NMR IN BIOMEDICINE

NMR Biomed. 2001;14:224–232

DOI:10.1002/nbm.707

Introductory article
Common processing of in vivo MR spectra

HJA in 't Zandt, M van der Graaf and A Heerschap*

Department of Radiology, Faculty of Medical Sciences, University Medical Center Nijmegen, 6500 HB, Nijmegen, The Netherlands

Received 19 March 2001; accepted 20 March 2001

ABSTRACT: This introductory article addresses approaches currently in use to process in vivo spectra. First, a brief
overview is given of the information content represented by the parameters of MR signals. Subsequently, common
steps in the processing of MR spectra such as pre-processing, normalisation and quantification and the use of prior
knowledge are described. Finally, some prospects for more advanced processing are given. Copyright  2001 John
Wiley & Sons, Ltd.
KEYWORDS: magnetic resonance spectroscopy; data processing; line fitting; quantification

INTRODUCTION can be distinguished for MRS data processing: (a) at the

One of the essential elements of an in vivo magnetic
resonance (MR) spectroscopy (MRS) examination is the
processing of the raw time domain data to quantities that
can be digested in further clinical evaluation or in
biochemical, physiological or morphological interpreta-
tions. This processing may range from Fourier transfor-
mation into the frequency domain and simple visual
inspection of MR spectra by the eye to the application of
highly advanced statistical approaches. Until now
research on MR spectroscopy methods has been focused
mostly on the optimization of acquisition techniques such
as those necessary for acquiring signals from well-
defined localized regions. Although continued efforts
will be made to achieve better localization and sensitivity
in MRS, in some areas of its application attention is
shifting towards optimal and efficient processing, espe-
cially in those areas addressing clinical problems.
Over the years MR spectroscopy has clearly proven to
be an important non-invasive tool in biomedical research
to study humans and animals. Although the potential of
MRS as a clinical tool has been obvious since its first
application to humans,1,2 only in recent years has this
started to be realized in a more routine clinical setting,
mainly as a result of the inclusion of a number of
automation steps in data acquisition. In particular for 1H
MRS of the human brain the major manufacturers of MR
systems now offer ‘push-button’ methods for data
acquisition. From this background two different needs
*Correspondence to: A. Heerschap, Department of Radiology/430,
University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The
Netherlands.
Email: a.heerschap@rdiag.azn.nl
Copyright  2001 John Wiley & Sons, Ltd.
clinical level, with minimal and easy user interface for
the MR technician and clinician who have no time to be
bothered by many technical details; (b) at the scientific
level, for the researcher interested in the selection and
exploration of various processing routes and parameters.
Ideally, both levels should be part of one software
package, so that any protocol developed under level B
can be easily transferred to level A.
Apart from this organizational aspect another important
issue to consider is the variable application of MRS,
which requires different data processing approaches. For
instance, MR spectra of the brain may need different
processing as compared to MR spectra obtained from
body tissues such as the prostate and liver or extremities
such as skeletal muscle. Likewise, MR spectra of different
nuclei may require different processing strategies.
It is not the intention of this paper to give a
comprehensive overview of cutting edge developments
in quantitative MRS. It rather presents an introduction of
some currently used approaches and views for common
MRS data processing.
MRS PARAMETERS AND THE INFORMATION
CONTENT OF MR SPECTRA
Most MR spectroscopy acquisition schemes that are
applied to the human and animal body will produce raw
data in the form of damped oscillations as a function of
time, mixed with noise. As direct visual inspection of
these time-dependent data is not straightforward, it is
common to analyze them in the frequency domain by
applying a Fourier transformation. The result is an MR
NMR Biomed. 2001;14:224–232
 COMMON PROCESSING OF IN VIVO MR SPECTRA
spectrum, which consists of noise and signals as a
function of frequency, the latter originating from specific
spin systems, mostly from small metabolites present at
tissue levels of about 0.1–1 mM or more in the body.3 The
raw data produced by MRS acquisition usually also
represent spatial information. Then, Fourier transforma-
tion may result in a single spectrum of one spatially
resolved volume of interest or in multiple spectra
associated with multiple locations.
