Note: Descriptions are shown in the official language in which they were submitted.
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AUTOMATIC MONOCHROMATOR-TESTING SYSTEM
BACKGROU~D OF THE INVENTION
The present invention relates generally to a
monochromator-testing system and method, and, more par-
ticularly, to a monochromator~testing system and method
Eor use in an automated chemistry-analyzing system for
analyzing blood or other body fluids.
The chemical analysis of blood or other body
fluids or serums is a vital part of medical diagnosis.
Testing fo~ various serum constituents, such as glucose
or heart enzyme~, for ~xample, or for some other medi-
cally significclnt Eactor, carl be performed in a manual
or automated process by adding specific amounts of
15 various reactive chemicals or reagents to a sample of
the serum in a specific sequence and under specified
conditions oE temperature and time. The light trans-
mittance value of the resulting test chemistry is then
measured, and this value can be used to determine the
amount of the particular constituent being measured in
the serum. ~he term "serum" is used to designate any
biological fluid.
More specifically, in analyzing a serum speci-
men, a sample of the specimen is typically placed in a
25 test tube or other appropriate container; and one or
more specific reagents are added, depending upon the
particular test to be performed. When the required
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chemical reactions have taken place, a sample of the
completed test chemistry is removed from the test tube;
and the ligh~-transmittance value of the test chemistry
is ascertained by using a spectrophotometer or the like.
This value can be used to calculate the optical density
of the chemistry; and from this, the concentration of
the constituent of interest in the serum can be ascer-
tained. Automatic systems for performing such analyses
are disclosed, for example, in IJ.S. Pztent No. 3,901,656
and Reissue No. 28,803.
Determination of the concentration in a serum
specimen of constituents of interest requires determina-
tion of the optical densities of the serum solutions
and the test chemistries. This determination is made
by passing light of a selected wavelength or wavelengths
through samples o the serum solutions and test chemis-
tries to measure their light-absorbance values at the
selected wavelength or wavelengths. The terms "optical
density" and "light absorbance" are used synonymously
to describe the efEect of the test solution Oll light
that passes through it. Light transmittallce may also
be used to describe serum solution testing. Correct
results re~uire accurate determinations oE the light-
absorbance values of the serum solutions and test chem-
istries. A variety of factors that can interfere withthe accuracy of these determinations. ~or example, the
presence of bubbles in the test solution can cause
measurement errors. Similarly, factors, such as
turbidity or settling, can also interfere with the
accuracy of the measurements.
Moreover, a number of constituents normally
present in varying amounts in the serum itself may
introduce significant errors into the measurements.
These endogenous transferring substances include
35 bilirubin, hemoglobin, lipids, and the likeO Such
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substances absorb light at various specific wavelengths;
and when the maximal absorbances of the interfering
substances are at wavelengths close to those wavelengths
at which the test chemistries are to be measured, the
optical measurements will be severly affected unless
correc-ted or compensated for in some way.
Techniques have been developed to reduce the
effect of these interferences with the accuracy of the
measurements. For example, where the effect of the
interference on the optical density of a solution is
known to be significant in one range of wavelengths and
nominal in others, several measurements of the same
test solution may be taken at different wavelengths.
One or more wavelengths may be selected to provlde a
measure of the significant determination of an inter-
fering factor without a significant contribution from
the factor being measured by the test chemistry~ Other
wavelengths may be selected to provide a significant
determination of the factor being measured by the test
chemistry with relatively nominal interference. From
such data, the contribution of the interference may be
determined and compensated for in analyzing the test
results.
Such techniques have generally been limited
~5 to manual laboratory procedures practiced by trained
technicians, although some automatic serum chemistry-
analyzing machines have the limited capability of
practicing such techniques with a few selected wave-
lengths. In such systems, this has been accomplished
by providing a plurality of filter elements and selec~
tively inserting them into the light path. Such systems
are limited to only a few light wavelengths and provide
little flexibility.
Where the system is capable of generating
data at only a few wavelengths within the milliseconds
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that represent substantially the same time for data comparison
and use, the few data points, while providing a measure of the
characteristics of a serum solution and test chemistry at the wave-
lengths of data points, provide no reliable information on the
light-absorbance characteristics of serum solution and test
chemistry between the data points. Isolated data points cannot
be used to reliably establish whether the light absorbance is
increasing or decreasing with varying wavelength adjacent the data
points and, of course, can provide no information on the rate of
change of any such variation. Three data points, for example, may
appear to lie on a straight llne that represents constant light
absorbance as a Eunction of wavelength when the light absorbance
actually varies substantially and significantly between the data
points. Determination and use o;E reliable inEormation on such
variations we~e not possible in automatic serum chemistry testing
systems.
Such systems have also used an intense polychromatic
("white") light passing through the test solution. Such intense
polychromatic li~ht includes wavelengths that can efEect changes
in the serum constituents, the reagents, and the test chemistry.
