Mid infrared spectroscopy as tool for Disease Pattern Recognition from

human blood
Gerhard H. Wernera; Johanna FrUha, Franz Kellerb, Helmut Gregerb, Ray Somorjaic, Brion Dolenkoc,
Matthias Ottod, Dirk BOcker'

aBoehringer Mannheim GmbH, 68305 Mannheim, Germany

bUnj of WUrzburg, Germany, 97080 WUrzburg, Germany
eNational Research Institute, Winnipeg, Canada
dTechn Univ. Freiberg, Germany, 09599 Freiberg, Germany

ABSTRACT

Disease Pattern Recognition ("DPR") is being developed as a reagent-free measurement technique for the diagnosis of
blood samples. The technological basis of this method is mid infra-red spectroscopy. For the analysis, 1 p.1 blood is
deposited on a disposable and dried before measurement. The infra-red spectra (wavenumbers between 500 to 5000 cm')
give rise to characteristic patterns in narrow wavenumber regions, which are modified in the presence of relatively small
pathophysiological changes. The spectral changes depending on the disease are always higher than those caused by the
deviations resulting from instrumental or handling errors. The information content of the spectra is reflected by the
standard error, which varies between differently "diseased" and "healthy" persons. The standard error of the first
derivative spectra is three times higher for "diseased" compared to "healthy" persons. This has been demonstrated for a
comparable population of "diseased" and "healthy" persons. In total, more than 2000 spectra from different individuals
were analyzed. Preliminary results using various mathematical algorithms indicate, that clear distinctions can be found
for a variety of different diseases compared to patterns of "healthy" spectra. This qualitative information about the blood
sample may be used as a quick and comprehensive diagnostic tool.

Keywords: IR-spectroscopy, disease pattern, human sera, blood samples

1. INTRODUCTION

More and more efforts are undertaken to implement new analytical technologies into the medical surrounding. Many
research groups around the world search for alternative methods to the routine wet chemical lab tests as well as to other
medical fields .2 Among these, infrared spectroscopy and Raman spectroscopy is one of the major fields of interest..
As analytical material all kinds of biofluids like blood (serum), urine, synovial fluid, saliva etc. have been taken into
account. There are many different foci of biochemical interesting substances like proteins , lipids 6 carbohydrates and
nucleic acids 8 As infrared spectroscopy is a fast technique combined with low costs it may become an alternative to time
consuming and expensive tests for special markers. One special example for this is the classification of arthritis patients
with infrared spectra of synovial fluid of different arthritis patients .
A different way to use this fast and cost effective infrared spectroscopy is the DPR approach, where the focus lies not only
on special details of the blood and serum, but on the possibility to find changes in the spectra that are typical for certain
diseases. This procedure gives the chance to use the whole information content of the spectra without restriction of the
view only to known substances. This includes additional information to the know parameter spectrum as in the region of
500 to 5000 wavenumbers each molecule gives its own fingerprint. Current work aimed at developing a new diagnostic
application of infrared spectroscopy is discussed.

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 2. EXPERIMENTAL

Sample generation:
The blood samples were taken from female and male volunteers b' venous puncture using standard MONOVETTES®.
After 30 mm the blood was centrifuged for 15 mm at 2000 g and the serum was stored at < - 20 °C. Before measuring the
samples were allowed to tow to room temperature. One microliter of serum was dispensed on the disposable and measured
after drying.
The serum samples were taken from different hospitals over a period of 2.5 years. The data analysis was made from up to
500 serum samples. In a database the clinical data of more than 2000 samples are stored which were generated in a quality
controlled laboratory.

Spectra generation:
The measurements were carried out on an FT-IR-Bruker IFS 66 Instrument with an optimized light housing and lamp.
The spectrometer was coupled with the commercially available Thin Layer Chromatography accessory, developed by
BRUKER and Prof. Kovar. head of the pharmaceutical department form the University of Tubingen, Germany.
The measurement of so small areas are complicated by inhomgeneous light beams.
Mid infrared spectra were recorded by a Bruker FT-spectrometer IFS 66® equipped with globar, KBr- beamsplitter and
MCT-detector (Grasby Inc.).
The measurements were carried out at room temperature. For the spectra 16 interferrograms providing a resolution of 4
cm' were added.

3. DIAGNOSIS

As the diagnosis of the diseases is the basis for a correct classification of the spectra a protocol was worked out together
with the involved clinics. Within this protocol the necessary clinical parameters and other examinations were fixed which
had to be turned out to generate an exact diagnosis. There still exists a rest probability of other diseases , but even in a
normal diagnostic process this uncertainty can't be excluded. To account this obstacle we decided to take at least 100
samples for a disease group. Within this group of samples the existence of overlapping diseases should be diminished to a
small risk.

