Infrared spectroscopy in the resection of gliomas
Elizabeth
1
Casselden,
1
Bulstrode,
1
Grundy,
Harry
Paul
2
3
1
Gray, Harvey Rutt, Diederik Bulters
Liam
1 Wessex Neurological Centre, Southampton, 2 Cardiff University, 3 University of Southampton
Optoelectronics Department
Results
Introduction
Gliomas are the most common primary brain tumour. Their infiltrative nature makes
complete resection difficult, and yet ‘gross total’ resection is believed to confer a
survival advantage of around 3 months over a subtotal resection.
Infrared absorption spectroscopy probes the molecular make-up of tissues based on
their absorption at different wavenumbers; a ratio at 2850/1655cm-1 representing
lipid:protein content has been shown to decrease incrementally from normal brain
tissue to grade IV astrocytoma (figure 1), and may offer a means of differentiating
grades of tumour and normal tissue. However, studies to date use small sample
numbers that are not paired with normal brain.
Spectroscopic differences, particularly at tumour borders, offer the potential for realtime analysis dependent on the ability to easily and rapidly analyse tissue. A method
of practical application might be through the use of the ultrasonic aspirator, used to
fragment and remove tissue during surgery. Analysis of the tissue stream in real-time
would offer immediate feedback to surgeons (figure 2), but depends on the assurance
that the spectroscopic properties of tissue are not altered by ultrasonic
fragmentation.
Figure 1: Previous results published by Krafft et al, who
demonstrated a decrease in the 2850/1655cm-1 absorption
ratio with malignancy.
Figure 2: A possible configuration of a device to attach to
the ultrasonic aspirator. As tissue is drawn through the
device, it is assessed by infrared light. There is potential for
immediate feedback.
Aims
 See if the infrared absorbance spectra of tissue collected through the ultrasonic
aspirator closely resembles that of tissue resected en bloc.
 See if these differences are maintained within tumours, and between patients.
 Reproduce the differences in infrared absorption reported between differing
tumour grades (including LGGs and non-astrocytic tumours) and tumour margins
at 2850/1655cm-1.
 See if other reference peaks offer discriminatory value.
 Otherwise no trends or significant differences were seen with
grade.
The effect of ultrasonic aspiration:
 12 paired samples (bloc-resected and aspirated from the same
tumour).
 Comparison showed:
• No significant difference in means at 2850/1655cm-1
• Absolute difference only 0.097 (aspirated mean±2SD:
0.465±0.288 vs .bloc resected 0.368±0.22, p=0.128, paired
t-test).
• High Pearson’s correlation, r=0.928, p<0.001 (figure 3).
Figure 3: Scatter graph showing correlation between paired aspirated and bloc-resected
samples. Pearson’s correlation coefficient r=0.928, p<0.001. n=12 in each group.
Differences at 2850/1655cm-1:
 By grade



Although numbers were very small for all non-grade IV
subgroups, there were no significant differences between mean
values for grade (figure 4).
Grade IV tumours showed a skewed distribution. When
considering median values, differences between grades are
smaller.
More paired normal and tumour samples (n=2) are needed to
show any consistent trend.
 Between white and grey matter

White matter showed a lower mean value at this ratio (n=2): a
counterintuitive result as this is lipid-rich and should be higher.
Samples: were taken during surgery from consented patients undergoing
craniotomy for suspected glioma in Wessex Neurological Centre. Ethical approval was
granted under REC08/H0505/165.
Sample preparation: Bloc-resected samples were cut from tumour destined for the
histopathology lab. Aspirated samples were collected from the stream of tissue
drawn through the Stryker Sonopet Ultrasonic Surgical System (UST-2001). Tissue
was processed for spectroscopy in KBr pellets, with blank KBr pellets prepared in the
same manner to establish a reference.
Data acquisition: Data were collected through an FTIR spectrometer, Varian 600-IR
in transmission mode, and assembled in Agilent Resolutions Pro v.5. Background was
recorded at 20 scans/min, samples at 15 scans/min. Resolution of 4cm-1 throughout.
The spectrometer was continuously purged with nitrogen, with regular background
spectra recorded.
Data analysis: Data were normalised to the peak around 1655cm-1 as per previous
literature. Peak maxima were identified. Corresponding values from a blank KBr
pellet were subtracted. Spectra were checked for ‘hidden’ peaks in the region of our
maxima. Ratios were compared according to histopathological diagnosis. SPSS was
used for statistical analyses.