Commonly, processing software packages provide
means to represent the data in the frequency domain,
although quantitative analysis can be done in either the
frequency (see Mierisova et al., this issue) or the time
domain (see Vanhamme et al., this issue) or a combination
of both.4 Individual signals in absorption mode MR
spectra are characterized by their resonance frequency,
line shape, line width, phase and integral (amplitude).3,5
The precise values of these parameters are determined by
the molecular structures and tissue levels of the corre-
sponding compounds, the local and global physiological
conditions in which they occur, and by particular MR
acquisition conditions. Any parameter may be of interest
and may uncover aspects of tissue morphology, metab-
olism and physiology or may serve as a clinical biomarker.
Normally the specific relative frequency of resonances,
or their chemical shift, is used to identify the biochemical
species from which these resonances originate. These
shifts are considered constant in most situations and can
be used as prior knowledge to improve the estimation of
other spectral parameters. However, the relative spectral
position of resonances may be affected by physiological
parameters like pH, Mg2‡-binding, oxygenation and
temperature. This possibility has to be taken into account
in the analysis of spectra. In turn, these specific shifts
may also be used to study variations in physiological
conditions. For instance, the shift of the water peak in 1H
MR spectra can be used to monitor temperature
changes.6,7 Monitoring high-energy phosphates using
31
P MRS during metabolic stress will reveal shifts of the
resonances of ATP and inorganic phosphate due to
changes in pH and Mg2‡ levels e.g. see8,9. The resonance
frequency of each peak in a spectrum obtained at a
particular location may also be shifted with respect to
those in spectra obtained at different locations in the body
due to B0 variations. Automatic processing of an array of
MR spectra in batch mode will be hampered by changes
of resonance frequencies as a function of time or of space.
In data processing most attention is usually focused on
the analysis of signal integrals as these integrals reflect
the tissue levels of metabolites, at least if MR spectra are
obtained under appropriate experimental settings. Under
particular (patho-)physiological conditions these levels
may change and signal integral variations thus may serve
as (clinical) biomarkers of these conditions. Moreover,
they may represent the cellular content of the metabolites
by which a more biochemical interpretation of these
changes becomes possible. However, the smallest
Copyright  2001 John Wiley & Sons, Ltd.
225
volume of interest measured by in vivo MR spectroscopy,
in most situations, contains many thousands of cells with
potentially various levels of heterogeneity. These vo-
lumes may also include blood vessels and other
intercellular spaces. Therefore tissue metabolite content
as deduced from MR spectroscopy reflects a mixture of
morphology and biochemistry. Further interpretations on
metabolic processes can only be made on the basis of
certain plausible assumptions such as the presence of
tissue homogeneity for certain biochemical processes and
minimal contribution of signals from extra-cellular
spaces.
Line width differences between resonances are largely
caused by the differences in the effective T2-relaxation of
spin systems. An important determinant of this type of
relaxation is the mobility of the molecular components, of
which these spin systems are part. Thus, while spin
systems of small metabolites have relatively long T2
values and small line widths, those of macromolecular
compounds usually have short T2 values and this may
cause the presence of broad overlapping components in
MR spectra. These components can be filtered out of the
spectrum using longer echo times for data acquisition,
which greatly simplifies quantitative analysis; albeit this
occurs at the expense of loss in available information and
signal-to-noise. Transient binding of small metabolites to
larger macromolecules such as proteins, nucleic acids or
lipids, or trapping by membranes or organelles also causes
a decrease in their T2-relaxation time, and this results in
broader peaks in the MR spectrum or even renders the
metabolites (partly) invisible.10,11 Other factors such as
(local) variations in susceptibility and chemical exchange
may also affect the line-width of resonances.
