The pressure is on: Imaging mass spectrometry for bedsore profiles

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  • Published: Nov 15, 2011
  • Author: Steve Down
  • Channels: Base Peak
thumbnail image: The pressure is on: Imaging mass spectrometry for bedsore profiles

Pressure ulcers profiling problems

One of the downsides for patients who are bedridden is the risk of pressure ulcers. Also known as bedsores, they develop when sustained pressure on a particular part of the body restricts the blood flow, resulting in tissue damage that can eventually lead to the formation of an ulcer.

Healthy people do not suffer from pressure ulcers because the continuous movement of their bodies prevents any part from being subjected to sustained pressure. However, those with certain health problems can be more at risk. For instance, people with type 2 diabetes have high blood sugar levels which can impair blood flow and those with chronic obstructive pulmonary disease have low blood levels of oxygen which make the skin more vulnerable.

In the UK, it has been estimated that about 500,000 people will develop at least one pressure ulcer in any given year, whereas US figures have indicated incidence rates of 0.4-38% and 2.2-23% for acute care and long-term care settings, respectively.

Although effective treatments are well established, they are provided without much knowledge of the molecular changes that are taking place in and around ulcers. This is partly because tissue from pressure ulcers is rarely sent for pathological examination, a practice that has also hindered the search for a modern diagnostic method.

One group of researchers investigating pressure ulcers circumvented the tissue shortage by collecting wound material that would normally have been designated as biohazard waste. It was taken from patients undergoing surgical removal of large pressure ulcers that were inhibiting their mobility.

Lillian Nanney and co-researchers from the Vanderbilt University School of Medicine, Nashville, Tennessee, USA, and the University of Calabria, Italy, wanted the tissue to test a novel approach to pressure ulcer analysis, which they described in Wound Repair and Regeneration.

Imaging mass spectrometry of ulcers

Imaging mass spectrometry has developed into a sensitive in situ technique for generating ion density maps of biological compounds distributed within tissue samples by measuring their molecular masses. It covers a wide mass range, encompassing peptides, proteins and lipids up to a maximum molecular mass of about 50,000.

The research team assessed the performance of imaging mass spectrometry for comparing the protein and lipid profiles of different regions around the ulcer. The bed and edge of the wound and the adjacent non-eroded tissue were examined, before comparing the upper and lower portions.

Tissue sections were mounted on a gold-coated plate for ion imaging on a matrix-assisted laser desorption/ionisation mass spectrometer in two modes. The first was profiling in which the samples were spotted with droplets of the matrix and those spots were analysed. For full imaging, the tissue samples were coated completely with matrix and mass spectra were acquired right across the sample.

The preliminary profiling studies for the proteins in each region of the wounds were unique. The distinctive profile of the wound bed had more peaks and higher abundances in the region m/z 2500-4000, whereas the adjacent healthy tissue displayed more peaks at m/z 4000-6000.

The diseased adjacent tissue had a unique profile too, with distinctive peaks at m/z 8000-12,000. Overall, many peptide/protein peaks were found to have variable intensities between the tissue types, with the alpha-defensins of particular interest.

The lipid profiles also varied with tissue type. For instance, several glycerophospholipids displayed different abundances whereas others were unique to particular regions. Some glycerophosphoserines and glycerophosphates were characteristic of the wound bed and others were unique to it.

Lipids and proteins differentiate pressure ulcer regions

The mass spectral profiles are informative but make it difficult to compare the spatial patterns across the ulcers. This is where ion imaging comes into its own. Following scanning in two dimensions, the associated software produced an image of the whole sample highlighting the locations of a particular ion.

Three of the defensins, known as HNP-1, -2 and -3, were prominent across the ulcers. In particular, HNP-3 was the most abundant in the wound bed for four out of six patients and the upper and outer portions of the ulcers were rich in HNPs, which is consistent with their exposure to microbes.

For some wounds, the lower bed appeared to be similar to the adjacent tissue as well as that of control patients as evidenced by the distribution of thymosin beta-4, a protein which is associated with mature intact granulation tissue. The exception to this observation was the low abundance of this protein for the "most stagnant wound in this cohort, suggesting little evidence of a return toward normal dermis in this specimen."

The ion maps for the lipids partitioned the wound bed into two specific areas corresponding to the upper and lower portions of the ulcer. The glycerophosphocholines and glycerophosphoinositols were concentrated in the upper wound bed whereas glycerophosphates were more likely to be abundant in the lower wound bed. Once again, the distributions allowed differentiation between the wounds and the surrounding tissue.

Although the ion images were revealing, a better visual representation was obtained by principal components analysis (PCA). When applied to the protein distributions, those from the upper and lower wound beds of one patient were clearly grouped into two clusters. For another patient, there was more overlap between the two clusters, indicating a more homogenous ulcer.

Cluster separation was also observed in both cases when comparing normal skin from a control subject with that from the hypertrophic epidermis at the wound margin, and with the skin immediately adjacent to the wound bed.

This pilot study has confirmed that the use of imaging mass spectrometry combined with the sensitivity of PCA will permit differentiation between the different degrees of ulceration. The researchers believe that technologies such as this are required to "advance the science and practice of wound care" beyond current practice of visual interpretation.

The study of many more biopsies should lead to the stage when "the molecular portrait of an individual wound is expected to classify wounds into those that are improving, remaining stagnant, or continuing to deteriorate."

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

The protein and lipid compositions of pressure ulcers have been visualised by imaging mass spectrometry to give a much-needed view of the molecular status of these chronic wounds that could lead to a prognostic test of their reparative status.Image: NHS UK

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