UMMARY OF THE INVENTION
In accordance with the present invention, there is pro-
vided in an apparatus for determining the concentration of a sub-
stance of interest in each of a plurality of test solutions,
comprising: means for supporting a plurality of test solutions
to be examined; means for passing light through said plurality of
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test solutions; means for detecting said light after being passed
through said plurality of test solutions and for generating signals
representative of the light-transmittance value of each of said
plurality of test solutions; and means for analyzing said signals
for providing a measure of the concentration of the substanee of
interest in each of said plurality of test solutions, the improve-
ment comprising wherein said means for passinq light through said
plurality of test solutions includes means for generating a
plurality of beams of substantially monochromatic light of different
wavelengths from a substantially continuous range of available
wavelengths, and means for directing said plurality of substantially
monoehromatic li~ht beams of difEerent wavelengths to different
ones of said plurality oE test solutions, said beam-directing means
eompris.ing a plurality oE opt:ical pathways or direeting said
plurality oE substantia.lly monochromatie l:ight beams to said
plural.ity of te~st solut:iorls, ancl optieal multiplexer means for
direeting eaeh of said plurali-ty of substantially monochromatic
light beams of difEerent wavelength to a selected one oE said
plurality oE optieal pathways for direeting eaeh of the sub-
stantially monochromatic light beams of different wavelengths toa selected one of said plurality of test solutions.
In accordance with another aspect of the invention,
there is provided an automatic method of testing a serum sample
which includes the steps of directing a substantially monochromatic
beam of light of selected wavelength to a test chemistry of the
serum sample being tested, and detecting the intensity of the
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substantially monochromatic beam of light passing through the
test chemistry of the serum sample to provide data on the light-
absorbance characteri.stics oE the serum sample, the method further
including the steps of storing first data on the light-absorbance
characteristic of the serum sample as a function of wavelength
at a multiplicity of selected substantially monochromatic wave-
lengths; taking the mathematical first derivative of the light-
absorbance characteristic of the serum sample defined by the first
data and storing second data on the mathematical first derivative
of the first data; taking the mathematical second derivative of
the light-absorbance characteristic of the serum sample defined
by the first data and storing third data on the mathematical
second derivative oE the E:irst data; and compariny the first,
second, and third data wlth stored data on the light-absorbance
characte:risti.cs o.E known interEerences as a function oE wavelength
and on the first and second derivatives of such known interferences
Eor analyzing said test chemistry :Eor interEerences.
In accordance with another aspec-t oE the invention,
there is provided an apparatus Eor analyzing test chemistries,
comprising means for rapidly generating monochromatic light of
differing wavelengths over a wide spectrum for analyzing test
chemistries, means for directing the monochromatic light of
differing wavelengths to an intensity monitor, wavelength cali-
bration means and a plurality of test detectors for generating test
data on tests taking place in the plurality of test detectors, a
test controller for analyzing the test data and a process controller
for controlling the process of operation of said apparatus, said
process controller and said test controller including means for
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identifying and storing test data points from sald plurality of
test detectors at a multiplicity oE differing wavelengths, and
for identifying and storing the wavelength of the monochromatic
light for each of the multiplicity of test data points, said
generating means, said directing means, said plurality of test
detectors, said intensity monitor, said process controller, and
said test controller all responding in real time for generating
such a multiplicity of test data that said test controller can
develop reliable mathematical derivatives of the test data as a
function of wavelength over the entire spectrum.
In accordance with another aspect o:E the invention,
there is prov.idcd an automatic apparatus :Eor determining the
concentration o.E a substance oE interest in a test solution com-
prising means .Eor support.ing a plural:ity o:E test solutions to be
examined; means :Eox passing monochromatic light through said
plurality oE test solutions; means Eor detecting said monochromatic
light a:Eter be:ing passed through said plurality of test solutions
and for generating data representative of the light-transmittance
value of said plurality of test solutions; and means for analyzing
said data for providing a measure of the concentration in said
plurality of test solutions of said substance of interest, said
means for passing monochromatic light through said plurality of
test solutions including means for rapidly generating a plurality
of beams of monochromatic light of different selected wavelengths,
each of said beams of monochromatic light being of substantially
any desired wavelength within a substantially continuous range of
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available wavelengths, and means for substantially simultaneously
directing said plurality of monochromatic beams of light of
different wavelengths to different ones of said plurality of -test
solutions.