As the solution condition of small MRS visible
metabolites may be approached as homogeneous and
freely distributed in cellular water, their MR resonances
are expected to have a Lorentzian line shape. However, a
pure Lorentzian line shape is seldom observed in MR
spectra from a living system and the shapes of peaks may
appear very irregular. The actual shape is influenced by
several experimental factors like variations in local
magnetic susceptibility of tissue, eddy currents caused
by switching field gradients and spatial variations in the
homogeneity of the magnetic field. It can also result from
tissue movement and compartmentalization or restricted
movement of molecules. The latter can even cause a
splitting of the peaks due to residual dipole–dipole
interactions such as has been observed for creatine,
taurine and lactate proton spins in 1H MR spectra of
skeletal muscle.12–14
Some lines may not occur as singlet peaks but as
multiplets because of splitting due to spin–spin interac-
tions, also known as J-coupling. These interactions may
affect relative frequencies, integrals and phases of the
peaks. As the J-coupling makes the integral and phase of
the multiplet peaks dependent on the echo time of
acquisition sequences, this may complicate data proces-
NMR Biomed. 2001;14:224–232
226 H. J. A. IN’T ZANDT ET AL.

Figure 1. (A) Flow scheme from raw time domain data, produced by an
MRS experiment, to clinical diagnosis or biomedical interpretation. The
raw data can be processed by appropriate software supplied by MR
manufacturers or by stand-alone software (e.g. LCModel, MRUI or
another). The quantitative result can either be used for further
interpretation or diagnosis, or as input in a pattern recognition routine
for classi®cation. Raw time-domain data may also be directly fed to
pattern recognition routines for classi®cation. (B) More details of
possible pre-processing methods, either in the time or frequency
domain that may be included in this evaluation software

sing. Knowledge of the associated phase modulation may
be needed to estimate the multiplet integral. Typical
multiplet characteristics may also be used to identify and
resolve signals of the corresponding spin systems, for
instance in special spectral editing sequences e.g. see5
such as employed to resolve the methyl lactate peaks
from strongly overlapping tryglyceride signals.
There are several other MRS parameters that may be
derived from MR spectra obtained in a time-dependent
manner by which further interesting metabolic or
physiologic information can be derived, but these will
not be discussed here.
of peak integrals from spectra plotted on paper by
triangulation.15 Processing of MRS data can either be
performed by programs available on commercial MR
systems or by stand-alone programs (see Fig. 1). For most
of the commonly available software packages the raw
time domain data serve as input to produce estimations of
spectral parameters or results on a higher level of
sophistication. This is usually achieved with some type
of pre-processing of the raw data and the frequency spec-
trum to improve the accuracy of the final evaluation.
Preprocessing

COMMON PROCESSING OF IN VIVO MR
SPECTRA
Quantification of the parameters of resonances in MR
spectra is currently performed by computer techniques
and this has completely replaced previous cumbersome
methods of manual assessment, such as the determination
Copyright  2001 John Wiley & Sons, Ltd.
A number of data corrections are related to the precise
experimental conditions by which the data have been
acquired such as RF inhomogeneity, off-resonance
effects, imperfect slice excitation profiles, outer volume
contamination, effect of the point spread function in
spectroscopic imaging experiments etc. Taking these
experimental conditions properly into account is a major
NMR Biomed. 2001;14:224–232
 COMMON PROCESSING OF IN VIVO MR SPECTRA
issue in comparative studies (e.g. of data acquired on
different MR systems). Some MRS pre-processing
features that have a more general use, and are included
in most processing software packages, are shown in Fig.
1(B) and will be briefly discussed below (details can be
found in several handbooks and monographs e.g.16,17).
Relevant pre-processing procedures in the time domain
are given below.
Zerofilling: this is the extension of the number of
sampled time domain points with points of zero
amplitude, mostly with the aim of obtaining an improved
digital resolution for a better spectral visualization. In
some cases spectral resolution and signal to noise may be
improved by these additions.17,18
Windowing and filtering: this is performed by multi-
plying the raw MR time domain signal by some function
with the aim of improving the signal-to-noise, or
inversely the spectral resolution, or to remove truncation
artifacts and/or broad spectral components in the
frequency domain. Numerous methods have been
proposed for this purpose but commonly filtering of the
time domain signal by window functions with exponen-
tial, Lorentzian, Gaussian or other shapes is performed to
improve the signal-to-noise ratio of the spectrum after
Fourier transformation. Optional frequency selective
filtering of specific disturbing spectral components (e.g.
dominating water or lipid signals) is a very useful pre-
processing feature,19 but not immediately available in
most current processing packages.
