was to summarise the procedural steps. A systematic literature search was conducted on 9 July 2021 using Medline, Embase, and Google Scholar databases. Investigators used McMaster's Critical Review Form for Quantitative Studies to assess quality, then performed a narrative synthesis reporting according to Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines. Twenty‐five studies were reviewed. Eighteen studies were of good quality, and seven were of moderate quality. These studies conducted skin and wound blotting on 176 animals and 1546 humans. Studies reported physiological and pathological states for diagnosis and prediction of conditions, including skin tears, wound healing, biofilm detection, and skin barrier function. The four steps for blotting are surface preparation, blot preparation, application and removal of blot, and analysis. This review demonstrates that blotting can determine the skin and wound surface chemistry using a versatile and reproducible technique. However, future research is needed to validate the technique and skin biomarkers identified.


| INTRODUCTION
Skin is a multifunctional organ that is responsive to the external environment and the internal systemic condition of the individual. 1 There is mounting evidence that the chemistry of the skin's surface can be correlated with underlying conditions and potentially with systemic changes. 2,3 Sampling the skin and wound surface for microbiological analysis is standard practise. A non-invasive sampling technique for biomarker analysis is anticipated to be of interest for diagnosing and monitoring both skin pathology and systemic conditions.
Biomarkers are objectively quantifiable and measurable characteristics resulting from biological processes. 4 Examples of skin biomarkers include testing of local (e.g., a swab to detect skin microbiota) or systemic status (e.g., biopsy to determine the level of myxovirus resistance protein A in cutaneous lupus). 5,6 Invasive tests, such as biopsy, remain a gold standard for comprehensive, full-thickness analysis. However, a biopsy requires a skilled practitioner and has associated complications.
A broad definition of blotting is the transfer of biological substances from one medium to another. 7 Blotting the skin to harvest material for surface chemistry analysis involves a nitrocellulose membrane applied to intact or broken skin for a period of 10 s to 10 min. 8,9 The blotting material is processed to identify the presence of biomarkers.
There has been no synthesis of the literature with respect to either skin or wound blotting. The primary objective of this study was to conduct a systematic review with narrative synthesis on blotting's applications for diagnosis and prognosis purposes. The secondary objectives were to present the typical blotting method with significance, variations, validity, and reliability to facilitate replication and guide future research. These results are anticipated to be helpful for wound care specialists, dermatologists and plastic surgeons working in clinical research.

| Blotting related to skin physiology
The skin is an effective barrier against irritants, pathogens and transepidermal water loss due to the presence of sebum, intercellular lipids, and keratinocyte tight junctions. 10 Under normal circumstances, only molecules <500 Daltons may penetrate the dermis. 11 Minematsu et al. demonstrated that large water-soluble molecules permeate in or out when the skin is over-hydrated. 9 Molecules within the deep dermis and subcutaneous tissue leak via the trans-follicular route, whereas those in the more superficial layer of the dermis and epidermis permeate via the trans-epidermal route. Minematsu 12 In the case of skin wounds, a fluid-rich sub-surface replete with cellular agents, messengers and pathogens is exposed. 13 Both techniques use nitrocellulose membranes, commonly used to fix proteins in Western blotting. These attract polar molecules and absorb proteins. 9 (see Figure 1). The commonly used steps are reported in Section 3.2.

| Search strategy
Following PRISMA-S guidance, a research librarian (CH) designed a systematic search strategy with investigators (see Appendix A). 15 Searches were conducted on the Ovid Medline, Embase, and Google Scholar databases on 9 July 2021. Citation tracking was used for known active authors. Forward and backward tracking identified key papers that the PubMed and Google Scholar algorithms assessed as similar. Citations related to skin and wound surface chemistry sampling were included for background information. Search results were aggregated in EndNote software, deduplicated and shared with team members for screening. 16 Through peer review and further citation searching, investigators found one eligible study and updated the synthesis to include this. 17 There were no other deviations from the protocol.

