Critical Reviews in Clinical Laboratory Sciences

ISSN: 1040-8363 (Print) 1549-781X (Online) Journal homepage: http://www.tandfonline.com/loi/ilab20

Applications of mid-infrared spectroscopy in the

clinical laboratory setting

Sander De Bruyne, Marijn M. Speeckaert & Joris R. Delanghe

To cite this article: Sander De Bruyne, Marijn M. Speeckaert & Joris R. Delanghe (2017):
Applications of mid-infrared spectroscopy in the clinical laboratory setting, Critical Reviews in
Clinical Laboratory Sciences, DOI: 10.1080/10408363.2017.1414142

To link to this article: https://doi.org/10.1080/10408363.2017.1414142

Published online: 14 Dec 2017.

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 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES, 2017
https://doi.org/10.1080/10408363.2017.1414142

REVIEW ARTICLE

Applications of mid-infrared spectroscopy in the clinical laboratory setting

Sander De Bruynea, Marijn M. Speeckaertb and Joris R. Delanghea
a
Department of Clinical Chemistry, Ghent University Hospital, Ghent, Belgium; bDepartment of Nephrology, Ghent University Hospital,
Ghent, Belgium

ABSTRACT ARTICLE HISTORY

Fourier transform mid-infrared (MIR-FTIR) spectroscopy is a nondestructive, label-free, highly sensi- Received 26 July 2017
tive and specific technique that provides complete information on the chemical composition of Revised 20 November 2017
biological samples. The technique both can offer fundamental structural information and serve as Accepted 3 December 2017
a quantitative analysis tool. Therefore, it has many potential applications in different fields of clin- Published online 14 Decem-
ber 2017
ical laboratory science. Although considerable technological progress has been made to promote
biomedical applications of this powerful analytical technique, most clinical laboratory analyses are
Downloaded by [RMIT University Library] at 12:02 16 December 2017

KEYWORDS
based on spectroscopic measurements in the visible or ultraviolet (UV) spectrum and the poten- Clinical applications; Fourier
tial role of FTIR spectroscopy still remains unexplored. In this review, we present some general transform infrared
principles of FTIR spectroscopy as a useful method to study molecules in specimens by MIR radi- spectroscopy; medical
ation together with a short overview of methods to interpret spectral data. We aim at illustrating laboratory science; mid-
the wide range of potential applications of the proposed technique in the clinical laboratory set- infrared absorbance
ting with a focus on its advantages and limitations and discussing the future directions. The spectroscopy; vibrational
reviewed applications of MIR spectroscopy include (1) quantification of clinical parameters in spectroscopy
body fluids, (2) diagnosis and monitoring of cancer and other diseases by analysis of body fluids,
cells, and tissues, (3) classification of clinically relevant microorganisms, and (4) analysis of kidney
stones, nails, and faecal fat.

Abbreviations and Glossary: ANN: artificial neural networks; ATR: attenuated total reflectance;
BCC: Burkholderia cepacia complex; CaOx: calcium oxalate; CaPh: calcium phosphate; CDA: canon-
ical discriminant analysis; CFS: chronic fatigue syndrome; CLS: classical least-squares; COD: calcium
oxalate dihydrate; COM: calcium oxalate monohydrate; CSF: cerebrospinal fluid; CVA: canonical
variate analysis; FTIR: Fourier transform infrared; HCA: hierarchical cluster analysis; HCC: hepatocel-
lular carcinoma; HDL: high-density lipoprotein; IBD: inflammatory bowel disease; ICU: intensive
care unit; ILS: inverse least-squares; IR: infrared; KNN: K-nearest neighbours; LDA: linear discrimin-
ant analysis; LDL: low-density lipoprotein; MCH: mean corpuscular haemoglobin; MCV: mean cor-
puscular volume; Micro-FTIR: Fourier transform infrared microspectroscopy; MIR: mid-infrared; NIR:
near-infrared; RMSECV: root mean square error of cross validation; PBMC: peripheral blood mono-
nuclear cells; PCA: principal component analysis; PCR: principal component regression; PLS-DA:
partial least squares discriminant analysis; PLS-R: partial least squares regression; POCT: point-of-
care testing; RAPD: randomly amplified polymorphic DNA; RBC: red blood cells; SIMCA: soft inde-
pendent modelling of class analogy; SNR: signal-to-noise ratio; UV: ultraviolet; XRD: X-ray
diffraction

Introduction spectrum shows which bonds have absorbed radiation

Fourier transform infrared (FTIR) spectroscopy is a
widely used technology for the analysis of biological
samples [1], as it provides complete information on the
chemical composition of samples and can both offer
fundamental structural information and serve as a
quantitative analysis tool [2,3]. In this technique, pho-
tons containing energy that completely match the
vibration energy of a covalent bond are absorbed if
there is a change in the dipole moment of the molecule
during the course of vibration [4,5]. An infrared (IR)
(wavelength), together with the absorption efficiency
(intensity) [4]. Conventionally, the IR region is subdi-
vided into three regions: the far-infrared (< 400 cm
1),
the mid-infrared (MIR, 4000–400 cm
1) and the near-
infrared region (NIR, 13,000–4000 cm
1). In this review,
FTIR spectroscopy refers to the study of the absorption
of electromagnetic waves in the MIR region. The
MIR region generally includes four regions: the X-H
stretching region (4000–2500 cm
1), the triple-bond
region (2500–2000 cm
1), the double-bond region

CONTACT Joris Delanghe Joris.Delanghe@UGent.be Laboratory Clinical Chemistry 2P8, Ghent University Hospital, De Pintelaan 185, 9000 Ghent,
Belgium
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
 2 S. DE BRUYNE ET AL.
(2000–1500 cm
1) and the fingerprint region

1
(1500–600 cm ). The fundamental vibrations of the
X-H stretching region are generally due to O-H, C-H and
N-H stretching, whereas vibrations of CC and CN
bonds are mainly observed in the triple-bond stretching
region. Absorption bands corresponding to C ¼ C, C ¼ O
and C ¼ N appear in the double-bond region [3]. The
fingerprint region of the MIR spectrum contains the fun-
damental vibrations of key chemical bonds, which are
characteristic of a specific molecule [3,6,7]. For analyz-
ing biological materials, the most important regions in
the IR spectrum are the fingerprint region and the
amide I and amide II region (1700–1500 cm
1) [5]. A
biological FTIR spectrum with typical molecular assign-
ments is shown in Figure 1.
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One of the main advantages of FTIR spectroscopy
lies in the high molecular sensitivity combined with a
spatial resolution down to a few micrometers.
Furthermore, almost any sample type can be studied
without the use of reagents. Solutions, liquids, fibers,
films, pastes, powders, gases and surfaces can be exam-
ined by using the appropriate analysis technique.
Additionally, modern FTIR spectrometers have a high
potential for applications in the clinical laboratory, as
they provide high resolution, high signal-to-noise ratios
(SNRs) and extensive data processing possibilities by
means of computer software packages. At last, FTIR
spectroscopy presents attractive features such as being
fast, easy-to-use, reagent-free, cost-effective and even
Figure 1. Typical MIR spectrum of biological material showing peak assignments from 4000–800 cm21. The spectrum represents
an in vivo measurement of the human skin (palmar surface of an index finger after cleaning with ethanol) obtained with attenu-
ated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). v: stretching vibrations, d: bending vibrations, s: symmetric
vibrations, as: asymmetric vibrations.
portable to work on-field [8]. Nevertheless, most clinical
laboratory analyses are based on spectroscopic meas-
urements in the visible or ultraviolet (UV) spectrum and
the potential role of IR spectroscopy in the clinical
laboratory still seems an underestimated topic.
Traditionally, FTIR spectroscopy is considered as the
gold standard for kidney stone analysis [4,9]. However,
today its application area extends far beyond that.
Different spectroscopic methods have been developed
to detect biochemical components in serum [10–14],
plasma [10,15,16], whole blood [10] and other biological
fluids [10,17–23]. The obtained spectra are rather com-
plex, but by the application of chemometric methods,
attempts to unravel them have often been successful
[16]. Furthermore, in recent years, this technique has
been used to study a range of various conditions
including neurological [21,24–26], mental [27], respira-
tory [28,29], gastrointestinal [30–36], kidney [37–39],
urinary tract [40], gynecological [2,41–44], hemato-
logical [45–49] and dermatological disorders [50].
Changes in the chemical composition and structure
must precede any symptomatic or morphological
expression of a disease. Because FTIR spectroscopy is
sensitive to those changes and allows rapid collection
of spectra obtained from millimeter-sized samples, it is
a very promising technique for detecting biochemical
signatures associated with the generation and progres-
sion of disease [7]. Therefore, IR spectroscopy is a
potential alternative for the early detection of diseases

