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BACKGROUND OF THE INVENTION
The invention relates to an instrument for determining the amounts of
metabolic products in the blood by means of a radiation source and a
radiation detection system delivering an output signal depending on the
intensity of radiation of the aforesaid source after it has been affected
by the blood containing the metabolic products.
Generally, the determination of amounts of metabolic products such as
polypeptises, urea, cholesterol, glucose, CO.sub.2 or ethyl alcohol in the
blood is carried out by withdrawing blood from the body and examining it
chemically. However, the time required for ascertaining the particular
amounts is relatively long and ranges from a few minutes to an hour.
On the one hand, there is danger in such extended periods of time that the
blood may alter resulting in spurious results. On the other hand, a
continuous testing of the metabolic products, as is desirable for instance
when examining the glucose content when suspecting diabetes, or when
determining the CO.sub.2 content during artificial respiration in the
course of an operation, is impossible. Again, it is impossible, in current
techniques, to ascertain transiently occurring, unknown metabolic
products.
It is furthermore known that the metabolic products in the blood absorb
infrared (IR) radiation, so that they may be ascertained by means of
absorption measurements. However, such infrared absorption tests suffer
from the difficulty that the blood acting as a solvent for the metabolic
products in itself represents an aqueous solution which is strongly
absorbing in the infrared spectrum, as is well known. Therefore, when
performing measurements by means of previously known IR spectometers on
the basis of transmission, very minute film thicknesses are required to
obtain measurement signals that are useful at all. This requires in turn
that the dissolved substances to be tested must be present at very high
concentrations so that a relative change in absorption can be detected at
all. Therefore only concentrations exceeding 1 percent can be ascertained
in fact when using previously known IR spectrometers.
As shown by the article "Infrared Absorption Spectroscopy of Aqueous
Solutions with a CO.sub.2 Laser", Applied Physics, Magazine 7, pp. 287-293
(1975), the measurement sensitivity in infrared absorption tests using the
transmission mode has been significantly improved by employing lasers with
an essentially higher intensity than the previously known light sources.
When lasers are used, however, there frequently occurs the undesired side
effect of appreciable heating of the substance to be examined due to the
strong absorption properties of aqueous solutions. This problem is rather
easily met when testing aqueous solutions in inorganic materials available
in ample amounts of solution. However, the tests are significantly more
difficult if the same transmission measurements must be carried out for
blood, which is available only in lesser amounts and which furthermore
already denatures when heated to 45.degree. C.
As was shown by applicant in an article in the book Modern Techniques in
Physiological Sciences, Academic Press, London and New York, 1973, blood
tests may also be carried out in vivo by means of laser beams. In that
experiment, venous blood was passed in an extracorporeal shunt through a
cuvette at a film thickness of 0.1 mm and at a flow rate of 30 cc per
minute, and examined by means of a CO.sub.2 laser beam of 2 watts. It was
found that the temperature of the blood being tested could be kept below
the critical temperature limit because of its high flow rate, and that
+/-0.5% changes in concentrations in ethanol or glucose could readily be
shown. However, this method suffers from the drawback that the examination
is exceedingly costly and practically is suited only for large operation.
This method furthermore suffers significantly from the problems of
achieving even flow through very thin cuvettes and then cleansing of same.
Again the ATR (Attenuated Total Reflectance) method described by J.
Fahrenfort in Molecular Spectroscopy, Proceedings of a Conference at
Brighton, 1968, Elsevier Publishing Company, Amsterdam, pp. 111-130, has
already been used. In this method, the radiation with which to examine a
sample is so beamed into a suitable plate as to be totally reflected
several times at oppositely located surfaces of this plate before being
made to pass out of it and examined for changes in intensity. The sample
to be tested touches one or both sides of the plate totally reflecting the
beam.
SUMMARY OF THE INVENTION
The present invention now addresses the task of providing an instrument
allowing rapid, simple and accurate indication of the amounts of metabolic
products in the blood.