| Screening, data extraction and statistics
Investigators paired up to independently screen abstracts and full-texts according to eligibility criteriathen extract data using a template (see Appendix B). In the case of unresolved disagreement, pairs reached a consensus through discussion. Descriptive statistics show population distributions with percentage frequencies, measures of central tendency and spread. No statistical testing was performed.

| Eligibility criteria
Including: • Publications in peer-reviewed journals.
• Using skin or wound blotting defined by Minematsu et al. 8,9 • Participant species are animals or humans.
• Articles in any language.
Excluding: Conference abstracts, review papers.

| Study quality
Using McMaster's Critical Review Form for Quantitative Studies, investigators independently assessed quality in pairs. This instrument determines the methodological quality of studies. 18 Where there was disagreement, pairs reached a consensus by discussion. The assessment covers the domains of purpose, literature, design, outcome, intervention, results, dropouts, and conclusions. Each study was assigned an overall score out of 15. Based on the previous reviews, 7-10 was moderate quality and >10 was good quality. 19

| Reporting of biomarkers
Organic compound types can categorise biomarkers: proteins, carbohydrates, lipids, and nucleic acids. They are further sub-categorised if there is a known association with a 'pro' or 'anti' effect in a recognised stage of wound healing. During processing, blots are stained or labelled to highlight biomarkers. Conventional histochemical dyes, such as Alcian blue, identify mucopolysaccharides, while immunostains recognise proteins such as tumour necrosis factor (TNF).
Visualisation is facilitated by traditional light microscopy, chemical luminesce, or immunostaining. A computer can count and calculate the relative number of stained/labelled pixels. The reported results determine whether a biomarker is present, the spatial distribution of that biomarker on the blot, the measured area of the blot, and the quantity of the biomarker using immunoreactivity intensity.

| Data synthesis
Some studies had two arms, a preliminary animal model and a human validation test. Both arms are reported in the text and tables. A summary of the skin and wound blotting steps is first presented, highlighting technique variations, validity, and reliability. This is followed by the role of blotting in associated skin physiology and disease, which has been categorised into skin or wound blotting.

| Quality assessment
The 25 included studies were assessed across nine domains. 18 In their overall score, 18 studies were deemed good quality, and 7 studies were of moderate quality.

| Blotting technique
The blotting technique can be divided into four interventions: (1) surface preparation, (2) blot preparation, (3) blot application and removal, and (4) analysis shown in Table 2. Technique variation and biomarkers studies are shown in Table 3.

| Significance and variations
Surface preparation: The anatomical location varied if a localised pathology necessitated blotting at its site of occurrence, for example, a pressure ulcer. In studies testing the skin in unlocalised pathology, the upper and lower limbs were frequently tested (8/14 or 57% of human studies). Koyano et al. verified no difference in the bilateral protein secretion on the intact skin of extremities for type IV collagen (COL-4), matrix metalloproteinase-2 (MMP-2), and tumour necrosis factor-alpha (TNF-α). 28 The paper demonstrated that systemic (e.g., age) and local factors (e.g. environmental exposures) influenced the intensity levels of COL4 and MMP-2 on the extremities while systemic factors influenced abdominal COL4 and MMP-2 (but not TNFα). Three skin blotting studies report controlling for cleaning or topical skincare agents, but this was not formally compared. Koyano et al. disallowed ointments or bathing the day before sampling to avoid disturbing protein balance. 28 Higuchi et al. allowed daily bathing but did not permit moisturisers. 29 Sari et al. reported blotting at least 30 min after ablutions or 1 h after bathing, and participants were asked to avoid skincare product applications. 41 There were 9/11 (82%) studies that described a protocol for cleansing wounds before blotting. This was performed with normal saline or chlorhexidine solutions, except for three studies that did not specify a cleansing agent.