[7,51]. Although the majority of applications are outside
the field of laboratory medicine, there has been a grow-
ing interest in FTIR spectroscopy in diagnostic labora-
tory medicine and the above-mentioned applications
are only a few examples within the present repertoire
of the medical application potential. The aim of this
review is to present an overall perspective about appli-
cations of MIR spectroscopy in clinical laboratory medi-
cine with a focus on its advantages, limitations and
future directions.
Measurement techniques
Traditionally, dispersive instruments have been used to
perform IR spectra, but their biggest disadvantage is
the fact that they cannot scan at speed. This limitation
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can be overcome with FTIR spectroscopy [52]. The main
benefit of rapid-scanning instruments is the ability to
increase the SNR by signal averaging. Thereby, FTIR is
commonly used and has significantly improved IR spec-
tra [53]. Three main sampling methods of FTIR spectros-
copy exists: transmission, transflection, and attenuated
total reflectance (ATR) (Figure 2) [5,7]. Each mode shows
convenience for some samples and challenges for
others [5].
Transflection-mode FTIR spectroscopy is based on
the absorption of IR radiation after transmission
Figure 2. Overview of the three main sampling methods used for Fourier transform infrared spectral acquisition. (I) transmission,
(II) transflection, and (III) attenuated total reflection.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 3
through the sample, reflection off the substrate and
transmission back through the sample [7,54].
Transflection has become a popular method due to the
relative low cost of substrates compared to transmission
windows and a higher absorbance due to a double pass
through the same sample. However, data from trans-
flection methods should be treated with caution
because spectra are distorted nonlinearly as a function
of wavenumber with respect to sample thickness. As a
consequence, qualitative information can be obtained
with relatively little danger, while quantitative informa-
tion such as peak heights and ratios are unreliable [55].
Transmission-based methods function by transmit-
ting IR radiation through the sample and substrate (e.g.
calcium fluoride) before the resulting radiation is
detected. Next to its relatively simple use and universal-
ity, the frequencies of bands and their shapes are dir-
ectly related to physical quantities [56]. Although these
methods are commonly used, there are a number of
technical difficulties commonly encountered with this
technique that can make collection of high-quality
spectra difficult. First of all, preparation of samples for
transmission measurements is a rather complex and
time-consuming task. Solid samples have to be diluted
with expensive IR transparent substrates, while liquid
samples have to be poured into a liquid cell with suit-
able path length. Analysis of liquid samples is restricted
 4 S. DE BRUYNE ET AL.
due to saturation effects of OH vibrational bands [56].
Furthermore, transmission spectra can be the subject to
a variety of physical effects such as interactions
between polar substrates and the sample, interactions
of surface water molecules (or other solvents) with the
window material, and the presence of air bubbles in the
liquid cells [7,56].
ATR-FTIR is able to overcome these potential prob-
lems. ATR-FTIR operates on the principles of total
internal reflection. A radiation beam entering a crystal
will undergo total internal reflection when the angle of
incidence is greater than the critical angle, which is
function of the refractive indices of the two surfaces.
The beam loses energy when a material that selectively
absorbs radiation is in contact with the internal reflect-
ing element (IRE) [7,53,56]. One limitation of this
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approach is the fact that samples have to be in close
contact with the IRE, which is sometimes difficult in the
case of solid samples. Because of the small light pene-
tration depth, the ATR technique is ideal for highly
absorbing samples, surfaces and thin-film measure-
ments [56]. The major benefits of ATR-FTIR, in contrast
to transmission and transflection experiments, are its
sample thickness independent measurements, the abil-
ity to probe highly IR absorbing materials without the
need for complex sample preparations and the
improved spatial resolution [57]. Furthermore, expen-
sive IR transparent substrates are not needed [7].
At last, FTIR microspectroscopy (micro-FTIR) can be
used as a technical variant of transmission measure-
ments. This technique is based on the coupling of a
spectrometer with a light microscope and has the
advantage of generating high-quality spectra with a
minimum of sample material [54,58]. Over the past
20 years, IR imaging using FTIR microspectrosopic tech-
niques has emerged from a proof of principle technol-
ogy to a widely accepted powerful analytical method
for the analysis of complex or heterogeneous biomed-
ical samples such as biopsy tissues, fixed cells, and liv-
ing cells (e.g. cancer cells) [1]. The technique can be
used to produce a two- or three-dimensional image of
the properties of a sample and offers great promise for
the improved diagnosis of the disease state [53,59].
However, applications of IR imaging are beyond the
scope of the current review, as excellent reviews con-
cerning this topic can be found elsewhere [1,57,59].
Spectral data analysis (general principle)
Pretreatment of spectra
FTIR spectra of biological material are often complex
and require data treatment to obtain reliable results.
However, several pretreatment steps have to be per-
formed before spectra can be used for analysis. First of
all, spectra should undergo a quality control by an
assessment of absolute absorption, SNR, and level of
water vapor [54,60]. Further steps of data pretreatment
are baseline corrections to correct spectra with sloped
or varying baselines, derivation to increase the amount
of useful information by dissolving complex and over-
lapping bands, smoothing to reduce background noise,
and normalization to compensate for differences in
absorption due to varying sample thickness or size [54].
Chemometric data analysis
After data pretreatment, the choice of an appropriate
chemometric technique for qualitative or quantitative
data analysis is a crucial factor. Chemometric data ana-
lysis involves the application of several statistical meth-
ods for drawing vital information from chemical data
[61]. A detailed description of different analytical techni-
ques is beyond the scope of the current review.
However, a short overview of several multivariate data
analytical methods, which are used for spectral analysis,
will be provided without detailing the associated math-
ematics. An illustration of a possible workflow in clinical
diagnosis based on IR spectral acquisition and chemo-
metric data analysis can be found in Figure 3. Samples
can be classified according to their IR spectrum by
using qualitative analysis techniques based on pattern
recognition methods. The classification techniques can
be divided into supervised and unsupervised methods
[54,62]. In unsupervised methods, such as principal
component analysis (PCA) and hierarchical cluster ana-
lysis (HCA), observations are classified without any prior
sample knowledge. They can be used to sort spectra
and to get a first impression of the complexity, hetero-
geneity, and similarity of the dataset. Furthermore, they
are useful tools to obtain an overview of the overall
separation of groups, to control reproducibility of the
measurements, and to check for outliers [54].
Supervised methods are recommended for the identifi-
cation of samples, as they use the category membership
of samples to define classes and optimize a model on
which identification is accomplished [54,62]. In other
words, a classification model is generated on a training
set of samples with known categories and an assess-
ment of the model performance is executed by compar-
ing the true categories with the predicted classifications
of validation (test) samples [62]. Examples of supervised
methods are linear discriminant analysis (LDA), soft
independent modelling of class analogy (SIMCA), partial
least squares discriminant analysis (PLS-DA), artificial
neural networks (ANN), K-nearest neighbors (KNN) and