In accordance with another aspect of -the invention,
there is provided an automatic apparatus for determining the
concentration of a substance of interest in a test solution com-
prising: means for supporting a plurality of test solu-tions -to be
examined; means for passing monochromatic light through said
plurality of test solutions; means for detecting said monochromatic
light after being passed through said plurality of test solutions
and for generating data representative of the light-transmittance
value of said plurality of test solutions; and means for analyzing
said data for providing a measure of the concentration in said
plurality of test solutions of said substance of interest, wherein
said analyzing means includes: means for storing data on the
light-absorbance characteristic of the test solution as a function
of wavelength at a multiplicity of selected substantially mono-
chromatic wavelengths; means for taking the mathematical first
derivative of the light-absorbance characteristic defined by the
data and storing data on the mathematical first derivative of the
data; means for taking the mathematical second derivative of the
light-absorbance characteristic defined by the data and storing
data on the mathematical second derivative of the data; and means
for comparing the characteristic defined by the data and the first
and second derivatives of the characteristic with stored data
corresponding to known interferences.
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~ he present invention provides an automated,
chemistry-analyzing apparatus in which a controlled intensity,
monochromatic light of substantially any desired wavelength can
be selectively directed through any one of a plurality of test
solutions in a spectrophotometer. A virtually unlimited
variety of t~sts can be performed on the test solution. The
test chemistries can be scanned with substantially mono-
chromatic light from a wide range of wavelengths in a very
short period
j,, .
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of time to provide data at a multiplicity of wavelengths.
With such data, the serum characteristic as a function
of wavelength and its derivatives can be reliably
determined and used to correct test data for interfer-
ences and to identify invalid tests. In referring tosuch a multiplicity of wavelengths, I mean more than
about 10 to about 20 wavelengths and as many as 250
wavelengths in a number of tests.
A presently preferred embodiment includes
10 means for providing substantially monochromatic light
of substantially constant intensity having any selected
wavelength from about 300 nanometers to about 800 nano-
meters. The optical system of such means comprises a
monochromator which includes a light source generating
a beam of polychromatic light and a diffraction grating
to produce substantially monochromatic light of
different wavelengths. Substantially monochromatic
light means light having a very narrow range of wave-
len~ths, t~pically 3-~ nanometers. The grating is
rotatable; and by rota~.incJ the grating, a beam of sub-
stantially monochromatic light o~ any selected wave-
length may be generated and directed to a test solution
to be evaluated.
The means for providing substantially mono-
c~lromatic light also comprises intensity-controlling
and calibratiny means. The intensity-controlling means
insures that the monochromatic light beams will be of
substantially constant intensity at all real times and
at all wavelengths. Such means comprises apparatus for
30 mon.itoring the light intensity at each wavelength, and
a variable aperture optical system that is adjusted in
real-time to control the intensity of the polychromatic
light directed to the diffraction grating and, hence,
the intensity of the selected monochromatic light
separated therefrom.
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The calibrating means insures that the se-
lected wavelength of light will be produced when it is
desired. The preferred calibration means includes a
holmium oxide filter through which monochromatic light
of each available wavelength and constant intensity is
sequentially passed. By producing a series of signals
that represent the absorbance of the filter at each
wavelength, and by comparing this series of signals
with the known absorbance characteristics of the holmium
oxide filter, the grating position can be correlated
with the wavelengths that it produces; and a reliable
selection of monochromatic light is possible.
Fiber-optical pathways are provided in the
system Eor directing the light beam from the mono-
chromator to each of a plurality of flow cells contain-
ing test solutions to be evaluated. The fiber-optical
pathways permit the direction oE substantially mono-
chromat:ic light to a plurality of flow cells at one
time. ~y addition O:e an optical "multiplexer", as part
o~ the optical interconIlect system, substantially mono-
chromatic light oE different selected wavelencJths can
be directed to di~erent flow cells at any time to per-
mit measurements to be taken. The light absorbance of
the test solutions is measured, preferably with the
25 photomultiplier tubes at each flow cell.
The system includes multiple microprocessors
to control the methods of system operation and testing.
The control processor provides control over the mechani-
cal componen-ts of the system and adjustment of the sys-
tem electronics. The control processor providesperiodic calibration of the monochromator means and
test chemistry light measurement means. The control
processor also provides a program for installation of
the system and for periodic component checking and align-
35 ment. The control processor stores the characteristics
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of important replaceable system components and permitsthe system to be realigned in the event the components
must be replacedO
Testing methods are controlled by a separate
microprocessor which can store the methods of 30 test
chemistries. The testing methods of selected test
chemistries are transferred to the control processor,
which runs the system to perform the tests. The multi-
plicity of resulting data from the tests is transferred
to the testing microprocessor for analysis and deter-
mination of serum characteristics as a function of wave-
length.