Starting with an instrument of the type initially mentioned, this problem
is solved by the invention in that the radiation source is an infrared
laser, and in that the laser radiation may be guided through an ATR plate
into the boundary-surface region of which may be brought the blood
containing the metabolic products.
Surprisingly it was found that such an arrangement for the first time
permits an extremely sharp separation of the individual metabolic products
which is the basic requirement for the quantitative determination of these
individual products. For instance, as discussed in greater detail further
below, the presence of contents in ethanol besides glucose can be
unquestionably shown, which was impossible with spectrometers known
previously.
Furthermore, this fact must be considered wholly surprising and
revolutionary, namely that the instrument of the invention for the first
time allows also determining the contents of metabolic products in the
blood without at all removing this blood from the body. This may be
achieved by placing the ATR plate directly against the skin and especially
against the tongue. It was wholly surprising therefore that no
difficulties due to tissue cell structure or overheating when locally
applying laser beams were encountered in the quantitative measurements.
It was found that the power of the laser used is only limited by the degree
of absorption of the ATR plate used.
The invention opens up wholly new feasibilities of examination and
simplifies those already known. For instance, reliable serial tests for
the early detection of diabetes can now be carried out for the first time.
When testing for glucose under stress, the essential advantage is obtained
that no blood need be taken at regular intervals from the patient.
Lasers that may be adjusted with respect to wavelength were found to be
especially suitable. Semiconductor-diode lasers, parametric lasers and
also gas lasers may be used. Parametric oscillators pumped by means of
pulsed neodymium and tunable depending on the crystals from 1.4 to 4
microns (LiNbO.sub.3) and from 1.22 to 8.5 microns (proustite) are
particularly suitable.
Furthermore, both pulsed and continuous wave lasers may be used.
Pulse-lasers offer the special advantage of introducing only minor
stresses in the form of heating the blood being tested despite the high
intensity available for such tests.
The angle of incidence of the laser beam with respect to the reflecting
surfaces of the ATR plate are appropriately selected as the function of
the ATR material. Preferably, however, they fall within the range of
45.degree.-60.degree..
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is explained below in greater detail, referring to the
drawings, wherein:
FIG. 1 is a diagrammatic representation of a total reflection process;
FIG. 2 is a diagram of an ATR plate;
FIG. 3 is a diagrammatic illustration of an instrument constructed
according to the invention;
FIG. 4 shows the relative transmission as a function of the wavenumber for
two different aqueous solutions, one containing 10% by weight of glucose
and the other 10% by volume of ethanol, measured with a conventional
spectrometer and on the same relative transmission scale; and
FIG. 5 shows the relative transmission as a function of wavenumber for two
aqueous solutions, one with 0.45% by volume of ethanol and the other with
0.5% by weight of glucose, the measurements having been performed with an
instrument of the invention.
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 merely shows diagrammatically the principle of total reflection,
which occurs when the incident light from an optically denser material of
index of refraction n.sub.1 is incident on an optically less dense
material of index of refraction n.sub.2.sup.* at an angle .theta.
exceeding the boundary total reflection angle obtained from the known laws
of physical refraction. Essentially the total reflection phenomenon is
characterized by no energy transfer taking place from the optically denser
medium n.sub.1 to the less denser optical medium n.sub.2.sup.* (n.sub.1
>n.sub.2.sup.*) on the average. The electromagnetic field, however, does
spread in a narrow boundary layer in the less dense optical medium. If the
less dense optical medium is not transparent, the equilibrium between the
incident and reflected light energies is disturbed by radiation absorption
in the boundary layer. This process is termed the so-called attenuated
total reflection, or ATR. This damped total reflection is used for
spectroscopic purposes with the ATR plate generally denoted by 2 and shown
in FIG. 2.