Blot preparation
Hydration: Pre-wetting the membrane was conducted in all skin blotting studies; however, the volumes and solutions used varied or were not specified. Wetting the blot membrane (and thus over-hydration of the skin) facilitates the passage of soluble molecules through the skin barrier. 9 3/9 (33%) wound blotting studies in humans wetted the membrane before application. shaped skin blots, with the former justifying this as minimising the effect of tape (adhesive) removal on human skin. 9,22 All other studies reported square or rectangular blot dimensions or did not specify shape.
Minematsu et al. was the first study to describe skin blotting and blotted the backs of mice for 1, 5, and 10 min. 9 The study found that immunostaining signals increased with increasing duration.
Adhesion: The method of using an adhesive backing to secure membrane adhesion to a surface was reported by 6/14 (43%) skin blotting studies. The use of adhesive backing 'tape' was most commonly used. 9 size, edge and shape may not provide a surface for adhesion to be applied.

Processing
Storage: Following the application of nitrocellulose membranes to wounds, 13/25 (52%) studies described storing the membranes at 4 C after blotting but before analysis.  39,40 Image processing software was used to evaluate densitometry. Twenty biomarkers were evaluated by skin (n = 14) and wound (n = 11) blotting studies (see Figure 3).

Animal and in vitro models
Five studies used models to generate hypotheses before validating them with human samples. Minematsu et al. demonstrated that a mouse model was valid for skin and wound blotting for TNF-α. 8,9 Tamai et al. validated the correlation of skin blotted albumin (Alb) with TEWL in rats before humans. 26  Six studies used histological samples to improve criterion-

| Pruritus
Sari et al. found that the signal levels of blotted Alb and NGF-β were significantly higher in the cohort with itching than those without (p ≤ 0.001 and p < 0.001, respectively) and proposed that blotting for these two biomarkers may be candidates for the non-invasive measurement of itch. 41

| Obesity
Three studies evaluated skin fragility associated with obesity and surface TNF-α level, suggesting this represents a possible test of skin's mechanical vulnerability in obese patients. Minematsu et al. found an association between TNF-α levels in healthy male and female volunteers (p < 0.01). 9 Ogai et al. conducted two studies using normalised TNF-α values in healthy Japanese male skin. There was a significant association of measured TNF-α levels with the severity of obesity measured by BMI, visceral fat rating, waist circumference, and body fat weight (p < 0.05). 21,23

| Wound blotting (non-intact skin)
Eleven studies used blotting for wounds. Seven studies examined biofilm detection, while the remaining four explored the detection of inflammatory biomarkers in wounds. week. 36 The use of iodine ointment was associated with a statistically significant reduction in biofilm area (p = 0.02).

| Healing
In 2020, Wu et al. aimed to modify the wound blotting technique to establish a fast and straightforward procedure that is more clinically applicable. 39

| Standardisation
Variations of the blotting method exist for skin and wound blotting, different pathologies and target biomarkers without apparent clinical reasoning. For example, the blot application duration was 10 min in 92% of skin studies versus 10 s in 91% of wound studies. Biomarkers in wound exudate pass to a blot membrane faster than through an intact epithelium. 8,48 The lack of blotting standardisation confers the advantage of the

| Limitations of included studies
Studies had a low level of evidencethe highest being level III. 57 Only seven were prospective, while the remainder were retrospective,

| CONCLUSION
Blotting is a versatile, non-invasive test of the skin and wound surface chemistry, which is valid and reproducible. This narrative synthesis systematically reviews its utility for diagnosing and making a prognosis in pre-disease, pathological and physiological states. Skin blotting biomarkers may predict skin tears, pressure injuries, newborn skin problems, pruritis, and evaluating skin barrier function and fragility associated with obesity. Wound blotting has been used for predicting healing, biofilm presence and non-visible inflammation. The steps for blotting are surface preparation, blot preparation, blot application and removal and analysis. Clinicians should be mindful that the blotting techniques have not been standardised across all settings. Further studies are needed to assess the effect of variation in technique to standardise the method, detect novel biomarkers, and appraise the technique against non-invasive surface chemistry tests.