The system of the present invention operates
at very high speed, permitting serum test solutions to
be scanned with a multiplicity of wavelengths of li-~ht
to provide extensive data on the characteristics of the
serum. The invention also provid~s substantial flexi-
bility and permits a wide variety oE t~sts to be more
reliably p~rformed. Further details and advantages of
the inverltion will hecome apparent hereinafter in con-
junction wlth th~ detailed d~scription oE the preferred
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates an overall bloc]c diagram
of a preferred embodiment of the invention;
FIG. 2 schematically illustrates the non-
electronic portion of the system of FIG. l; and
FIG. 3 illustrates hypothetical test results
30 and a serum function and its derivatives.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
FIG. 1 illustrates an overall block diagram
of a preferred system of this invention. The system
includes a means 10 to provide substantially
monochromatic light of substantially constant intensity
at any selected wavelength from about 300 nanometers to
about 800 nanometers. Means 10 includes a monosource
11, an intensity monitor 12, a wavelength calibrator
13, and control processor 14 of the system electronics.
Means 10 generates a substantially monochromatic beam
of light at essentially any selected wavelength. This
monochromatic light beam is directed via fiber-optical
pathway 15 to an optical interconnect system 16, which
directs the light beam to one or more detectors, for
example, 17, 18, 19 20, via fiber-optical pathways, for
example, 21, 22, 23, 2A. The test detectors are capable
of presenting a particular test chemistry or serum solu-
tion to be analyzed in the substantially monochromatic
light. In particular, as will be described later in
greater detail, the system is designed to permit up to
four di~erent test solutions to be analyzed simulta-
neously i.n four separate spectrophotometers; and the
optical-interconnect system 16 can direct a beam o~
light o:~ a d:i~ferent selected wavelength to each of the
~our test solutions.
The system electron.ics of FIG. 1 includes, in
addition to the control processor 14 for generally con-
trollincJ the operation of the various components of the
optical-illumination system, electronics for analyzing
the detector outputs, for example, 26, 27, 2~, 29.
Microprocessor 25 may produce an output 51 indicating
the results of the test being conducted and may be
instructed by any conventional data input system or
30 other computer 52 upon the tests to be conducted. The
system electronics, through the control processor 14,
has the capability of controlling the operation of the
monosource 11 via lines 31 and 32, the optical inter-
connect 16 via line 33, and the test detectors 17-20
via lines 34, 35, 36, and 37. The control of these
components is described later in greater detail.
As shown in FIG. 1, the intensity monikor 12
is coupled to the monosource 11 via fiber-optical path-
way 38 and the wavelength calibrator 13 is coupled tothe optical interconnect 16 via fiber-optical pathway
39. The function of the intensity monitor 12 is to
monitor the intensity of the light from the monosource
and develop signals to maintain the monochromatic light
beam at a substantially constant intensity notwith-
standing its wavelength. The function of the wavelength
calibrator 13 is to calibrate the monosource which
generates the monochxomatic beam of light to insure
that the selected wavelength of light will be produced
when desired. The signal outputs of the intensity
monitor 12 and the wavelength calibrator 13 are trans-
mitted to process controller 14 by lines 40 and 41,
resp~ctively. The signals on lines ~0 and 41 are
developed into driving outputs to control the monosource
20 via lines 31 and 32.
FIG. 2 schematlcally illustrates the various
components of the pre~erred means to pxovide substan-
tially monochromatic light of substantially constant
intensity. As shown in FIG. 2, the monosource 11 is
comprised of a plurality of components including a
source 81 of polychromatic light, a variable aperture
system 82, an order filter 83, and a monochromatorl
generally designated by reference numeral 84. The poly-
chromatic light source 81 preferably comprises a Halogen
lamp o~ conventional type. Light from the ~lalo~en lamp
is receive~ by variable aperture system 82. The vari-
able aperture system includes a collimating lens system
86 to concentrate the light at the entrance slit 87 of
the monochromator 84 and a variable aperture 88. The
35 aperture structure includes a shutter system which is
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driven by a galvanometer movement 88a to maintain the
monochromatic beam o light at a constant intensity
notwithstanding its wavelength by varying the intensity
oE the polychromatic light beam directed at the mono-
chromator 84. After passing through the variable aper-
ture system 82, the polychromatic light beam then passes
through order-separation system 83. The system is con~
trolled by a rotary solenoid and comprises two order-
separation glasses. The rotary solenoid rotates one or
the other of the two glasses in the light path. The
operating range for one of the glasses is preferably
from about 300 to about 500 nanometers, while the other
glass has an operating range of from about 400 to about
800 nanometers. There is some overlap of the bandwidth
due to the test chemist:ries. Either one glass or the
other glass is in the light path, allowing either 300
to 500 nanometer light or 400 to 800 nanometer light to
pass through. Selection oE the glasses is handled auto-
matically ~Ipon identi~icat.ion of the test to be
conducted.