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Figure 3. Possible workflow in clinical diagnosis based on IR spectral acquisition and chemometric data analysis. In the case of
complex spectra, chemometric data analysis may be helpful in medical decision making.
canonical variate analysis (CVA) [62,63]. IR spectroscopy
is most often used for qualitative analysis. However, it is
also well-suited for quantitative analyses, which can be
useful to know more precisely and to what extent sam-
ples differ from each other [62]. Quantitative methods,
such as classical least-squares (CLS), inverse least-
squares (ILS), principal component regression (PCR) and
partial least squares regression (PLSR), are now widely
used in the development of IR analytical methods. CLS
and PLS are least-squares methods involving matrix
operations. When very complex mixtures are investi-
gated, factor analysis methods, such as PLSR and PCR,
are preferred. These methods use functions to model
the variance in a dataset [53].
Direct spectroscopy of body fluids
Direct clinical biochemistry of body fluids forms a utile
tool for the diagnosis of diseases and monitoring of
medical treatments. Most clinical laboratory analyses
are based on a spectroscopic measurement in the vis-
ible or UV spectrum with the use of direct measure-
ments of pigment absorption or indirect measurements
based on a dye, enzyme-catalyzed reaction, or antige-
n–antibody interaction with the use of high amounts of
specific reagents [10]. The main disadvantages of these
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 5
methods are the reagent costs, which are out of the
economical possibilities of many countries, the time-
consuming handling procedures (i.e. purchasing, stor-
age, reconstitution and inventory control), analytical
errors caused by the reagents and errors occurring dur-
ing assay execution [11,16]. FTIR spectroscopy offers a
promising alternative technique for the determination
of clinical parameters in body fluids since these mole-
cules, regarding both major and minor compounds,
have characteristic fingerprint bands in the MIR spectral
region. The exclusivity of a molecular structure can
determine the quality of analysis. As an example, urea
can be predicted precisely in whole blood samples
because its chemical structure is rather unique in com-
parison with other blood constituents and the carbonyl
group is a strong IR absorber [10]. Several research
groups have tried to accomplish reagent-free determin-
ation of clinical parameters in serum, plasma, whole
blood and other body fluids by using FTIR spectroscopy
in combination with multivariate data analysis in order
to eliminate matrix effects. Besides the chemometric
processing of data, calibration of FTIR methods is
another crucial point in method development.
Calibration must be executed with high quality refer-
ence methods and must include carefully selected sam-
ples that cover the greatest possible concentration
 6 S. DE BRUYNE ET AL.
Table 1. Overview of clinically relevant parameters analyzed in body fluids with FTIR spectroscopy.
Matrix Analytes
Serum Albumin, creatinine, glucose, HDL,
LDL, total cholesterol, total pro-
tein, triglycerides, urea, uric acid
Plasma Albumin, apo-A1, apo-B, apo-C3,
cholesterol, fibrinogen, glucose,
haptoglobin, immunoglobulins
(IgG1, IgG2, IgG3, IgG4, IgA, IgM,
IgD), lactate, transferrin, triglycer-
ides, total protein, urea, a1-acid
glycoprotein, a1-antitrypsin,
a2-macroglobulin
Whole blood Albumin, glucose, total cholesterol,
total protein, triglycerides, urea
Urine Creatinine, inorganic phosphate, total
protein, urea, uric acid
Amniotic fluid Albumin, lecithin, sphingomyelin,
surfactant
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Saliva a-amylase, cortisol, phosphate, IgA,
total protein, urea
ATR: attenuated total reflection; FTIR: Fourier transform infrared; HDL: high-density lipoprotein; IR: infrared; LDL: low-density lipoprotein; POCT: point-of-
care testing.
range because the representativeness of the calibrator
set determines the prediction capability of a model for
external samples [11,16]. The calibration procedure is
quite labour-intensive, but once successful calibration
algorithms are acquired, they will be valid indefinitely
because IR spectra do not change over time. IR spectra
obtained from identical samples at different geographic
locations should be the same. As a consequence, IR
spectra acquired at a remote location (e.g. satellite labo-
ratories, pharmacies) can be electronically transmitted
to a central laboratory, where several analyte concentra-
tions can be simultaneously determined on the basis of
a single IR spectrum by using the appropriate calibra-
tion algorithms [16]. Table 1 provides an overview of
clinically relevant parameters analysed in body fluids
with FTIR spectroscopy. The fact that total protein, albu-
min and other proteins, such as the different classes of
immunoglobulins, could be quantified separately shows
that even very small spectral signatures are sufficient to
discriminate between proteins. However, next to gen-
eral challenges such as background absorption and
environmental factors (e.g. degree of humidity), IR spec-
troscopy of biological fluids is faced with some specific
challenges. First of all, a routine clinical laboratory analy-
ses samples with a highly variable composition and ori-
gin. The matrix complexity and variability within
samples can cause difficulties in multivariate calibration
FTIR Techniques Used Remarks References
ATR In the case of creatinine, uric acid and [11–14]
Transmission triglycerides, the method suffers from a
lack of sensitivity.
ATR FT-IR spectroscopy is a useful tool for [10,15,16]
Transmission determining the concentration of several
analytes in microsamples of plasma at
the same time, even for biomolecules
belonging to the same family.
ATR The possibility to analyse small sample vol- [10]
umes (< 5 lL) of whole blood without
sample preparation is especially promis-
ing for POCT applications.
ATR Urinary protein concentrations are often [10,17]
too low for accurate quantification using
FTIR spectroscopy.
Transmission IR analysis of the lecithin/spingomyelin [18,19]
ratio and the surfactant/albumin ratio is
an alternative option for the assessment
of foetal lung maturity since IR uses
minute amounts of amniotic fluid, leav-
ing sufficient material for other associ-
ated clinical tests.
ATR FTIR analysis of saliva opens a new level of [20]
non-invasive out of the laboratory
diagnostics.
of measurements. Moreover, the low concentration lev-
els of some analytes could hamper the determination of
clinical parameters in body fluids. Therefore, the use of
chemometric algorithms is normally required for elimi-
nating matrix effects and a representative calibration
set must be selected [11]. Furthermore, the large
amount of water within biological matrices makes it the
most serious interferential element for MIR spectral ana-
lysis due to strong O-H absorption caused by funda-
mental O-H stretching and H-O-H bending vibrations
[7,53]. Figure 4 illustrates the strong absorbance of
water in the MIR region. Elimination of the intense
water absorption can be done in several ways. These
methods include purging with dry air or nitrogen during
spectral acquisition to obtain dried films, subtraction of
reference water, and using the combined approach [64].
Direct transmission measurements of aqueous solutions
require that the cuvette windows are made of water-
insoluble substances such as calcium fluoride [10–14].
This difficulty can be overcome by using an ATR device,
which is currently highly favored in the community [65].
ATR-FTIR is an easy manageable technique to record IR
spectra and has optimal characteristics to be used as a
screening tool, because this method provides the com-
plete spectra of samples in an easy, fast, and direct way
without requiring any chemical pretreatment [11,16].
Therefore, ATR-FTIR in combination with multivariate