This ATR plate 2 is shown in FIG. 2 is of essentially trapezoidal
cross-section and has two opposite surfaces 6 and 7 which are essentially
parallel to each other. Beam 3 used for testing is coupled into the plate
by means of one of the trapezoidal end faces. The beam then is totally
reflected several times at surfaces 6 and 7 before exiting at the opposite
trapezoidal end face in the form of beam 4. The intensity of beam 4
exiting from plate 2 now may be affected by depositing the substance to be
tested, which in this instance is schematically shown as 5, on one or both
boundary surfaces 6 and 7, or by bringing it into contact with either or
both.
The essential advantage in affecting beam 3 by the substance 5 to be tested
consists in the latitude of arbitrarily selecting the layer thickness for
all practical purposes without thereby influencing the result obtained,
obeying merely the relation d>3.lambda., where d is the layer thickness
and .lambda. the wavelength of the test beam.
Several ATR plates for infrared spectroscopy already are known. Plates made
from Geranium, Irtan 2, Irtan 6 or KRS 5 are preferred. The only essential
feature for this procedure is that the particular ATR plate absorb as
little as possible of the beam being used.
The information in beam 4 leaving the ATR plate is the higher, obviously,
the larger the number of reflections taking place at the boundary layer
touching the tested substance. On the other hand, the number of
reflections clearly must be so chosen so that the signal obtained from
beam 4 can be unambiguously measured and processed. ATR plates with
dimensions 15 mm by 40-50 mm, and 1-2 mm thick, are used. The number of
total reflections at the boundary layer with the tested substance was from
3 to 14. Good results are obtained when there were 5 total reflections at
the boundary layer touching the tested substance.
FIG. 3 shows a diagrammatic embodiment of an instrument of the invention.
In this instrument, as in conventional spectroscopy when measuring
absorption, the method uses a reference beam. The radiation source is
generally denoted by 9 in FIG. 3. This source consists of a laser 10, a
tuning system 11 for the wavelength, an electronic Q-switch 12, and an
output stabilizer 13.
A beam 18 generated by laser 10 is split by a semi-transmitting mirror into
two half-beams 16 and 17 which are made to pass through the measurement
cell 27, the latter containing an ATR measurement plate 14 and an ATR
reference plate 15, both in the shape of prisms. The two half-beams 16 and
17 after exiting from the measurement cell are combined by means of
mirrors 22 and 24 into a common beam 19 which then passes through a lens
26 and is incident on detector 25 of a signal processing system generally
designated by 8.
In order to obtain higher sensitivity, the two half-beams 16 and 17 are
chopped in a known manner by means of a chopper 28 comprising a chopper
wheel 29. Chopper wheel 29 is provided with a varying sequence of
apertures and stops on two different concentric circles so that the two
half-beams 16 and 17 are converted into alternating light of relatively
different frequencies. The frequency of rotation of a chopper motor can be
varied in order to select the most favorable frequency range for the
further processing of the signals obtained at detector 25.
Three types of detectors may be used in the spectral range of 10 microns:
the photo-conductive Germanium semiconductor detectors Ge:Cu, Ge:Hg or
Ge:Zn; the thermistors or pyroelectric triglycine-sulfate (TGS); or
BaSr-Tio.sub.4 based detectors. The processing of the signals obtained
from detector 25 may be carried out in a known manner so that the
potential U.sub.a (t) obtained at the output of the detector is split by
two selective amplifiers in synchronism with the pertinent chopper
frequencies f.sub.m and f.sub.r into the respective proportional
potentials U.sub.m and U.sub.r corresponding to the light outputs P.sub.m
of the reference beam and P.sub.r of the measuring beam.
A differential amplifier then forms .DELTA.U=U.sub.r -U.sub.m. If the
conditions in the reference and measuring channels are the same, .DELTA.U
must be zero. Prior to each measurement, the control unit sets the null
point by automatically balancing the differential amplifier. The
difference in potential .DELTA.U occuring during the measurement procedure
is proportional to the difference in light power caused by the absorption
of the measured medium,
.DELTA.P=P.sub.r =P.sub.m.