~ Eter passing through the oxder-separation
glasses, the light entexs the monochromator 84 which
includes an entrance slit 87, an exit slit 89, and a
diffraction grating 91. The grating 91 is mounted on
and rotated by a galvanometer movement 9la. The gratiny
is used to separate different wavelengths of light from
the light entering the entrance slit 87. When incident
light strikes the grating, different wavelengths will
be reflecte~ from the grating at different angles (the
angle is determined by the wavelength). By rotating
the grating 91, substantially monochromatic light of
different wavelengths within the spectrum of the poly-
chromatic light bearm can be directed to the exit slit
89 of the monochromator. The galvanometer 91a, which
is like a meter movement but more massive, can rotate
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the grating 91 through a range of angles and scan the
spectrum of the polychromatic light acro~s the exit
slit 89 of the monochromator, producing a multiplicity
of substantially monochromatic light beams. The grating
is enclosed ancl sealed in a housing to prevent the entry
of dust which can destroy its background characteristics
(i.e., light is scattered by striking dust particles,
thereby increasing background radiation which affects
the linearity of the measurement system). It is impera-
tive to have as little background radiation as possible,particularly because of the higher absorbance readings
that may be accommodated with this system. The grating
itself is capable of separating light into wavelengths
of about three nanometers; and the term substantially
15 monochromatic, as used throughout this application,
therefore, is intended to comprise a light having a
spectrum widt.h o:E approxlmately three nanometers.
Thu~" with the system of this invention, it
is possible to qene.rate substantially monochromatic
ligllt o.E substantially any wavelength within the range
of from about 300 to about 800 nanometers as passed by
the order-separation glasses. From the exit slit 89 of
the monochromator 8~ (i.e., the output) of the mono-
source 11, the light impinges upon a bifurcated optical
25 fiber bundle. Approximately, 20 percent of the light
is directed to intensity monitor 12 via fiber-optical
pathway 38 where it is used to monitor the intensity of
the monochromatic light. About 80 percent of the light
goes to optical interconnect 16 via fiber-optical path-
30 way 15. The reason for the 20/80 split is to providesubstantially equal light to the test detectors and
wavelength calibrator notwithstanding any loss of light
in the light-directing system. The test detectors 17-20
use, preferably, photomultiplier tubes to determine
35 light absorbance of the test solutions, and it is
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advisable that the light intensity be substantially
constant at each photomultiplier tube to obtain similar
gain and response time.
As mentioned above, the intensity monitor 12
monitors the intensity of the monochromatic light pass-
ing through optical fiber 38 to maintain substantially
constant intensity monochromatic light for transmission
to the test detectors 17-20 and the wavelength cali-
brator 13. Intensity control of the monochromatic light
emanating from the monosource 11 is effected by con-
trolling in real time the variable aperture 88 in the
path of the polychromatic light leaving the light source
81. Basically, the monochromatic light passes through
fiber-optical pathway 38 and is sensed by photomulti-
plier tube 102. The photomultiplier tube 102 generatesa signal which is a function of the light intensity.
This intensity signal is delivered on line 40 to the
process controller 14. The process controller 14 then
develops a drivi.n~ siynal which is delivered over l.ine
31 to the galvanometer movemcnt 88a which operates the
shutter mechanism of the variable aperture means 88.
The variable aperture means 88 there~y adjusts the
intensity of the polychromatic li.ght beam in real time
to maintain a constant intensity monochromatic light
25 output at all wavelengths within the 300 to 800 nano-
meter spectrum of the polychromatic light.
The optical interconnect 16 may be a direct
optical coupling of 80 percent of the substantially
monochromatic light to the four fiber-optic pathways
30 21-24 in a manner known in the art. Another embodiment
of an optical interconnect, shown schematically in
FIG. 2, includes an "optical multiplexer".
The optical interconnect multiplexer of FIG. 2
includes a lens assembly 92 connected to the input fiber
15 from the monosource, a mirror 93 mounted to a stepper
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motor 94, four optical fiber transmitters 21-24 going
out to the test detectors 17-20, and one optical fiber
transmitter 39 going to the wavelength calibrator 13.
The function of the mirror 93 is to direct the light
beam from the monochromator 84 to each of the four out-
put optical Eibers 21-24 and to the optical fiber 39
Alignment of the optical interconnect mirror is per-
formed by system-generated software. When the optical
interconnect system 16 is first initialized, the stepper
motor steps to the "home" position at the optical fiber
39. "~Iome" position is indicated generally by a refer-
ence sensor on the sensor disk, which is actually about
two to three motor steps wide so that it is easy to
find. Alignment is completed by moving in half steps
lS to locate the maximum light intensity on the optical
fiber 39. Once the mirror is aligned w:ith "home" posi-
tion, the process controller calculates the number of
steps to each of the other optical fibers. Actual motor
aligIlment iB accomplished automatically every time the
system is i.nitialized.
The wavelength calibrator 13 insures that the
desired wave:Length of light will be passed through the
exit slit 89 when called for. A holmium oxide calibra-
tion filter 103 is used to calibrate the monochromator
84. Ilolmium oxide glass has a very complicated light
absorbance spectrum with a number of absorbance peaks
and valleys at known wavelengths. The entire spectrum
of polychromatic light can be "fingerprinted" using
this holmium oxide filter. The calibration filter 103
is mounted on a solenoid 104, which, when activated,
puts filter 103 in the path of the light from optical
fiber 39 when the monochromator 84 is to be calibrated.