Our results suggest that there are
differences in absorption from paired
samples taken from solid HGG and their
margins.
 These differences were observed at
2850/1655cm-1 as well as
2959/1655cm-1.
We were unable show any previously
described differences between HGG and
low-grade tumours or normal brain.
However with n values of 2, further
investigation is required.
Figure 4: Graph of absorption at 2850/1655cm-1. There appears to be a trend to an
increasing value at the 2850/1655cm-1 ratio with increasing malignancy (n=2 for normal
tissue; n=2 for grade II tissue; n=4 for grade III tissue; n= 16 For grade IV tissue).
Mean value for samples taken from the margins of HGG (n=4) appears lower than of solid
tumour.
 With astrocytic tumours alone
Materials and methods
We have shown that the effects of high
intensity, low frequency ultrasound on the
spectroscopic signature of gliomas are
limited:
• We showed a high correlation between
aspirated and bloc-resected tissue.
• Our spectra demonstrate the presence
of the same lipid and protein bands as
bloc-resected samples (as in figure 5).
Pending the production of statistically
significant data demonstrating large
enough differences between tissue types, it
is possible that a device could be developed
to analyse tissue in an ultrasonic aspirator
as a means of supplying real-time feedback
to operating surgeons (figure 2).
Intratumoural and intertumoural differences:
 2 pairs of samples taken from different parts of the same
tumour.
 Comparison showed:
• No significant difference in means at 2850/1655cm-1
• Absolute difference only 0.070 (mean±2SD: 0.857±0.032
vs. 0.787±0.066) and 0.001 (mean±2SD: 1.457±0.036 vs.
1.457).
• Substantial overlap of error bars for both pairs.
 One-way ANOVA demonstrated differences across the grade IV
cohort (n=16).
• Differences remain when justifiably outlying values
excluded.
• This might represent tumour heterogeneity or analytical
limitations.
Conclusions
After excluding oligodendroglial tumours, a trend in line with
that found by previous groups is seen; astrocytic tumours have
a lower value (both median and mean) at this ratio than normal
tissue. Differences did not reach significance.
Tumour margins:
 Samples were taken from the margins of 4 high-grade gliomas.
 Paired comparisons showed a difference of 0.143 (p=0.337,
paired samples t-test) between marginal tissue and tumour
proper.
 The difference in means was not significant (figure 4).
Other reference peaks:
References
Sanai N, Berger MS. Glioma extent of resection and its impact on
patient outcome. J Neurosurg 2008;62(4):753-766.
Krafft C, Sobottka SB, Schackert G, Salzer R. Analysis of human brain
tissue, brain tumors and tumor cells by infrared spectroscopic
mapping. Analyst 2004;129:921- 925.
Krafft C, Thummler K, Sobottka SB, Schackert G, Salzer R.
Classification of malignant gliomas by infrared spectroscopy
and linear discriminant analysis. Biopolymers 2006;82:301305.
Gaigneaux A, Decaestecker C, Camby I, Mikatovic T, Kiss R,
Ruysschaert JM et al. The infrared spectrum of human glioma
cells is related to their in vitro and in vivo behaviour. Exp Cell
Res 2004;297:294-301.
Ahmadi F, McLoughlin IV, Chauhan S, ter-Haar G. Bio-effects and
safety of low-intensity, low-frequency ultrasonic exposure.
Progress in Biophysics and Molecular Biology 2012;108:119138.
Acknowledgements
 We examined peaks at 2929cm-1 and 2959cm-1.
 These appeared more consistent in their wavenumber than
2850cm-1.
 At 2929/1655cm-1, tumour margins demonstrated a difference
of 0.709 (p=0.04) from normal tissue.
Figure 5: Example absorbance spectra from various tissues demonstrating the consistent
presence of the various peaks, and our ability to at least reproduce spectra broadly comparable
to those in the literature.
Mr. Jonathan Duffill and the Wessex Neurological Centre
theatre teams.
Tess de Leon, Optoelectronics Department, University of
Southampton.
Dr. Sandrine Willaime-Morawek, Elodie Siney, Alex
Holden, Clinical Neurosciences, University of
Southampton.