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Figure 4. MIR spectrum of liquid water obtained with ATR-FTIR. The strong absorbance at 3314 cm21 is due to fundamental O-H
stretching vibrations (change in bond length), while the absorbance at 1638 cm21 is due to H-O-H bending vibrations (change in
bond angle).
analysis is considered as a promising alternative tech-
nique for expensive clinical laboratory analyses [11].
However, there is a concern over water-associated dis-
tortions that remain evident in the ATR spectra of cer-
tain samples. Furthermore, unless a multi-ATR crystal
device is developed, the technique is not likely to be
implanted in a high-throughput setting due to the time
required for each sample to dry one by one. In compari-
son with ATR, sample preparation for transmission
measurements is clearly faced with more challenges,
but each method has its advantages [65]. A simple,
robust and fast transmission method for the simultan-
eous determination of glucose, triglycerides, urea, chol-
esterol, albumin and total protein in unmodified human
plasma was reported with stable and accurate results
[16]. The interassay imprecision met international qual-
ity criteria and determination coefficients between the
six FTIR methods and corresponding reference methods
were between 0.87 and 1.00. Furthermore, linearity of
the method extended over clinically significant concen-
tration ranges. ATR-FTIR has been evaluated in a study
based on cross-validation parameters by using a large
number of calibration samples [10]. Validation studies
were executed on a separate set of samples and PLSR
was used to predict the concentration of albumin, chol-
esterol, glucose, total protein, urea, and triglycerides in
whole blood or plasma samples, and the concentration
of urea, uric acid, phosphate, and creatinine in urine
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 7
with volumes as small as 5 lL. The authors concluded
that the absolute precision and reproducibility of the
reached prediction was sufficient for routine clinical
analysis and was only limited by the precision of the ref-
erence analysis used for calibration. However, the high
number of latent variables (6 to 18) employed in this
study can cause an overfitting of models and subse-
quently can have a negative influence on the prediction
capability. A study [11] evaluated ATR-FTIR as a point-
of-care testing (POCT) tool on a large number of serum
samples from different origins (primary care, hospital
and pre-dialysis). Total protein and albumin were deter-
mined successfully, while glucose, urea, HDL, LDL and
total cholesterol were predicted with relative errors
between 15% and 23%, which is acceptable for applica-
tion as a screening tool. However, creatinine, uric acid,
and triglyceride analysis suffered from a lack of sensitiv-
ity. Taken together, FTIR spectroscopy of body fluids
seems to have the potential to become the clinical
method of choice for several clinically relevant parame-
ters, especially in depressed economic areas and in
POCT applications, since this method allows an immedi-
ate, reagent-free, and simultaneous determination with
a minimum of sample volume. Therefore, IR spectros-
copy can save both time and money.
However, FTIR spectroscopy cannot be seen as a uni-
versal tool for the quantitative analysis of all constitu-
ents in body fluids for several reasons. The lack of
 8 S. DE BRUYNE ET AL.
sensitivity and band overlapping restrictions of
untreated samples cannot be ignored. Very low param-
eter concentrations cannot be reliably detected due to
the relatively low extinction coefficients associated with
IR spectroscopy (50–500 times smaller than those of
electronic transitions). Enzymes, hormones, and trace
compounds are beyond the detection limit without the
use of enrichment procedures. Furthermore, ions that
are not bound in a molecular structure cannot be
detected due to the vibration based measurement prin-
ciple of IR spectroscopy [10]. One of the main future
challenges will be the expansion of the number of con-
stituents in body fluids that can be measured by FTIR
spectroscopy.
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FTIR spectroscopy of body fluids, cells and
tissues for the diagnosis and monitoring of
cancer and other diseases
FTIR spectroscopy has the ability to differentiate body
fluids, cells, and tissues based on characteristic spectral
properties reflecting their chemical composition and
structure. It also has the potential to function as a diag-
nostic tool in the detection and discrimination of differ-
ent diseases or disease progression states due to
biomolecular alterations [2,7,35,40–42,46,50,58,66,67].
Diagnostic applications of FTIR spectroscopy for body
fluids offer a number of advantages. The technique is
often non- or minimally invasive, is affordable, and can
be repeated conveniently during disease evolution.
Moreover, composition of serum or plasma is assumed
to reflect the health status of the body because blood
perfuses all organs and tissues. In the case of malig-
nancy, proteins and peptides are released from the
tumor microenvironment into the newly formed micro-
circulation. Consequently, tumor markers can be found
in the circulatory system as whole functional proteins or
cleaved products, which can give rise to specific spec-
tral patterns [36]. Furthermore, for the majority of can-
cers, the only way to make a definitive diagnosis is to
perform a biopsy to collect cells for closer examination
in order to determine the specific cell lineage and to
make appropriate therapeutic decisions. A histopatho-
logical diagnosis using an adequate sample of tumor
tissue will typically involve light microscopic examina-
tions, immunohistochemical investigations, and evalu-
ation of other markers or receptors, sometimes
supplemented by more specific investigations such as
electron microscopy or genetic studies [68]. This pro-
cedure is associated with several limitations such as
delaying provision of diagnostic results and being
rather subjective. Over the last years, FTIR spectroscopy
has become an alternative tool in tissue cancer
diagnosis since this method exhibits the potential to
overcome those limitations. This has opened a way
toward clinical applications such as a method that scans
samples to assess the presence of malignant cells in
biopsies, which allows pathologists to better character-
ize cells that are suspicious, but not diagnostic for can-
cer. Furthermore, ATR-FTIR is gaining increasing interest
as a diagnostic tool for detecting cancer using body
fluid samples. This technique forms an excellent candi-
date for rapid POCT applications, due to its simple oper-
ation handlings and minimal sample requirements [67].
Pioneering research has been performed with FTIR for
the diagnosis and prognosis of several types of cancers
and other diseases. This section will highlight some
recent applications (from 2012 till 2017) of FTIR spec-
troscopy for rapid, reliable and minimally (or non-) inva-
sive screening of various diseases (Figure 5).
Neurological disorders and mental illness
Alzheimer’s disease (AD) is the most common cause of
dementia, especially in the elderly. However, at this
moment, no specific diagnostic tests for AD exist and
the clinical diagnosis is particularly difficult in the begin-
ning stage of the disease. Several research groups
explored the potential role of FTIR spectroscopy as a
simple and inexpensive method for the diagnosis of AD
[21,24–26]. Recently, it has become possible to differen-
tiate between mild, moderate, and severe AD, and con-
trols with reasonable success rates (85% accuracy when
using WBC spectra, 77% when using plasma spectra), in
the time span of a few minutes, by using FTIR micros-
copy combined with multivariate analysis [24]. The
potential role of FTIR spectroscopy for the monitoring
of spectral differences occurring in plasma and mono-
nuclear leukocytes of patients with AD has already been
reported earlier [25,26]. Moreover, FTIR analysis of cere-
brospinal fluid (CSF) is proposed as a new diagnostic
tool in AD with an overall good sensitivity (88.5%) and
specificity (80%) [21]. Since all CSF specimens are pre-
cious, FTIR spectroscopy forms an interesting alternative
technique for the analysis of CSF because it only
requires minimal amounts of fluid, leaving sufficient
material for other associated clinical tests. Next to AD,
FTIR spectroscopy is also employed by several research
groups in the study of brain tumors [69–71]. The use of
IR light provides a spectral signature from human serum
to detect cancer versus noncancer, metastatic cancer
versus organ-confined cancer, brain cancer severity, and
the organ of origin of metastatic disease from the same
sample, which enables stratified diagnostics depending
upon the clinical question asked [69]. Furthermore, the
application of FTIR spectroscopy as a complimentary