In calibrating the monochromator 84, the calibrating
~ilter is placed between the optical fiber 39 and photo-
35 multiplier tube 105 of the wavelength calibrator 13.
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The grating is then rotated by the galvanometer movement
91a to scan the full spectrum of constant intensity
monochromatic light (i.e., from about 300 nanometer
wavelength thxough about 800 nanometer wavelength) at
the output 89 of the monochromator 84. The photomulti-
plier tube 105 generates a signal from each wavelength
that represents the absorbance of the holmium oxide
filter at that wavelength. The set of signals are
transmitted to the process controller 14 over line 41.
The process controller, pursuant to the calibration
program, compares this signal data with stored data on
the light-absorbance spectrum of the holmium oxide fil
ter 103 to correlate the wavelength of the monochromatic
light with the rotational position of the grating 91 of
the monochromator 84. The rotational positions of the
grating 91 are then stored for a multiplicity o wave-
lengths in the 300 to 800 nanometer spectrum. In opera-
tion, Eor example, if light at a wavelength of 340
nanometers is requested, the process controller 14 can
determine ~rom the stoxed calibration data the position
to which yrating 3~ must be driven so that light of
that wavelength will leave exit slit 89. The mono-
chromator 84 can be calibrated with a single pass
through the spectrum. After the calibration is com-
pleted, another pass is generally made based on thecalibration to verify its accuracy by matching the
original table. This calibration procedure is part of
an alignment protocol for the system every time the
instrument is powered up and before every test, once
30 every 7.2 seconds.
In testing serum samples and test chemistries,
monochromatic light of selected wavelength is directed
by the optical interconnect 16 on one or more of the
desired fiber-optical pathways 21-24 and is directed to
35 one or more test detectors 17-20 which comprise
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essentially spectrophotometers. ~lore specifically, the
test detector 17 includes a housing 96 which contains a
flow cell 97, an input optical fiber 98, and an output
optical fiber 99. The light from the output optical
fiber 99 is received by a photomultiplier tube 101 which
is supported within a housing with the photomultiplier
tubes of the other test detectors and the photomulti-
plier tubes for the wavelength calibrator and the
intensity monitor. In FIG. 2, however, the photomulti~
plier tubes are separated for the purpose of clarity.
The flow cell 97 is designed to be customer replaceable.
Specifically, each flow cell has a number etched on its
side, which number is a calculation of the actual light
path length through the flow cell. This number is
entered into the process controller 1~ of the system
electronics. The path length of the flow cell is stored
because a correction is made for the dif~erent path
lengths of each photocell. With this information stored,
the raw ~ata that comes out of the measurement system
is automatically corrected to minimize error. The cor-
rection calculations are done in a mathematical process
within the process controller 1~. The path length of
the flow cell is defined by in-house testing. Specifi-
cally, a standard flow cell, kept in-house, is used as
a reference; and all other flow cells are compared with
the standard flow cell to provide ~or the system a light
path length for every photocell based on the reference
number placed on it.
The photomultiplier tubes or PMTs used in
this system are small and have a very fast response
time (faster than a silicone detector) in the order of
nanoseconds due to their very low internal capacitance.
Although the sensitivity of the photomultiplier tubes
will deteriorate with time, the deterioration can be
compensated for through appropriate software by
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adjusting the high voltage to maintain a proper gain.
This procedure will prolong the life, which can be a
couple of years with this compensation, of the photo-
multiplier tubes . ~ shutter is preferably provided in
the front of each PMT to protect -the PMT from direct
ambient light if an optical fiber is disconnected while
there is power to the PMT and also to provide dark cur-
rent testing. Since the flow cells are replaceable,
when an optical fiber is pulled from one of the housings
and a high voltage (typically 650 volts) is applied to
the PMT, ambient light conditions or any other light
source can be high enough to overload the PMT, degrading
them or severely burning the plates out, making the PMT
inoperable.
Dark current is also a common phenomenon of a
PMT. Dark current drifts ~uite a bit; even ambient
temperature is enough to cause drifting. Because of
the need to use logarithmic amplifiers, even a small
change in dark current can produce a significantly
erroneous output signal. In order to compensate for
this phenome~non, a test i~ performed to determine the
dark current of the photomultiplier tuhes every 1.5
seconds in order to recorrect or re~ero the logarithmic
amplifiers so drift error is eliminated.