Following the normalization
.DELTA.U/U.sub.r .about..DELTA.P/P.sub.r,
a signal will be available which is proportional to the absorption constant
.chi. of the measured medium and hence to its concentration in the
solution, for instance blood.
In principle, any infrared laser may be used, but frequency-tunable lasers
are particularly advantageous. For the embodiment shown in FIG. 3, tests
were carried out in particular with a 2 watt CO.sub.2 laser and with a 5
watt model XB-5 by Apollo-Lasers, Inc. (USA). It was found in the course
of the measurements that measurement accuracy is highly affected by the
laser. In order to obtain high measurement accuracy, care must be paid to
using a laser of high stability regarding frequency, mode and power. The
aforesaid Apollo laser essentially meets these requirements.
The measurements carried out by means of the instrument described in FIG. 3
in principle involves deposition of the solution to be tested on one
surface of the ATR measuring prism, while a control solution lacking the
materials being tested--or in the case of blood, distilled water--was
deposited on the corresponding surface of the ATR reference prism.
The comparison between FIGS. 4 and 5 is merely intended to provide an
example of the wholly unexpected capabilities of the instrument of the
invention. The curves of FIGS. 4 and 5 are plotted on the same abscissa
scale, the abscissa being in wavenumbers. FIG. 4 shows two different
absorption curves, 30 and 31, recorded by means of one of the best
previously conventional infrared spectrometers. The first curve, 30, shows
the relative transmission of an aqueous solution containing 10% by volume
of ethanol as a function of a wavenumber. The second curve, 31, shows the
relative transmission of an aqueous solution containing 10% by weight of
glucose. Both curves evidence a marked peak of absorption between the
wavenumbers 1000 and 1050. The expert immediately sees that when measuring
an aqueous solution simultaneously containing 10% by volume of ethanol and
10% by weight of glucose, the two absorption peaks can no longer be
unambiguously distinguished, so that neither clear cut qualitative nor
flawless quantitative conclusions would be possible from a corresponding
absorption measurement.
FIG. 5 also shows two absorption curves, 40 and 41, in the same
representation. Curve 40 shows the absorption curve of an aqueous solution
containing 0.45% by volume of ethanol. Curve 41 shows an aqueous solution
containing 0.5% by weight of glucose. The measurement of the glucose
absorption curve unfortunately had to be terminated at a wavenumber
slightly over 1000 because of being carried out with a CO.sub.2 laser.
However, the two curves clearly show that even in the presence of
superposition of the curves, clearly separate evidence both of ethanol and
of glucose is possible.
When measuring metabolic products in the blood, tests were performed in
which the blood removed from the body was allowed to run over the
measurement surface of ATR prism 14 or be let to dry, and also in which
the ATR measurement prism 14 was made to lie with its boundary surface
against the patient's tongue. In every case quantitative measurements of
an accuracy of 5 mg% or 50 ppm. was obtained for metabolic products such
as glucose, cholesterol and uric acid. These values were verified by
corresponding measurements of conventional type. When using the aforesaid
Apollo laser of especially stable characteristics, a further very
significant increase in sensitivity was obtained, by means of which
concentrations of 1 mg% ppm could be ascertained.
The invention has been described in respect to measuring the amounts of
metabolic products in the blood. It is apparent that the invention may
also be used similarly to measure minute impurities in aqueous solutions.
This can also be applied to monitoring and controlling ecological
pollution, including industrial waste waters and for monitoring and
controlling industrial processes in general.
The invention may be embodied in other specific forms without departing
from the spirit or essential characteristics thereof. The present
embodiment is therefore to be considered in all respects as illustrative
and not restrictive, the scope of the invention being indicated by the
appended claims rather than by the foregoing description, and all changes
which come within the meaning and range of equivalency of the claims are
therefore intended to be embraced therein.
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