4. SPECTRAL INFORMATION

The Mid infrared spectroscopy detects the information of all molecular groups incorporated in the serum as well as their
complex interactions. As there are several hundred substances included in the serum of which only a part is known a direct
transformation of a signal to a special ingredient is only possible if this substance is one of the main components of the
blood like glucose. cholesterol or triglycerides. The result of this fact is a complex spectrum which has twomain regions
m.a.u

1350 1250 1150 1050 950 850

wavenumber cm1
Figure 1: Information content of spectra from ,,normal" (black) and ,,diseased" (gray) volunteers

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 '
where composition changes of the serum are detected. This is the region between 2800 cm and 3500 cm' called the C-H
stretch region and the even more complex fingerprint region between 800 cm' and 1800 cni' . Figure 1 shows the
information content of two groups of volunteers. The larger group comprises 1000 spectra of more than 20 different
disease classes, whereas the second group comprises 300 spectra of healthy blood donors. This illustrates the need of a very
precise measurement of the spectra.. We realized this with achieving a sufficient difference of the standard error of the
first derivative spectra. For "diseased" compared to "healthy" persons it is three times higher in the most described regions

Because of the corn-

plexity of the spectra it
is not possible until now
to distinguish between
spectra from patients
belonging to two dii-
ferent disease classes
according to known
regions which corres-
pond with the change of
typically known sub-
stances. For this reason
it is necessary to
evaluate the spectra not
by eye but by high
sophisticated
Figure 2: Characteristic spectral variances of healthy volunteers mathematical methods
that can find out typical
patterns for different
disease classes. In some
cases different regions
may also be fixed by eye
(figure 2 & 3).

Figure 3: Characteristic spectral variances of volunteers with Metabolic Syndrome

5. MATHEMATICAL THEORY

5.1. Regularized Discriminant analysis (RDA)

In contrast to Fisher's discriiuinant analysis classification, RDA is carried out by Bayes's theorem 10 The assignment of a
sample, x, characterized by d features (here clinical parameters or points in the JR spectrum) to a class (disease) k of all
classes K is based on maximizing the posterior probability

P(kI x) fork=1,..,K. (1)

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 Application ofBayes's theorem for calculation ofthe posterior probability reveals:

P(klx) = p(xI k)P(k) (2)

p(x)
According to equation (2) the posterior probability is computed from the probability density function for the considered
class, p(xlk), the prior probability for that class P(k) and the probability density function over all classes p(x). A sample x
is then assigned to that class k. for which the largest posterior probability is found.

For computation of the class probability density, pxJk), the multidimensional normal distribution is assumed:

p(x 1k )= (2)d/2Is I0.5xP[_ O.5(x — k )S'(x - Xk )T ] (3)

where the covariance matrix Sk based on the class centroid Xk is obtained by:

ki
I
Sk (x — ik )T (x — Xk ) (4)

Xk =—-tx
k i=1
(5)

flk describes the number of samples in class k.

Maximizing the posterior probability is related to minimizing the so-called discriminant scores obtained by:

dk (x) = ( — )S1(X Xk )T + 1Sk 2JINP(k) (6)

An unknown sample is assigned to that class k, with the shortest distance to its class centroid. The first term in equ. (6)
represents the Mahalanobis distance between the sample x and the class centroid Xk
Compared to the linear and quadratic discriininant analysis in RDA the covariance matrix is regularized by two
parameters A and yaccording to:

Sk()t) =(1-2) S + ,S (7)

Sk(A,y)=(l-y)Sk(2) +Ttr[Sk(2)]/pJ (8)

This assures that even in the case of ill-conditioned systems, e.g. in case of very similar spectra, good results for the
estimation of the inverse covariance matrix in equ. (6) and the subsequent classification of unknown samples is to be
expected.

Outliers may be detected on the basis of the probabilities again computed for a single sample again on the basis of Bayes's
theorem. The probability of a sample x to be contained in class k, pxIk), is given from equ. (3). For estimation of the prior
probability for the K classes, P(k), the number of samples in a class, k, is considered in relation to the total number of
samples n, i.e.:

(9)

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 The probability density over all classes px) is then obtained from the sum of the class probability densities and the prior
probabilities according to:

p(x) = p(x ic)P(k) (10)

For each sample the posterior probability to be a membership of class k is according to the Bayes rule (cf. equ. (2)):

IxVc,i—
\_ K
p(xv)P(k)
(11)
>p(x )P(k)
For a posterior probability a value of 1 represents membership to class k, and 0 stands for non-membership to class k. On
the basis of the individual probabilities for each sample multiple assignments to several classes are also possible since the
posterior probability according to equ. ( 1 1) of a sample x is always calculated for all classes. For example, in case of three
classes a sample may be members of class 2 by 70%, of class 2 by 20% and of class 3 by 100-70-2010%.