The shutters are solenoi.d controlled and
mounted on top of the P~T housing. A set of comparators
in the measurement system monitors the preamplifiers
for the photomultiplier tubes. If the output of any
one of the preamplifiers exceeds about 10.5 volts, which
30 is the limiting factor, it will automatically trigger
the comparator and activate the shutter to close. This
protective action is accomplished by software within
process controller 14. Upon "power down", the shutters
are in the normally closed position.
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The process controller 14, which controls the
operation of the components of the monosource, the opti-
cal interconnect, the intensity monitor, the wavelength
calibrator, and the test detectors, is, for example, an
~,, 5 Intel 802~ microprocessor which has an 8085A-~ central
processing unit. In addition to the process controller
14, the system electronics to control the optical com-
ponents includes an analog processor unit (APU) with a
high-voltage supply, a data acquisition unit (DAU), two
general control units (GCU), and a fluorometer control
unit (FCU) and associated high voltage supply. These
four units are all software controllable.
The analog processor unit (APU) is an analog
system that takes the inputs from the test and inten-
sity-monitoring photomultiplier tubes, transforms the
signals to current, and amplifies them in a three-stage
amplifier. The test analog signals are manipulated
throuyh software by programmable gain and oEfset cir-
cuitry. The test ancl intensity-monitoring signals per-
form two different Eunctions. The signals go from thephotomultiplier tube module -to the daka acquisition
units. The signals are also multiplexed, amplified,
and fed to a difference amplifier which then transfers
the resulting signal to the data acquisition unit. The
signals are also fed into two comparator circuits which
determine if the shutters of the PMT tubes need to be
closed because of exposure to an overload of bright
light (ambient or otherwise).
The data acquisition unit (DAU) contains a
16-channel multiplexer which is used to take the analog
signals from the analog processor unit (Test, Intensity
Monitoring, Difference signals and analog ground), and
other inputs for system diagnosis. These signals are
multiplexed together and sent through an anti-aliasing
35 filter, which filters out other noise and quickly
ark -17-
~11 2~
-18-
throughputs the analog data, onto a 16-bit A/D Converter.
The signals are then presen-ted to the data buss for
software interpretation. The two shutter control
signals from the analog processing unit are carried
through to the data buss. A thermistor input is used
-Eor flow cell temperature. There are also four expan-
sion or auxiliary inputs to the multiplexer. Two of
the channels are voltage inputs from Ion Specific
Electrodes. When the system tests a solution using the
Ion Specific Electrodes, it is programmed to read those
devices in order to obtain their output values. The
other two channels are for future expansion.
The measurement system has the capability of
reading percent Transm:ittance (%T) as well as logarith-
15 mic output or Absorbance (ABS). These two referencesreside within the data acquisition unit. There is
another internal reference within the data acquisition
unit which .is used to chec]c the scaling of the ~/D
Converter to see if it is accurate. The A/D Converter
has ban]c~d in ~15 volts or -15 volts to guarantee that
all the bits to the A/D Converter operate. The -~5 volts
line and the absolute values of each of the other con-
nected power supplies are checked. The voltage ranges
of each power supply are stored in the process control-
ler l~, and a faulty power supply can be detected bymeasuring power supply voltage through the A/D Converter.
The general control units (GCUs) contain the
current drivers for the scanner (grating 91 on the
galvanometer 91a) and the variable aperture (the shutter
30 on the galvanometer). The general control units also
contain the drivers that control the stepper motor for
the mirror on the multiplexer (optical interconnect)
and, in one of the GCUs, the drivers for the order-
switching, calibration glass and shutter solenoids.
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"~_~ 7t~
--19--
There are also sensor inputs; two are from the sampler
stepper motor sensor disk.
The fluorometer control unit (FCU) consists
of a unit for controlling the filter wheel, shutter
solenoid for the Test photomultiplier tube, lamp power
supply and CW light source for the fluorometer. The
ECU has a set of discriminator photon counters that it
uses for the photon counting mode, and other supporting
fluorometer electronics.
The analog processor unit, data acquisition
unit, general control UIlitS, and fluorometer control
unit are interfaced to the control processor and testing
processor via two interfacing boards; the Standard
Transceiver Board and the ISBX Transceiver Board. These
two interEclce boards permit communication between the
two types of buses (STD and ISBX).
q`he testing processor is an Intel 8086 16~bit
microproce~sor which has an 8086 central processing
unit and 80a6 and 80~7 math coprocessor boards. The
20 math coprocessor provid~s all o~ the data reduction,
data correction, and correctiorls for path length varia-
tion and alinearity on the log ampliEiers, etc. The
system generates a multiplicity of data that needs to
be analyze~ and precondensed before it can be used.