Corrections using a reference signal: time or spatially
dependent experimental artifacts which are difficult to
assess directly from signals of metabolite spins with low
signal-to-noise ratio, may be corrected using the much
stronger signal of water. This signal can be obtained from
the same volume of interest, for instance in a separate
measurement using no water signal suppression. This is
in particular useful in localized 1H MRS to correct for
eddy current artefacts caused by switching magnetic
gradients.20,21 The point-wise division in the time domain
by the phase of the signal of this water reference is also
useful for unbiased zero-order phasing of 1H MR spectra.
Furthermore, frequency shift corrections using either the
water or another major signal as a reference are helpful to
correct for frequency variations between voxels in
spectroscopic images22 or in spectra obtained as a
function of time.23,24
In practice, the water 1H MR signal should be used
with care as a reference, because unwanted interference
may spoil the correction.25 For instance, if next to water a
substantial amount of lipid signal is present, the latter MR
signal should be removed using filtering techniques like
HLSVD26 before the eddy current correction is executed.
Furthermore, in the time between the acquisition of the
water reference signal and the metabolite spectrum, slight
changes in experimental conditions may occur, such as
frequency shifts, which may spoil the phase correction.
An additional problem occurs in clinical spectroscopic
Copyright  2001 John Wiley & Sons, Ltd.
227
imaging exams where the separate acquisition of a water
reference data set may be too time-consuming. Several
alternative methods have been proposed to deal with
these problems in the correction of line shapes e.g.
see.27,28
To obtain good quality frequency domain data the
following procedures, to be performed after the Fourier
transformation, are relevant.
Phasing: for correct quantification, proper phasing of
the spectrum is essential to obtain a pure absorption mode
MR spectrum. Most of the commonly available software
packages provide means for automatic phasing of MR
spectra.
Baseline correction: experimental artifacts and
broad resonances of large molecules may contribute to
a rather ill-defined ‘baseline’ that may hamper proper
quantification of the resonances of the small meta-
bolites. MRS processing software packages also provide
different means to correct for this background signal.
For instance, most packages have the capability of
estimating the baseline using spline or polynomial
functions and to subtract it from the original spectrum.
Especially in ‘short’ echo time proton MR spectra, this
baseline may pose a severe quantification problem. A
significant overlapping background component may
occur due to residual (broad) water and lipid signal
components and due to the presence of broad macromol-
ecular signals more prominently present at shorter echo
times.29
Quanti®cation
After proper pre-processing the spectral data can be
quantified. This may comprise simple approaches such as
computer integration of signal areas after manual
indication of its spectral extent and the base line.15 Data
may be processed effectively in this way, but the outcome
is biased by the knowledge of the operator of how an
MRS signal or the baseline should look, and it is
laborious. Many less operator-dependent evaluations
have been proposed up to complete operator-independent
‘black box’ types of analysis based on approaches
derived from chemometrics and related fields e.g. see.30
Knowledge of the line shape of the resonances in MR
spectra is a very helpful asset that can be used as prior
information in data processing. However, as the line
shape function of an in vivo MR resonance often is not
well known, it is a matter of debate whether peak
quantities should be estimated with a fit using a model
line shape function or by model free approaches e.g.
see.15,31,32
The fitting of peaks by a line shape model function
often requires further pre-processing. For example, it
may be advantageous to convert line shapes to Gaussians,
which have relatively narrower feet, for improved peak
integration and curve fitting in the case of overlap.33 To
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228 H. J. A. IN’T ZANDT ET AL.