5.2. Binary Classification

Several binary (pair-wise) classifiers for healthy vs. disease(s) or disease vs. disease(s) pairs that are based on IR spectra
ofblood serum are developed. The classifiers use spectral information from both the fingerprint (868-1343 cm, 250 data
points ) and the C-H stretch region (28 12-3002 cm1 , 100 data points). All spectra are first normalized (to unit area in the
region considered) and then their first derivatives are calculated. Additional smoothing on the first derivative spectra is
generally beneficial. This is done by averaging S ( 2, 3, 4, . . . , etc.) adjacent data points.
The classification strategy consists of preprocessing (feature space reduction) and crossvalidated classifier development.
Any classifier outcome is reported as a probability. Classifier robustness is achieved by partitioning the classes into
training and validation sets and carrying out crossvalidation. Our crossvalidation approach (RBS, robust bootstrap) is
closely related to the bootstrap 12
Feature reduction is essential, because for classifier robustness and reliability the number of spectra per class must be at
least 1 0 times more than the final number of features selected. Our optimal region selector (ORS) was specifically
developed to find features that retain their spectral identity. It is a sliding Linear Discriminant Analysis (LDA) procedure:
in the spectral region of interest (e.g., the 250-point fingerprint (FP) region), the first R adjacent points 1,2,3, ..., R (called
an R-window) are used as an R-feature input to a weighted LDA classifier, crossvalidated by our PBS method (250-500
RBS iterations). The classification results for this R-window are saved and then the window is advanced by K 1 steps:
the new window consists of data points K+1,..., K+R. The results of the new classification are again stored. This K-step
window ,,sliding" and classification is repeated until the end of the region is reached. The resulting R-windows are
ranked, based on the best kappa value for the validation set. These best R-feature subregions provide the starting point for
additional processing. {S, R, K} are at our disposal, enabling us to reduce R as much as possible.
The final classifier can be produced in two ways: either by creating a new feature set from subregions of length R, +R +
Rk + . . . selected exhaustively from the ranked {R1 , R2, . . . , R,, m 8}, or by combining the outcomes of several classifiers
via Wolpert's Stacked Generalizer 14 (WSG). The former is simpler but less satisfactory because the total number of
features is increased. WSG uses the output class probabilities obtained by the individual classifiers as input features to
another classifier. For 2-class problems the number offeatures is 2 per classifier (i.e., 2K for K classifiers), typically much
less than R + R + R + . . . , hence increasing the ultimate reliability. Whichever combination approach is used, the overall
classification quality is generally higher. The crispness of the classifier, (i.e., the fraction of the total number of spectra
that are assigned with >75% probability to one of the two classes) is invariably greater. This is important in a clinical
environment because fewer patients will have to be reexamined.

The two classification procedures only differ in one aspect. The regularized discriminant analysis takes more than two
classes simultaneously into account whereas the binary classifier distinguishes between only two classes at one time.
Because we focused on only a few disease classes the first results presented are obtained with the binary classification.

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 6. RESULTS

As already mentioned four different volunteer groups (Healthy, Metabolic Syndrome, Diabetes, Rheumatoid Arthritis) had
to be evaluated. This was done by utilizing the procedure of the binary classification. For this some important terms have
to be defined:

Definitions (2-Class Classifiers):

TN: True Negative
TP: True Positive
FN: False Negative
FP: False Positive
SE: Sensitiyjy : TP I (TP + FN)
SP: Specificity : TN I (TN + FP)
PPv : Positive Predictive Value : TP I (TP + FP)
NTPV : Negative Predictive Value : TN I (TN + FN)
Kappa (K) : SE(1) + SE(2) — 1 (Agreement Measure Beyond Random)
0: Overall Accuracy : (TN + TP) I (TN + TP + FN + FP)
C: % Csps : (Class assignment Probability > 75%)
Q: (Classifier Qality): 25(O + K + PPV (1) + PPV (2))
Complete and partial results for a number ofbinary classifiers are presented below.