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Figure 5. Overview of recent (2012–2017) diagnostic and monitoring applications based on MIR-FTIR.
diagnostic tool for the detection and discrimination of
mental illnesses has been suggested. Ogruc Ildiz et al.
[27] used FTIR spectroscopy on plasma samples for the
first time in the classification of bipolar and schizo-
phrenic patients with excellent sensitivity and specificity
values.
Respiratory disorders
The potential of ATR-FTIR spectroscopy for the detec-
tion of malignant lung tissue has been demonstrated
[28]. Compared with the histologic diagnosis, 96.7%
of original grouped cases were correctly classified by
ATR-FTIR spectroscopy and canonical discriminant
analysis (CDA). An important advantage of ATR-FTIR
spectroscopy in lung cancer differentiation includes
its non-invasive nature, which allows precious sam-
ples to be analyzed many times without damaging
the tissue. As it is reagent-free, it can save the sam-
ple for further permanent histologic examination,
which is particularly useful for small peripheral lung
lesions.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 9
The combination of FTIR spectroscopy and multivari-
ate data analysis might also be useful to find novel bio-
markers of pulmonary hypertension. In an animal model
[29], the plasma spectral signature of pulmonary hyper-
tension was significantly different from that of systemic
hypertension in terms of lipid composition/metabolism
as well as in the content of RNA and glucose.
Gastrointestinal disorders
The early detection of colorectal cancer (CRC) can
reduce both mortality and morbidity. However, most
available screening tests, such as colonoscopies, are
invasive and the rate of non-compliance among eligible
patients is high. The fecal occult blood test and fecal
immune test are the only approved noninvasive screen-
ing methods with sensitivity values ranging from 60%
to 80% and specificity values ranging from 85% to 95%.
Several research groups [30–32] have reported the
application of FTIR spectroscopy in the detection of CRC
by direct measurement on colonic tissue samples.
A recent study [32] employed a FTIR spectrometer and
 10 S. DE BRUYNE ET AL.
ATR fiber probe for diagnosing colorectal cancer on
freshly removed tissue samples with an accuracy of
93.8% compared with histological diagnosis. The use of
an optic fiber attached to an ATR probe could achieve
intraoperative in vivo detection and differentiation of
malignancies. This approach could help guide surgeons
for a fast diagnosis, determining the extent of dissec-
tion, and avoiding unnecessary surgical trauma [28].
Next to the analysis of tissue samples, the potential of
FTIR spectroscopy as a simple and low-cost blood test
for early diagnosis of CRC by analyzing the entire bio-
molecular profile of peripheral blood mononuclear cells
(PBMCs) and plasma has been investigated [2]. PBMCs
present a new route for cancer detection based on the
immune system response to the tumour, rather than on
analysis of the tumor cells themselves [2]. Satisfactory
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sensitivity and specificity values were obtained when
spectral biomarkers from both PBMCs and plasma were
combined. These findings suggest a promising prospect
of blood-based IR spectral analysis as a screening tool
for CRC.
Besides CRC, FTIR spectroscopy shows promising
results in the detection of inflammatory bowel diseases
(IBDs). The two major forms of IBDs, ulcerative colitis
and Crohn’s disease are debilitating gastrointestinal
tract disorders that can lead to serious complications
such as colorectal cancer. Assessment of intestinal
inflammation with colonoscopy is rather difficult and
not ideal for monitoring since it is expensive and inva-
sive. Recent research [33,34] demonstrated the applica-
tion of ATR-FTIR spectroscopy as a safe and cost-
effective technique for initial screening of colitis in mice
serum by protein secondary structure analysis along
with glucose and mannose signatures. Another recent
example of FTIR analysis is the discrimination of sera
from cirrhotic patients with and without hepatocellular
carcinoma (HCC) with an overall accuracy of 82–86%
[36]. At last, FTIR spectrometers can also be used in
combination with a microscope for the analysis of both
formalin-fixed paraffin-embedded tissue or snap-frozen
tissue. Profound alterations of the biochemical compos-
ition of the pathological liver have been detected with
FTIR microspectroscopy, which allows discrimination
between cirrhotic nodules, dysplastic lesions and hepa-
tocellular carcinoma [35].
Kidney and urinary tract disorders
More reliable biomarkers using POCT are needed to
improve early diagnosis and intervention for patients
with renal disease [37]. During recent years, several
research groups have suggested applications of FTIR
spectroscopy in nephrology such as for the diagnosis
and monitoring of renal failure. In the routine clinical
laboratory, analyses such as determination of urea and
creatinine in serum samples enable physicians to iden-
tify kidney failure. However, ATR-FTIR has also been
used to discriminate between serum samples obtained
from healthy patients and patients with renal failure
[38] with an overall accuracy of 95%.
Hemodialysis is the most common method used to
treat acute kidney failure and end-stage renal disease.
Despite effective treatment and minimized side effects,
hemodialysis in intensive care units requires regular
control of the dialysis process. Current methods such as
in-line monitoring of the dialysis liquid by conductome-
try or the use of a urea sensor immersed in the dialysate
do not allow precise patient monitoring. A new method
for real-time and in-line monitoring of hemodialysis has
been reported [39]. A flow cell using ATR-FTIR spectros-
copy was coupled downstream of the dialysis filter and
a quantitative determination of urea, glucose, lactate
and creatinine was performed at a precision only lim-
ited by the off-line reference analysis required to estab-
lish a calibration model. This study demonstrates that
precise and efficient monitoring of hemodialysis is pos-
sible with little manpower and no consumables at all by
using ATR-FTIR spectroscopy.
Another application of ATR-FTIR spectroscopy is
the label-free detection of sensitive biomarkers of glom-
erulonephritis in urine. Several characteristic mid-IR
spectral markers in urine samples have been
detected in rodent models of inflammatory glomerulo-
nephritis as well as in patients with crescentic glomer-
ulonephritis [37].
At last, the applicability of ATR-FTIR as a high-
throughput diagnostic screening tool has been demon-
strated by developing an automated approach for the
analysis of blood samples from patients with urinary
bladder cancer [40]. Thin dried films were robotically
prepared for transmission mode measurements. The
proposed method yielded a sensitivity of 93 ± 10% and
a specificity of 46 ± 18% for bladder cancer. The low
specificity was attributed to the unbalanced and small
number of control samples.
Gynecological disorders
The surgical management of ovarian tumors mostly
depends on the histopathological diagnosis, because
screening for ovarian malignancies with tumor markers
and radiological investigations has a low specificity.
Furthermore, preoperative biopsies are associated
with important risks such as intraperitoneal dissemin-
ation. Intraoperative frozen sections are accurate in dis-
criminating tumors according to their histological type