correct irregular line shapes it is possible to convert them
to Lorentzians as proposed in the QUALITY procedure
using the water signal as a reference.34 By partly
combining this approach with the eddy current correction
procedure as proposed in Klose20 this line shape
correction method has been further improved.35 As
model functions are often only approximations of the
actual line shapes their use in fitting may introduce
model-dependent structural errors in the estimation of
signal integrals. For the estimation of peak integrals often
only part of the wings of the peaks is included to avoid
too much interference with baseline and noise. The
choice of the cut-off point for the selected part will have
an effect on the outcome. For these reasons it is essential
to use the same line shape function and processing
procedure in a complete analysis of series of spectra
obtained in an examination.3,15
The software programs for MRS processing provided
by the major MR manufacturers can be used to estimate
signal integral, width, phase and resonance frequency
from fits of the measured data to model functions. An
advantage of using these packages is that the data can be
processed on the same measurement console and full
support of the input of the data into the analysis routines
can be expected. However, this software mostly lacks
advanced algorithms which may be needed for proper
signal analysis and many groups have developed signal
fitting packages for improved performance, either
processing data in the time domain or frequency domain
or both. Some of these packages are widely used. For
example the so-called LCModel (linear combination of
model spectra of metabolite solutions in vitro) program,36
which processes data in the frequency domain, is often
used today to evaluate (short echo time) 1H MR spectra of
the brain, the major clinical target tissue of MR
spectroscopy. The parametric MRS processing package
called MRUI (magnetic resonance user interface),37
which processes data in the time domain,38,39 has broader
possibilities. It is often successfully used to process 31P
MR spectra of various sources and also (long echo time)
1
H MR spectra of the brain and other tissues. It contains
some useful features like frequency selective signal
removal (e.g. of water and lipids). As currently available
packages require off-line processing and integration, they
are not optimal for routine clinical purposes. Further-
more, as they initially were developed to process MR
spectra of single locations in the body the evaluation of
multi-voxel or spectroscopic imaging data may be
cumbersome, in particular in the absence of dedicated
spatial visualization tools. A possible work-around is to
combine the use of several software packages and exploit
the strongest features of each package e.g. see.40 Then
transfer of data between packages is needed, which may
be impractical and home-written conversion software
may be required, as well as different computer platforms
and software support tools.
An important feature of any package should be that it
Copyright  2001 John Wiley & Sons, Ltd.
provides an estimation of the error in the determination of
spectral parameters so that the user has a tool available to
discriminate between good and bad data. The Cramér–
Rao lower bound as error estimation is mostly used for
this purpose e.g. see.4,38,41,42
USE OF PRIOR KNOWLEDGE
MR spectra, obtained in vivo, often suffer from an intrinsic
low signal-to-noise ratio and overlap of resonances which
hamper proper signal assessment and quantification. This
situation can be significantly improved by invoking a
priori knowledge in the data evaluation process. Already
some pre-processing manipulations may assume general
features of the data. In line fitting procedures assumptions
on the line shape model itself constitute a basic type of
prior knowledge.38 If other signal parameters are known
such as relative resonance frequencies, amplitude ratios,
scalar coupling and phases, any of these can be
incorporated in fitting routines to reduce the number of
model parameters and thus to enhance the robustness and
speed of the fit. One of the first successful applications of
this approach to in vivo MR data was in time-domain
fitting of signals in 31P MR spectra of muscle and brain by
the so-called variable projection method VARPRO, using
J-coupling characteristics of the ATP signals and assum-
ing equal signal phases.43
The use of prior knowledge in the fitting of signals
occurring in 1H MR spectra is less obvious. For example,
the resonances of J-coupled spin systems from metab-
olites like glutamate, glutamine and myo-inositol, which
are visible in 1H MR spectra of the brain, may show
complicated multiplet patterns. They strongly overlap
with each other and with singlet peaks of other MRS
visible compounds, especially at the low clinical field
strength of 1.5 T. The appearance of these patterns is very
sensitive to some 1H MRS acquisition parameters such as
the echo time. For these reasons de Graaf and Bovée33
have introduced the measurement of individual metab-
olites in vitro at similar experimental conditions as the in
vivo measurements to derive parametric information on
multiplet structure, chemical shifts, relative amplitudes
and line widths and the degree of J-modulation. This
parametric information was used as prior knowledge for
improved quantitative assessment by frequency-domain
fitting of signals in rat brain MR spectra.33
A different implementation of a comparable approach
was introduced by Provencher and is available in the
software package LCModel36,44 (this issue). It also makes
use of experimentally determined MR spectra of the
individual metabolites, which are expected to contribute
to the in vivo MR spectra. The spectrum of each
metabolite in solution is acquired separately under the
same experimental MR conditions in which the in vivo
spectra are obtained. These spectra are not further
parametrized but added as such to a basis set. A linear
NMR Biomed. 2001;14:224–232
 COMMON PROCESSING OF IN VIVO MR SPECTRA
combination of these spectra is used to fit the experi-
mental data in the frequency domain and to estimate
metabolite levels. As the program does not rely on
parameter information of separate peaks or of some
related peaks but on the near-complete spectral pattern of
each metabolite, including effects of MR machine
specific experimental settings on the spectra, it exploits
a fairly extensive range of available prior knowledge.