Binary Classifier:
I
Healthy vs. Other Diseases 94.0 97.2

Healthy vs. Rheumatoid Arthritis 97.0 93.5

Rheumatoid Arthritis vs. Other Diseases 95.9 100.0

Healthy vs. Metabolic Syndrome* 90.4 80.8

Healthy vs. Diabetes (I & II) (FP & CH (W)) 98.1 100.0

Diabetes I vs. Diabetes II (FP Region)* 96.2 92.6

Diabetes (I & II) vs. Metabolic Syndrome* 94.6 91.3

Diabetes vs. Other Diseases (FP Region) 94.0 98.3

Diabetes vs. Other Diseases (CH Region) 93.0 94.0

* Incomplete results.
+
Other Diseases: mixture of several diseases concerning liver, kidney, heart, pancreas and metabolic disorders

These samples are all obtained from volunteers that are in the fasting stage, are non smokers and do not get medication
except for Rheumatoid Arthritis patients. The age range of the volunteers is from 18 to 60 years and there is no preference
of sex or other conditions. Children haven't been subject of our studies because in Germany we would have need the
acceptance of the parents which is often hard to get. On the other hand the diseases mentioned are not very common under

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 children despite diabetes so there should not be a big effect not having included children in this studies. For children other
diseases have to be taken into account which would be more useful to have a general diagnostic tool for. This has to be
proofed in further studies.

7. DISCUSSION

The results of the binary classification show very high quality rates combined with a high crispness. Especially the combi-
nation of the fingerprint region with the C-H region leads to increasing crispness rates. For Metabolic Syndrome these
high crispness values could not be reached. But this isn't an astonishing result because Metabolic Syndrome as a precursor
for Diabetes 2 patients is a totally asymptomatic status which can only be diagnosed with very high diagnostic efforts
because there are not many changes in the metabolism at that stage.
As these results are very preliminary a check for separating different age groups has not started yet. This together with
many other side effects like nutrition, smoking etc. may have an influence on the spectra that can be separated. Up to now
the samples are taken from patients in a fasting stage, so there could not be seen an influence of nutrition so far. As the
studies are turned out exceptionally in Germany more than 90 % of the volunteers were Europeans. This is also an
influence that has to be checked in further studies with volunteers from different ethnic groups in different countries. It
also has to be proofed whether differences might be caused by different nutrition habits or by real ethnic inluences, because
many diseases are caused by nutrition.

8. SUMMARY

With the classification of all four groups with more than 90 % quality and more than 90 % crispness (except 80 % for
Metabolic Syndrome) the first results are very promising. These results are very preliminary because a sample size of 100
per class is at the lower limit of a statistically representative population. On the other hand the results should even become
more crisp with increasing the number of samples. This together with studies checking for the influence of other important
factors like nutrition etc. will be done in further studies.

REFERENCES

1. M. Jackson, H. H. Mantsch, Biomedical Aplications of Spectroscop", ch. 5, pp. 185, R. J. H. Clark, R. E. Hester,
Wiley, New York, 1996
2. H.M. Heise, ,,Potential application of infrared spectoscopy in clinical chemistry", Lab. med., 15, pp.470 -476, 1991
3. Robert J. Dampsey, D. G. Davis, R. G. Buice, R. A. Lodder, ,,Biological and Medical Application of Near-Infared
Spectroinetry", App. Spec., 50 (2), ppl8A - 34A, 1996
4 A. Mahadevan-Jansen, R. Richards-Kortum, ,,Raman Spectroscopy for the Detection of cancer and Precancer", J.
Biomed. Optics, 1 (1), pp. 31 - 70, 1996
5. M. Jackson, H. H. Mantsch, ,,The use and misuse of FTIR spectroscopy in the determination of protein structure" CRC
Crit, Rev. Biochem. Mol. Biol. 30, pp. 95, 1995
6. H.L. Casal, H. H. Mantsch, ,,Polymorphic phase behaviour of phospholipid membranes studied by infrared
spectroscopy" Biochim. Biophys. Acta 779, pp. 381- 401, 1984
7. M. Mathlouthi. J.L. Koenig, ,,Vibrational spectra ofcarbohydrates", Adv. Carbohydr. Chem. Biochem. 44, pp. 7, 1986
8. E. Taillandier, L. Liquier, J. A. Taboury, Advances in Spectroscopy 12, pp. 65, R. J. H. Clark, R. E. Hester, Wiley,
NewYork, 1985
9 R. A. Shaw, S. Kotowich, H. H. Eysel, M. Jackson, G. T. D. Thomson, H. H. Mantsch, Rheumatol. mt., 15, pp. 159,
1995
10 J. H. Friedman, ,,Regularized Discnminant Analysis", J. Am. Stat. Assoc. 84, pp. 165, 1989
11 M. Otto. Chemometrie, VCH Weinheim, pp. 171, 1997
12 B. Efron, R. J. Tibshirani, ,,An Introduction to the Bootstrap", Monograms of Statistics and Applied Probability, 57,
Chapman Hill, New York, 1993
13 R. L. Somorjai. B. Dolenko, Proceedings of the European Society of Magnetic Resonance in Medicine, Prague, Czech
Republic, #801, 1996
14 D.H. Wolpert, Neural Networks, 5, 241, 1992

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