but have the disadvantage of prolonged operation
times [41]. Recently, several research groups
[41,42,72,73] set out to determine whether ATR-FTIR
combined with chemometric analysis can be used to
differentiate between normal and malignant ovarian tis-
sues. Patients with ovarian and endometrial cancer
were discriminated from healthy controls with a remark-
able accuracy of up to 96.7% in the case of ovarian can-
cer and a relatively high accuracy of up to 81.7% for
endometrial cancer using ATR-FTIR on serum and
plasma samples [42]. ATR-FTIR has also been used on
serum and plasma samples to discriminate between dif-
ferent stages of ovarian cancer (stage I vs. stage II–IV),
between different histological types (serous vs. non-ser-
ous carcinoma) and between different age groups
(60 years vs. >60 years) with sensitivity and specificity
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levels ranging from 91.6% to 100% [73]. Normal, border-
line and malignant ovarian tissues have been discrimi-
nated and ovarian carcinoma subtypes have been
successfully been classified based on ATR-FTIR analysis
of dewaxed ovarian tissue blocks [41]. This novel diag-
nostic approach has the potential to assist surgical deci-
sion-making and avoid under- or overtreatment, with
minimal impact for the patient.
The potential value of FTIR analysis in the early
detection of breast cancer has also been investigated.
Mammographic screening has several disadvantages
such as a lack of sensitivity (about 66%) and the need
for additional evaluation with ultrasound or magnetic
resonance imaging in women with dense breasts [2].
The utility of FTIR spectroscopy on PBMCs for early
breast cancer screening in conjunction with the gold
standard diagnostic methods such as mammography
and ultrasound has been explored [2]. PBMCs of healthy
subjects, including patients with benign tumors, were
biochemically different from the PBMCs of patients with
breast cancer. A sensitivity of 90% and specificity of
80% for breast cancer detection were reported, which
could suggest a potential role of FTIR spectroscopy in
cost-effective and minimally invasive mass screening.
Another interesting application of FTIR spectroscopy in
gynecology is found in the analysis of cervical cytology.
Cervical screening programs have significantly reduced
the burden of disease. Nevertheless, conventional cer-
vical screening still lacks sensitivity and specificity.
Therefore, there is a need for the development of a
cost-effective screening technique [43]. FTIR spectros-
copy has been touted as a technique capable of dis-
criminating between different grades of dysplasia.
Several spectral differences have been reported
between normal, low-grade and high-grade specimens
[43]. However, a degree of crossover between classes
was noted, which could be associated with imperfect
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 11
conventional screening, resulting in an inaccurate diag-
nosis or incomplete transition between the different
classes. In another study [44], ATR-FTIR spectra derived
from cervical cytology were not able to differentiate in
a diagnostic fashion, when classes were based on con-
ventional screening. However, when histology-based
categories were used to perform analyses, the IR spectra
displayed a markedly better segregation. In other
words, histology demonstrated that ATR-FTIR spectros-
copy of cervical cytology detects underlying atypia or
diseases missed with conventional cytology screening.
FTIR spectroscopy combined with chemometrics could
be invaluable in screening programs in the developing
world, which is plagued by cervical cancer [43].
However, there is a need for future studies where cate-
gories are based on histology to allow validation of FTIR
spectroscopy as a screening tool [44].
Hematological disorders
Malaria is still a major public health problem in terms of
morbidity and mortality in countries where the risk of
contracting the infection with one or more of the
Plasmodium species exists [45]. Because many of the
malaria endemic countries have limited diagnostic
resources, there is an urgent need for new cost-effective
diagnostic tools that can detect malaria parasites. The
detection and quantification of malaria parasites was
first reported by Martin et al. [46], based on ATR-FTIR
spectroscopic analysis of wet-packed RBC samples.
Analysis on heparin samples was able to generate a lin-
ear calibration by using spiked RBCs (0–1% parasitemia).
However, ATR-FTIR spectroscopy has also been applied
as a point-of-care test for identifying malaria parasites,
blood glucose and urea levels from a single drop of
spiked blood on a glass microscope slide [47]. This mul-
tianalyte/disease diagnosis is utile because hypogly-
cemia and renal dysfunction are well-described
complications of severe malaria, especially with
Plasmodium falciparum infection. The specificity of
the PLS-DA was found to be 98% for parasitemia levels -
>0.5%, but a rather low sensitivity of 70% was
achieved, which was attributed to the small number of
negative samples in the model. In PLS-R the root mean
square error of cross validation (RMSECV) for parasite,
glucose and urea concentrations were, respectively,
0.58%, 16% and 17%.
In addition to malaria diagnosis, the application of
ATR-FTIR spectroscopy for the screening of thalassae-
mias has been evaluated. Thalassaemias are widely dis-
tributed in some areas of the (sub)tropics, and
screening and genetic counselling are essential for the
prevention and control of severe disease. The gold
 12 S. DE BRUYNE ET AL.
standard for diagnosis depends on DNA testing, but dir-
ect DNA testing for all individuals is rather unrealistic
(expensive and time-consuming). However, hemoglobin
(Hb), mean corpuscular volume (MCV) and mean cor-
puscular hemoglobin (MCH) are important indicators for
the diagnosis of thalassaemias [48]. The effectiveness of
ATR-FTIR spectroscopy on whole blood lysate for the
hematological analysis of thalassaemias has been eval-
uated, based on appropriate cut-off values of predicted
MCV and predicted MCH [48]. Obvious positive correla-
tions of Hb, MCV and MCH were observed between rou-
tine methods and ATR-FTIR spectroscopy. The best cut-
off value of predicted MCV and predicted MCH for
phenotypic-positive subjects yielded sensitivity values
of 100% and specificity values of respectively 100% and
96.8%. Next to screening and diagnostic purposes in
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hematology, FTIR spectroscopy has also potential to
study the impact of drugs in malignant cells. Micro-FTIR
in combination with HCA for identifying drug-resistance
or sensitivity in chronic myeloid leukemia cells exposed
to tyrosine kinase inhibitors (TKIs) resulted in an accu-
racy >95% in the classification of viable (drug resistant)
and apoptotic (drug sensitive) cells [49]. The authors
concluded that the method potentially represents a
rapid, convenient and robust screening approach to
study the impact of drugs in leukemic cells as well as in
peripheral blasts from patients in clinical trials with new
anti-leukemic drugs.
Dermatological disorders
The most common form of malignancy in humans is
non-melanoma skin cancer, which represents 95% of
cutaneous neoplasms. Squamous cell carcinoma is the
more aggressive form and shows a pattern of destruc-
tive growth with a metastatic profile [50]. The role of
ATR-FTIR spectroscopy as a useful tool to complement
histopathological analysis for the diagnosis of cutane-
ous squamous cell carcinoma by assessing biochemical
changes in normal skin caused by squamous cell carcin-
oma, induced by multistage chemical carcinogenesis in
mice, has been investigated [50]. Clustering analysis
showed 86.4% accuracy in differentiating the spectra of
neoplastic and normal skin tissue.
Classification of clinically relevant
microorganisms
The classification of microorganisms is generally based
on morphologic and biochemical characteristics.
Disadvantages of this approach are the time-consuming
handling procedures and the need for trained techni-
cians. While recent molecular techniques have led to
faster characterisation of microorganisms, they rely on
already know DNA sequences. Furthermore, the high
costs per test in combination with the need for highly
specialized equipment render them rather impractical
for use in the routine clinical laboratory [74]. Nowadays,
modern FTIR spectroscopy in combination with chemo-
metric data analysis forms a fast and consistent tech-
nique that provides very specific whole-organism
fingerprints, which can be used in the reliable typing
and classifying of microorganisms even below species
level. Therefore, FTIR spectroscopy is gaining increasing
interest in the microbiology field. The technique has
been applied in many different fields like food micro-
biology and microbial ecology [54] and is extensively
used in medical diagnostics. Research has shown that
FTIR spectroscopy in combination with chemometric
data analysis can give a fast, user friendly and inexpen-
sive screening with the ability to classify clinical patho-
gens such as bacteria [63,74–78] and fungi [79–82] at
different taxonomic levels. As an earlier example,
Maquelin et al. [79] explored the potential of FTIR spec-
troscopy for the identification of bacteria and yeasts
that are most often isolated from blood cultures such as
Staphylococcus aureus, Escherichia coli, Enterococcus sp.,
Pseudomonas aeruginosa, Streptococcus sp., and Candida
sp. by using models based on LDA and ANN. In com-
parison with the phenotypic identification, a high iden-
tification accuracy of 98.3% was achieved. Whereas
routine identification has a typical turnaround time of
1–2 days, FTIR spectra of microcolonies were collected
6–8 h after microbial growth was detected by an auto-
mated blood culture system. Another example is the
rapid identification of nonfermenting gram-negative
bacteria isolated from sputum samples from cystic fibro-
sis patients based on FTIR in combination with ANNs
[83]. Pseudomonas aeruginosa, Stenotrophomonas malto-
philia, Achromobacter xylosoxidans, Acinetobacter sp.,
Ralstonia pickettii, and Burkholderia cepacia complex
(BCC) bacteria were identified with a success rate of
98.1%. The four most clinically relevant species of BCC
bacteria (B. cepacia, B. multivorans, B. cenocepacia and B.
stabilis) were classified with a correct identification rate
of 93.8%. Furthermore, the ability of FTIR spectroscopy
and HCA, in comparison to randomly amplified poly-
morphic DNA (RAPD), to discriminate between strains of
C. albicans isolated from patients in the intensive care
unit (ICU) has also successfully been investigated [80].
Although these examples demonstrate the strong
potential of FTIR spectroscopy in the identification of
microorganisms in routine clinical microbiology labora-
tories, these methods have the disadvantage that isola-
tion and culture are still needed [63], which can be
labor-intensive when many different isolates need to be