This reduction in freedom of the model significantly
contributes to its robustness. LCModel performs opti-
mally at short echo and long repetition times. In this way
possible problems due to differences in relaxation times
between proton spins of metabolites in solution and in
vivo are avoided e.g. see.45,46 The application of these
timings may not always be practical and alternatively
relaxation time corrections may be applied.
The inclusion of specific parametric information in
frequency domain fitting33 has been further explored for
the analysis of 1H MR spectra of human brain.47
Parameter data derived from in vitro metabolite measure-
ments have been applied at an increasing level of
sophistication together with knowledge of the contribu-
tion of broad macromolecular components to the
spectra.48 In parallel VARPRO-type time-domain fitting
procedures including parametric prior knowledge has
been applied to 1H MR spectra of the brain, first including
some rudimentary information e.g.49,50 and later at a
more robust and advanced level.39,51 The latter advances
are now part of the MRUI package.37 In order to
overcome the specific limitations of fitting signals either
in the time or the frequency domain, a processing strategy
combining both domains has been developed with the
possibility of including prior knowledge.4 For instance,
frequency-selective fitting in assessing small resonances
in 13C MR spectra, such as the C1 signal of glycogen
between dominating signals for lipids, may be easier in
the frequency than in the time domain.
Often the prior knowledge that is used in the approaches
described above is derived from specific measurements for
a particular MR experimental condition. This means that
the complete set of basis data or parameters may have to
be obtained again after changing to another MR sequence
or field strength. In principle this problem can be avoided
if the necessary spectral information and/or the line shape
pattern of each metabolite could be simulated for each
experimental MR condition. This is possible if informa-
tion is available on the chemical shifts of each singlet or
multiplet group, J-couplings, field strength and the
acquisition pulse sequence and its timing. For instance
in the quantification of citrate levels in the human prostate
from its methylene 1H MR signals, the scalar coupling and
the relative phase of the multiplet peaks were derived from
in vitro measurements and quantum mechanical calcula-
tions52,53 and used as prior knowledge.39 A general
approach for the formation of a priori knowledge by
spectral simulation of metabolite spin systems has been
described54 and applied to automated spectral analysis of
Copyright  2001 John Wiley & Sons, Ltd.
229
1
H MR spectroscopic imaging data of the human brain.55
Extensive spectral information on many metabolites
needed for these simulations was recently presented.56
In all these procedures one has to be careful that the
prior knowledge that is applied is a good representation of
the real situation in vivo, otherwise it may spoil the data
evaluation instead of improving it. To some extent the
spectral patterns and parameters depend on particular
physiological conditions. Some of these may be mimicked
in the in vitro measurements such as temperature and pH,
but for others this is difficult, such as physiological
conditions in muscle causing the presence of MR-visible
dipolar interactions or other cell specific conditions (e.g.
interactions of metabolites with cations). This complicates
the input of prior knowledge and may require the use of
soft constraints for fitting. The inclusion of information on
macromolecular spectral components and other non-
parametric elements as prior knowledge to improve the
overall quantitative assessment of MR spectra is also of
concern. As the identity of the signals from macromol-
ecular compounds is not precisely known, quantitative
assessment of their levels is difficult.
