Biochemical markers for early detection of superficial pressure ulcers

Pressure ulcers are areas of soft tissue breakdown resulting from sustained mechanical loading of the skin and underlying tissues. They are painful, difficult to treat, and represent a burden to the community in terms of health care and finances. The prevalence of pressure ulcers is unacceptably high and varies from 13% in hospitalized patients to 35% in institutions for the physically handicapped. This high prevalence is partly due to limited risk assessment. Currently, risk assessment is mainly performed by questionnaires or scales like the Norton, Braden, and Waterlow scale. Most risk assessment scales are based on expert opinion or literature review and have a limited scientific background. Furthermore, they do not predict the degree of susceptibility with the required accuracy and indeed, up to 30% of the hospitalized patients with pressure ulcers are still misclassified. Accordingly, patients with high risk might not receive adequate preventive measures. The Dutch National Prevalence Survey demonstrated that grade I ulcers, the first stage of superficial ulcers in the skin, accounted for approximately 50% of the prevalence of pressure ulcers. Grade I ulcers are classified as non-blanchable erythema (NBE) of intact skin. Currently, the transparent disk method is commonly used for visual observation of NBE, in which the disk is pressed into the erythematous tissue. If the skin under this disk does not blanch, it is regarded as a grade I ulcer. The mentioned technique aims for measuring the consequences of ‘harm’ already done to the skin (i.e. inflammation). Whereas pressure ulcer prevention should aim for earlier detection of developing ulcers. Improved pressure ulcer risk assessment is, therefore, necessary. A first step in developing a new risk assessment tool is directed towards the detection of skin reactions that precede grade I ulcers. Cytokines and chemokines are of interest for pressure ulcer detection, since they are known to mediate inflammatory responses. The present thesis focuses on cytokines and chemokines as biochemical markers for early detection of superficial pressure ulcers. A loading device has been developed to study the damaging effects of mechanical loading on an in vitro model of the epidermis. The commercially available EpiDerm cultures were employed as human epidermal equivalents. The general morphology of the EpiDerm cultures is comparable to human epidermis. Various degrees of epidermal damage were obtained by increasing either the magnitude or duration of loading. Cytokines and chemokines, such as IL-1®, IL-1RA, TNF-®, CXCL8/IL-8, CCL20/ MIP-3®, CCL2/MCP-1, and CXCL1/GRO-®, were evaluated as biochemical markers for mechanically-induced epidermal damage using this custom-built loading device. IL-1®, IL-1RA, IL-8, and TNF-® were shown to be released as a result of mechanical loading. MIP-3®, MCP-1, and GRO-® could, however, not be measured. The release profiles of cytokines and chemokines were studied at various magnitudes and durations of mechanical loading. After 24 hours of loading with pressures ranging between 0 and 200 mmHg, an increase in the release of IL-1®, IL-1RA, IL-8, and TNF-® was observed at 75 mmHg and beyond. At the relatively low pressure of 75 mmHg, structural epidermal damage could barely be detected. So there seems to be a threshold for these markers, beyond which visible tissue damage occurs. Furthermore, as early as 1 hour after loading with 150 mmHg and before the onset of structural damage, the levels of IL-1®, IL-1RA and IL-8 were raised. Therefore, these markers are suitable for early detection of mechanically-induced epidermal damage in an in vitro setting. On the other hand, TNF-® seemed less suitable for early damage detection, since this marker was only raised when the first signs of structural damage were already apparent. Furthermore, the amount of TNF-® was very small and could hardly be detected using a ‘high sensitive’ immunoassay technique. Urinary incontinence is widely recognized as an important risk factor for pressure ulcer development, although scientific proof was lacking. Therefore, biochemical markers, such as IL-1® and IL-1RA, were used to study the effect of synthetic-urine on the susceptibility of the epidermis to mechanical loading. An increase in epidermal damage, as well as in the release of IL-1® and IL-1RA was observed when both synthetic-urine and pressure were applied. Although these findings are obtained in vitro, they might indeed imply that urinary incontinent patients have a higher risk of developing pressure ulcers. A clinical study was performed to determine whether IL-1®, IL-1RA, and IL-8 could also be detected in an in vivo setting. Sebutapes, adhesive films, were used to collect cytokines and chemokines from the skin surface in a non-invasive and painless way. Patients with a grade I ulcer at the sacrum, as well as patients without pressure ulcers participated in this study. For both patient groups, Sebutapes were applied to the sacrum as well as to the volar aspect of the left forearm (i.e. control site). Currently, only IL-1® could be detected using the Sebutape sampling method. For both patient groups, an increase in the absolute level of this cytokine was found at the sacrum compared to the volar aspect of the left forearm. This increase in IL-1® might be caused by sustained mechanical loading of the sacrum upon supine lying and sitting and was observed in spite of a high inter-subject variability in IL-1®. For patients with a grade I ulcer at the sacrum, the relative increase in IL-1® median ratio was higher when compared to the patient group without pressure ulcers. This result is promising and might again imply that a threshold is present for IL-1® beyond which visible tissue damage, as evidenced by NBE, occurs. In conclusion, cytokines and chemokines seem promising biochemical markers for early detection of superficial pressure ulcers. A combination of different markers, rather than a single marker, is however required to sufficiently monitor the status of soft tissues (i.e. skin). Currently, the field of biosensor technology is emerging. A biosensor might eventually be developed that is able to monitor various biochemical as well as physiological markers to determine the patients risk for pressure ulcer development

Diffusion measurements in epidermal tissues with fluorescent recovery after photobleaching

Background/purpose: Pressure ulcers are areas of soft tissue breakdown, resulting from sustained mechanical loading of the skin and underlying tissues. Measuring biochemical markers that are released upon mechanical loading by the epidermis seems a promising method for objective risk assessment of the development of pressure ulcers. This risk assessment method will better determine the risk of a patient to develop pressure ulcers than the risk score lists currently used. So far, experimental studies have been performed that measure the tissue response in the culture supernatant. To elucidate the transport of the biochemical markers within the epidermis, the diffusion coefficient needs to be established. Methods: In the current study, fluorescent recovery after photobleaching (FRAP) is used to determine the diffusion coefficient of fluorescent-labeled dextran molecules in human epidermis, porcine epidermis and engineered epidermal equivalents. These dextran molecules have a similar weight to the biochemical markers. Results: Similar diffusion coefficients were found for human and porcine epidermal samples (6.2 × 10-5±1.2 × 10-5 and 5.9 × 10-5±6.1 × 10-6 mm2/s, respectively), whereas the diffusion coefficient of the engineered epidermal equivalent was significantly lower (2.3 × 10-5±1.0 × 10-5 mm2/s). Conclusion: The diffusion could be measured in epidermal tissues using FRAP. In the future, the diffusion coefficients obtained in the current study will be used to study the difference between the transport in EpiDerm cultures and in human epidermis