analyzed. A high-throughput methodology has been
developed that allows microcultivation of bacteria from
low volumes (typically 200 lL) to be coupled with FTIR
spectroscopy [74]. This method is fast, easy to perform,
and enables separation of different isolates of E. coli
involved in urinary tract infection with respect to both
genotype and resistance to the antibiotic ciprofloxacin.
The authors stated that the high-throughput nature of
this approach is able to analyze several hundred or
even thousands of bacterial samples per day.
Additionally, spectra are sensitive to small variations in
culture parameters and environmental stress [80]; there-
fore, the necessity exists to follow a strict protocol for
sample preparation. Due to the latter, a worldwide
accessible database of reference spectra is currently
lacking [54]. Furthermore, inter-replicate variations to
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standardize and compare results have been reported by
several research groups [80–82]. However, among all
techniques available, FTIR spectroscopy combines some
unique properties in the identification of microorgan-
isms. A rapid and accurate identification of microbial
pathogens with FTIR spectroscopy has the potential to
reduce infection-related morbidity and mortality of hos-
pitalized patients in a very cost-effective way without
the need for consumables and with virtually no sample
handlings, which is in sharp contrast with expensive
molecular techniques. Furthermore, it uses open data-
bases that can be fitted to user-specific needs and
changes in the microorganism nomenclature. At last,
pathogen typing can be carried out without the need
for additional experiments [54].
Kidney stone analysis
Renal stones (nephrolithiasis) are a relatively common
problem and can be caused by a variety of underlying
disorders. In Europe, prevalence and incidence of uro-
lithiasis has increased markedly during the last decades.
These days, it affects approximately 10% of the Western
population [9], presumably due to changes in dietary
habits (e.g. increased intake of animal proteins and salt)
[4,9] and the increased incidence of obesity, metabolic
syndrome, and type-2 diabetes mellitus [4]. Knowing
the composition of a stone is important in order to
reveal the etiology of stone formation, to offer a specific
treatment, and to provide an optimal prophylactic treat-
ment to prevent recurrence. Approximately 50% of
nephrolithiasis patients will suffer from a new attack
within 5 years of their first episode. Chemical and phys-
ical techniques can be used for the identification of
stone components [4,9]. However, wet chemical meth-
ods are generally deemed to be obsolete for several
reasons [84,85]. Firstly, they do not allow the
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 13
quantification of each component in mixed stones [9].
Secondly, crystal structures cannot be identified, which
means that a differentiation between the various crys-
talline phases of calcium oxalate (CaOx) or calcium
phosphate (CaPh) is not possible. Finally, they are
unsuccessful in identifying rare purine stones resulting
from genetic disorders (e.g. 2,8-dihydroxyadenine) and
drug-induced calculi, which is an important drawback
that could lead to underdiagnosis [4,9]. Considering
those disadvantages, physical methods, i.e. IR spectros-
copy, X-ray diffraction (XRD) and polarization micros-
copy, are recommended techniques for kidney stone
analysis [84–88]. However, the combination of lower
instrument costs, rapid analyses, easy data interpret-
ation and reliable results even with very small amounts
of sample have made IR spectroscopy the gold standard
for kidney stone analysis [4]. Because kidney stones may
remain months or years in the urinary tract, they are
often composed of several components [9]. FTIR spec-
troscopy allows identification of each component in
mixed stones (Figure 6) and provides the relative con-
tent percentage of the different components within the
stone [4,9]. An identification of minor components can
be clinically relevant to gain insight in the lithogenic
process or to determine the influence of environmental
factors (e.g. diabetes mellitus type 2). FTIR spectroscopy
is suitable for CaOx and CaPh stones as well as non-cal-
cium stones such as cysteine, xanthine, struvite, 2,8-
dihydroxyadenine, uric acid, urates, proteins, lipids, and
drugs [9]. Furthermore, information on crystalline
phases of the same species can be obtained, which
means that it is possible to distinguish between, for
example, CaOx monohydrate (COM) and CaOx dihy-
drate (COD) stones. This is important as COM stones
may be linked with hyperoxaluric conditions such as
primary hyperoxaluria, while COD stones are often
related to hypercalciuria [9,89].
Nail analysis
The human nail plate is a rather impermeable biological
structure made up of layers of flattened keratinized cells
that are fused into a dense and somewhat elastic mass
[90,91]. The main component of the nail plate is a-kera-
tin, which forms the stratum corneum. Alterations in
physical and chemical properties may be reflected in
the molecular structure of nail proteins [90]. FTIR spec-
troscopy is a powerful method to detect structural
changes of nails. The nail is an excellent target for non-
invasive IR spectroscopic investigation as it is a readily
accessible structure with a low water content compared
to other tissues, which largely eliminates the interfer-
ence of water in the spectrum [91]. However, the
 14 S. DE BRUYNE ET AL.
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Figure 6. MIR spectrum of a calcium oxalate monohydrate stone (COM, red line), calcium phosphate stone (CaPh, blue line) and
a mixed stone composed of 60% COM and 40% CaPh (black line) in the 1750–400 cm21 region obtained with ATR-FTIR.
potential of FTIR spectroscopy for nail characterization
may be restricted by the shallow depth of penetration
into tissue (typically <10 mm) [92]. Upon examining nails
in vivo and ex vivo using ATR-FTIR, the ATR spectrum
showed the presence of lipids on the nail surface with
the appearance of the lipid ester carbonyl peak at
1745 cm
1 and the lipid methylene stretching vibrations
at 2924 and 2853 cm
1, which are typical of unordered
lipid acyl chains [91]. Furthermore, protein absorption
was identified at 1650, 1540 and 1250 cm
1. Generally,
the condition of nails can be affected by several dis-
eases; therefore, nails can be an indicator of health
[93–99]. As an example, fatigue is supposed to affect
the condition of nails. Possible differences in the MIR
spectra of nail plates from chronic fatigue syndrome
(CFS) patients and healthy donors have been investi-
gated [95]. a-Keratin is known to have abundant a-heli-
ces. The secondary structural content of proteins
observed in nails indicated a decrease in a-helices and
an increase in b-sheets in CFS nails in comparison to
healthy counterparts including both males and females,
suggesting reduced levels of the normal elements of
the nail plate. As the a-helix contributes to the stabiliza-
tion of the protein structure, a decrease in a-helices
may cause destabilization of the proteins in nails.
Furthermore, diabetes mellitus also supposedly affects
the condition of nails. As the protein content in kerati-
nized structures, such as nails, is approximately 80% of
the total mass, glycation of proteins has been
observed in several studies [96,100–102]. A recent study
demonstrated the successful application of ATR-FTIR
spectroscopy for measuring glycated nail proteins in
human finger nails [93]. The spectrum between 970 and
1140 cm
1, which is the region associated with carbohy-
drates, was identified as the zone of interest. A good
diagnostic performance for the diagnosis of diabetes
mellitus was reported (specificity: 82%; sensitivity: 90%).
Analysis of protein glycation in finger nails with ATR-
FTIR spectroscopy is an interesting alternative for the
diagnosis and monitoring of diabetes mellitus in third
world countries because of its much lower psycho-
logical threshold for sampling without the need for
medically trained personnel. No reagents are needed
and the preanalytical phase is extremely robust. A short
communication also reported differences in the spectra
of controls and diabetics [90]. Amide II bands were
found to be absent in the nails of nondiabetic patients.
However, the study of Coopman et al. [93] could not
confirm this finding. Another study used ATR-FTIR spec-
troscopy on finger nail clippings from patients with
psoriasis and healthy donors to understand the degrad-
ation mechanism induced by psoriasis in human finger
nails [94]. A significant reduction of the a-helix content
coupled with an increase of b-sheet and random coil
content was observed in psoriatic fingernails. This illus-
trates that the damage produced by psoriasis in finger-
nails is associated with the a-b transition, followed by a
b-sheet mediated protein aggregation, while the ran-
dom coil increase is indicative of protein denaturation.
Moreover, increased absorption bands at 1170 and
1040 cm
1, which are specific to the S-O bonds of the
cysteic acid unit, were observed. A higher concentration
of S-O bonds in psoriatic nails is indicative of nail matrix
degradation. Taken together, these findings could allow