NORMALIZATION AND (ABSOLUTE) QUANTI-
FICATION
MR spectral analysis results in arbitrary levels for the
time domain amplitudes or frequency domain peak areas
of metabolite spin systems. This may be sufficient for
some purposes: in clinical diagnosis it may be useful to
consider only signal ratios such as those of the methyl
proton spins of N-acetylaspartate, creatine and choline in
1
H MR spectra of the brain to assess the presence of
pathological tissue, for instance tumor tissue e.g.57,58 For
general clinical purposes a local reference data set with
normal ratios for healthy tissue is often required. In focal
pathologies, this reference spectrum can be acquired
from the patient, e.g. the acquisition of a spectrum from
the contra-lateral part of the brain. By the use of signal
ratios some problems, such as partial volume differences
arising from different cerebrospinal fluid contributions to
the selected volumes, can be largely avoided. However,
with changes in ratios of metabolite peaks it remains
uncertain which component actually changes. To uncover
this information it is necessary to normalize individual
peaks with respect to a stable standard signal. A control
set of spectra measured in healthy tissue (e.g. in
volunteers) under institutionally defined experimental
conditions is usually required. In principle the determina-
tion of absolute tissue levels of metabolites, taking into
account further experimental factors affecting signal
intensities (such as echo and repetition times), would
provide institutional independent values. Normalization
and absolute quantification using an external or an
internal reference signal requires the performance of a
calibration procedure e.g. see.15,45,55,59–63 The signal of
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230 H. J. A. IN’T ZANDT ET AL.
tissue water is often used as an internal reference
assuming a particular H2O content. The calibration
procedure needed for the determination of absolute
values may introduce more (site-specific) practical and
experimental problems, which may affect the absolute
accuracy of the final results. Thus, although this
procedure has the advantage that clinical values can be
compared directly between different hospitals, indepen-
dent of the acquisition protocol and MR machine used, it
may turn out to be more difficult to identify small spectral
abnormalities. In that case, an institutional reference set,
better reflecting the actual experimental conditions, is
necessary to uncover these finer details.
Absolute quantification requires minimal T1 and T2
relaxation time weighting of the spectra, which means
short echo times and long pulse repetition times. When
longer echo times and/or short pulse repetition times are
used, the precise values of these relaxation times should
be known for proper signal correction, which may be
difficult to assess for pathological tissue. Apart from
experimental MR parameters like T1 and T2 relaxation
times, other effects may also have to be taken into
account for the determination of molecular levels. For
instance, the amplitude of the creatine methyl resonance
in 1H MR spectra may be subject to magnetization
transfer effects due to magnetization exchange between
water and creatine spin systems.64 The methyl proton
resonance intensity of creatine may decrease because of
pre-saturation of the water signal. This can be minimized
by keeping the length of the water suppression scheme as
short as possible.
It is common to discuss absolute tissue contents in
terms of ‘concentrations of metabolites’. This may be
misleading as it suggests that real biochemistry of these
metabolites in a homogeneous solution is applicable,
while tissue or cellular compartments with different
concentrations will contribute to the same metabolite
signal, as in vivo MR usually is performed on tissue at a
macroscopic level. Therefore, without prior information
on cellular reference compounds (e.g. ATP levels) or on
tissue composition, it is preferable to use more neutral
expressions such as ‘tissue levels’.