development of a non-invasive method for the early
diagnosis of psoriasis.
Fecal fat analysis
The human diet consists of fats with a large spectrum
of fatty acids with varying chain lengths and degrees of
saturation. Triacylglycerols are the main components of
dietary fats (92–96%) and are composed of long-chain
fatty acids. Absorption of these fats is dominated by
two processes. First, hydrolysis of triacylglycerols pre-
dominantly by pancreatic lipolytic enzymes (¼ lipolysis)
leads to the formation of fatty acids and 2-monoacyl-
glycerols. Second, mixed micelles composed of bile
components and lipolytic products are formed for
intestinal uptake [103]. Fecal fat analysis has its clinical
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utility for the diagnosis of fat malabsorption or maldi-
gestion. An increased excretion of lipids in feces (stea-
torrhea) can be caused by malabsorption in patients
with small intestinal disease such as Crohn’s disease,
coeliac disease and gastroenteritis or by decreased tri-
glyceride degradation in patients with pancreatic or bil-
iary disease such as cystic fibrosis [104–106]. Attention
has been paid to FTIR spectroscopy for fecal fat analysis
as a simple and elegant alternative for the laborious
and time consuming classical Van De Kamer approach
[103]. The Van De Kamer method was introduced in
1949 and is still considered as the gold standard for
fecal fat analysis. However, important interlaboratory
differences have been reported due to the lack of
standardization [105]. FTIR spectroscopy has the poten-
tial to quantify the amount of fecal fat in feces and to
provide detailed information about the chemical nature
of fats by molecular fingerprinting. A rapid method,
based on ATR-FTIR with PLSR, for FTIR measurements of
fecal lipids without the use of reagents and need for
sample preparation has been described [104].
Homogenized stool samples were spread directly on
the transparent crystal, IR spectra were acquired, and
multicomponent analysis was done over several spec-
tral bands. This procedure prevents prolonged handling
of stools using extraction procedures. However, the
authors noted that preselection of samples was neces-
sary. Only homogeneous stools (with no visible food or
other fragments) with a normal consistency (not liquid,
not too tough) were used. Stool consistency is an
important factor to ensure complete spreading on the
crystal surface because IR beams have an extremely
small penetration depth (maximally 5 lm) with this
sampling method. As a consequence, an incorrect sam-
ple adherence on the crystal causes unreliable quantita-
tive results. Furthermore, liquid stools were also
discarded due to major interference of water in the IR
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 15
spectrum, which causes relative poor repeatability and
reproducibility coefficients. In addition, measurements
on untreated stool samples with a large number of
interfering substances resulted in complex spectra. As a
consequence, secondary reference samples, which are
samples from which the sample concentrations have
been determined by means of a generally accepted
method such as the Van de Kamer method, are needed
for calibration [107]. For these reasons and as the
results of the Van de Kamer method are poorly
exchangeable [105], several studies [103,105–107]
reported methods based on a simple extraction proced-
ure of fecal fat. In order to improve standardization,
Jakobs et al. [105] developed a method for fecal fat
determination by using a transmission cell.
Quantification was based on the absorbance band of
the CH2 group (2855 cm
1) of free fatty acids and fatty
acid glycerol esters after an extraction procedure with
acidified petroleum ether-ethanol. The extraction pro-
cedure has the advantage that it prevents the interfer-
ing influence of water and water-soluble interfering
substances. Furthermore, primary standards (e.g. ste-
aric-palmitic acids) can be used for calibration instead
of secondary reference samples. The method was found
to be more precise with respect to interlaboratory vari-
ation than the Van de Kamer method and enables rapid
diagnosis and monitoring of steatorrhea. A method
based on a simple hexane extraction procedure having
the advantage that both fatty acids and triglycerides
can be quantified in one run has also been published
[106]. Quantification was based on the measurement of
peak height absorbances of both the C ¼ O stretch
vibration of the carboxylic acid residue of the fatty
acids (1714 cm
1) and the C ¼ O stretch vibration of the
triglycerides (1751 cm
1). In addition to the mentioned
studies above, De Koninck et al. [103] demonstrated
the application of FTIR spectroscopy on stool samples
in the determination of lipase hydrolysis efficiency, fatty
acid chain length, and trans-unsaturated fatty acids. To
elucidate the efficiency of fat digestion by pancreatic
lipase (by hydrolyzing triglycerides), fatty acid/triglycer-
ide ratios in feces were calculated using the absorbance
ratio observed at 2855 cm
1 (C-H bond symmetric
stretch vibration): 1746 cm
1 (C ¼ O vibration of gly-
cerol esters). The fatty acid chain length
(CH3(CH2)xCOOH) was calculated using the ratio of the
absorbance observed at 2855 cm
1 (C-H bond symmet-
ric stretch vibration): 1709 cm
1 (C ¼ O bond stretch
vibration). The C ¼ O absorption band is very similar for
each kind of fatty acid because each fatty acid contains
one aldehyde group (-COOH), while the absorbance at
2855 cm
1 represents the fatty acid chain length
((CH2)x). At last, trans-double bonds provoke a
 16 S. DE BRUYNE ET AL.
characteristic absorbance peak at 966 cm
1 due to the
out-of-plane CH deformation. This information is rather
difficult to obtain with other techniques and might be
useful to monitor diets in different clinical settings.
Trans fats are unsaturated fats produced industrially by
the chemical reduction of unsaturated bounds with
catalytic hydrogenation. They are used to solidify fats
and improve taste, texture, and shelf life of certain
foods. Nutritional authorities consider all trans fats as
equally harmful for health, and therefore, an elevated
pressure is carried out on food manufacturers to reduce
the use of synthetic trans fats. The FTIR assessment of
trans fatty acids in stool samples could provide an
interesting tool for epidemiological research.
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Future directions
Recently, the field of FTIR spectroscopy has expanded
into new and exciting clinical applications due to signifi-
cant improvements in instrumentation and the develop-
ment of new techniques. As a consequence, the
number of vendors and the variety of commercially
available instrumentation has increased. Generally, FTIR
spectroscopy tends to follow two major paths: more
capability with an increased performance, and the
development of smaller, simplified and less expensive
devices. Due to the latter, FTIR spectroscopy has made
a transition from the laboratory setting to on-site appli-
cations. Portable and handheld FTIR spectrometers are
currently used in different fields such as biodiesel ana-
lysis, soil analysis, and quality assurance of food ingre-
dients. In terms of medical applications, integration of
this technology in a portable battery powered device,
like the current glucometer, could offer a point-of-care
platform to monitor multiple health parameters and
thereby facilitate the creation of bedside technologies
for diagnostic and treatment monitoring purposes for a
various range of diseases [34]. The portability and bat-
tery operation of the devices enable the application to
be carried out in remote locations, where power and
road transport are issues, which is of particular interest
to developing countries [47]. Ongoing miniaturization
of the technique could lead to the development of a
personalized diagnostic tool in which patient-to-patient
differences in molecular signatures would allow the
assessment of disease status and personalized drug
management [34]. In recent years, there has been an
increasing interest in on-chip MIR sensors. Recent pro-
gress on advanced MIR light sources, waveguides, and
device concepts predicts next-generation optical sens-
ing platforms, which are suitable to overcome the chal-
lenges of in situ diagnostics [108]. Therefore, it is
anticipated that label-free integrated MIR-sensing
schemes will readily complement existing chem-/bio-
sensor technologies and may have a broad impact
upon personalized healthcare. Several advantages of
this new technique such as inherent molecular selectiv-
ity, real-time monitoring capability, ease of operation,
cost efficiency, and a compact device footprint promise
reliable optical diagnostics [108]. As an example, MIR
sensors are a promising candidate for clinically deploy-
able breath analyzers. Significant changes of constitu-
ents within exhaled breath (e.g. NO, CO, CS2, COS, NH3)
may serve as an indicator for pulmonary diseases or
other diseases such as schizophrenia, cancers,
Helicobacter pylori infection, and diabetes mellitus [108].
Furthermore, with further innovations and translational
development, implantable MIR photonic devices could
offer a pathway to improve (continuous) health moni-
toring and diagnostics. Due to the complexity of IR
spectra, interpretation of data is the major hurdle in the
long and winding road to clinical applications.
Therefore, integration of chemometrics into IR equip-
ment will facilitate the introduction of the technology
in clinical laboratories. Finally, spectra of biological sam-
ples represent a mine of information. The growing col-
lection of this type of information might be used for
providing an a posteriori diagnostic analysis in novel
applications.
Conclusion
The main limitations of MIR-FTIR spectroscopy can be
found in its relatively low extinction coefficients and
a certain strain to analyze wet specimens. However, it
can be stated that the benefits outweigh the disad-
vantages. FTIR spectroscopy is a simple, nondestruc-
tive, label-free, and cost-effective technique that
allows acquisition of biochemical information of
molecular composition with minimum amounts of
sample and can offer qualitative, quantitative and
multicomponent analyses, even at remote locations.
The development of ATR-FTIR analysis has strongly
reduced the time needed for sample preparation,
which makes the technology attractive for routine
clinical laboratories. Despite the huge potential of the
proposed technique in clinical laboratory sciences,
the level of skepticism of many clinical laboratory
managers and pathologists still remains too high. The
MIR absorbance spectrum fits all the characteristics
for the development of clinical diagnostic or monitor-
ing applications and can be considered as a promis-
ing analytical technique complementing traditional
laboratory-based analytical devices (e.g. chemilumines-
cence, gas chromatography, mass spectrometry) in a
compact way with extremely low costs per test.

Disclosure statement
The authors report no declarations of interest.
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