Shifts of resonances may be assessed more accurately
than their amplitudes. For instance, a precise relative tissue
pH value may be obtained from 31P MR spectra using the
shift of the inorganic phosphate peak with respect to
creatine phosphate. For an absolute determination of pH
values a calibration is needed which is less accurate
because of the required assumptions.3 The Pi peak may get
broadened in a series of measurements and in some cases it
even splits in several peaks, a situation which requires
more care in the choice of prior knowledge e.g.65
ADVANCED PROCESSING
The sections above dealt with an introduction to various
Copyright  2001 John Wiley & Sons, Ltd.
issues commonly encountered in the processing of in vivo
MRS data. Many more aspects have been investigated,
such as the robustness and the validation of various
processing approaches, issues that are beyond the scope
of this paper. Furthermore, progress towards more refined
and automated data evaluation has been made, of which
two are briefly mentioned below.
After one has obtained useful values for spectral
parameters, the further clinical or biomedical interpreta-
tion is often done on the basis of an educated selection of
single or multiple metabolite peaks as markers to
characterize the metabolic condition of the tissue. For
this purpose additional analytical software programs may
be used. However, the separate quantification of signal
parameters may be by-passed and the (pre-processed) MR
spectroscopy data can be assessed directly using pattern
recognition methods, e.g.66,67 in order to classify results in
for example tumor and non-tumor tissue e.g.68 The
difference with parameter estimation is that, in pattern
recognition methods, the program itself estimates the
important parameters that distinguish between different
types of tissue pathologies. The disadvantage of the latter
method is that large data sets of normal and diseased tissue
should be available to train the program. It is also possible
to use the results obtained from signal quantification
programs as input to classify MR spectra using pattern
recognition type of approaches e.g.69 [Fig. 1(A)]. In this
way pattern recognition can be performed with less noise,
but delicate spectral features, which may help in the
classification, may be lost. Fully automated classification
based on in vivo MR spectra is being explored although it
is not yet routinely available; in tumor diagnosis important
progress in this direction has been made.70–72 Multi-center
trials, such as those on brain tumors, e.g.58,73 can provide
large data sets to explore the features necessary for a
proper classification of these kinds of spectra.
The evaluation of MR spectroscopy data in clinical
diagnosis is much enhanced by the combination with MR
imaging results. This is most obvious in the assessment of
partial volume effects. For instance, the volume fraction
with cerebrospinal fluid contributing to the voxels of
interest in 1H MRS of the brain may strongly affect
quantitative results of individual signal intensities or
absolute metabolite levels e.g.61,74 This contribution can
be assessed in various ways; most conveniently this is
done by automatic segmentation of the corresponding
structures in MR images e.g.75 The proper combination
with MR imaging becomes inevitable in focal disease
like, for example, cancer or in diseases specific to white
matter. In the latter case it is important to know what the
ratio of white and gray matter in the selected volume is.
Further, when the effect of a therapy on the disease is
monitored using MR spectroscopy, the selected volume
in the brain should be of identical size and position in
each exam, which may be problematic in practice. The
method of choice in these situations is the combination of
MRS and multi-spectral MR imaging analysis. For
NMR Biomed. 2001;14:224–232
 COMMON PROCESSING OF IN VIVO MR SPECTRA
instance, from MR images of the brain the ratio of white
matter, gray matter and CSF can be determined using
segmentation techniques e.g.76 In this way partial volume
effects can be assessed better, such as the percentage of
white and gray matter in a selected volume.
These segmentation techniques make a separation
between areas in the brain based on distinct MR
parameters arising from a dominant cellular or tissue
fluid feature. For example gray matter contains mainly
cell bodies while in white matter mainly myelinated
axons are found. However, the brain contains many
different cells, each contributing at various levels to
specific regional functions. Thus detailed anatomical
identification and segmentation, beyond white and gray
matter in brain, will become more important for the
characterization of localized MR spectra of specific tissue
regions. Moreover, as pathological tissue may interfere
with standard MR segmentation procedures further
refinement is needed taking spatial inhomogeneity of
both normal and lesion tissue into account.
Acknowledgements
We thank A. W. Simonetti (MSc) and Dr L. Buydens for
useful discussions. We thank the EU for support (IST
project – 1999-10310 (INTERPRET) and TMR project
ERBFMRXCT970160 (Advanced Signal Processing for
Medical MRI/MRS)).
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