Each chemistry test has a set of software parameter
blocks, sequence control blocks, data analysis blocks,
etc. This information is used to tell the control pro-
cessor what type of math is to be used, ratios to be
calculated, output data format, and so forth. Since
30 this data is frequently more than what can be trans-
mittedl it must be precondensed in the 8086 micropro-
cessor before it is transmitted. The A/D Converter in
the data acquisition unit is capable of making one con-
version every 15 microseconds. Since the system can
35 run spectrum scans as fast as 100 milliseconds per scan,
the front ends and all of the analog circuitry of the
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system must be fast. Because of this fast operating
speed, certain types of noise cannot be filtered out
using hardware, but must be filtered through software
using digital-filtering techniques within the 8086
microprocessor.
The system is set up by downloading, to the
8086 central processiny unit, everything it needs to
run the chemistry tests. As many as 30 such test
chemistry processes can be stored by the testing pro-
cessor 25. The testing processor 25 then provides thecontrol processor 14 a sequence control block that
describes the procedure of a test chemistry to be per~
formed. With this control block, the appropriate wave-
length is selected; and a multiplicity of readings is
taken. This data then goes through digital processing
in the 8086 micxoprocessor. Thus, in a sense, the
testing processor 25 te:Lls the control processor 1~
what tests it wants to run. 'rhe control processor con-
trols all the mechanical operakions, collects and then
transfers the data to the 8086 mernory. The testiny
processor 25 is then informed that a particular flow
cell test i9 finished. The testing processor 25 then
provides the information needed for testing the next
flow cell. While the testing is being conducted, the
testing processor 25 operates on the data from the
earlier tests; and the data is reduced for use.
By downloading the information needed to run
any of 30 chemistries, the only system inputs needed
are the identification of the tests to be conducted.
30 The system automatically knows what the output format
should be. The control processor has all of the align-
ment tables (i.e., what grating positions to use, what
light source intensities to use, what gain ranges to
use, etc.) that are needed to run the selected chemistry.
No manual adjustments are needed in the
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-21-
electronics system. The electronic units are processor
controlled. Each time the system is turned on, and
anytime it is operated, the system will go through its
alignment process and flag anything that it finds mis-
aligned or that it cannot align correctly. Thus, ifthe CRT screen indicates a detected defective data
acquisition unit, the user can pull the defective DAU
out of the chassis, insert a spare one, and re-initiate
the alignment process to automatically align the new
DAU. The self-diagnosis of the system can be as spe-
cific as detecting a defective log amp, preamp~ or a
photomultiplier tube that has an improper response.
However, there are some areas where human judgment may
have to be made. For example, the CRT screen may indi-
cate a bad test photomultiplier tube; but since the PMTis part of the preamp circuitry of the analog processing
unit, the defect could be the preamp itse]f. A new PMT
tube can be installed to see if the same error message
continues on the CR~' screen (process of elimination).
If not, the deEect is In the preamp circuitry.
With the system, a test can be completed in
about 7 seconds on a sincJle index and in about 14
seconds on a double index. Each of the four flow cells
can be read in 200 ms for each pass. During that 200 ms,
25 any wavelen~th or any combination of wavelengths can be
selected; multiple scans (reads) can be taken; and as
many as 3000 data points per scan can be recorded.
In one test, each flow cell can be scanned
five times, yielding 15,000 data points. On a double
30 index test, each flow cell is scanned ten times, yield-
ing 30,000 data points. All of this data is condensed
in the 8086/8087 testing processor. Depending upon the
chemistry, all of the data can be averaged; or a digital
filter of all of the data can be conducted first with a
35 subsequent averaging. Also, 250 different wavelengths
-21-
-22-
can be used in 200 ms per flow cell if needed ~i.e.,
run a full spectrum at one time). This is very advan-
tageous when running a kinetic test, such as seeking
how much of the reaction is settling and turbidity, and
how much is the actual reaction itself. The system can
even determine how much enzyme is turned over into
another product. To obtain correct data, the system
has the capability to detect bubbles by inspecting the
spectrum to determine if there is a bubble present in
the flow cell.
Such a multiplicity of test data permits the
use of mathematical numerical analysis techniques on
the data to determine test-solution, light-absorbance
characteristics as a function of wavelength. FIG. 3a
indicates a hypothetical test chemistry characteristic
as a function of wave]ength. Such a comple~ function
can be reliably cletermined with a multiplicity of data
points while a few data points will provide little
reliable inEormation on the variations oE light absor-
bance as a Eunction of wavelength or the rates of change.By furtll~r numerical analysis of the test data, the
first and second derivatives of the light absorbance
characteristic can be derived.
The testing processor can, for example,
determine the first and second derivatives of the serum
light absorbance as a function of wavelength. FIGS. 3b
and 3c indicate, for example, such derived first and
second derivatives of the FIG. 3a test solution charac-
teristic. Analysis of the test chemistry for interfer-
30 ences and for faulty tests can permit purification oftest chemistry data by subtracting the influence of the
interference from the collected data. Eligher order
derivatives can also be developed and may be usable in
such analysis.
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23-
Information on the light absorbance of the
test solution as a function of wavelength and on the
derived first and second derivatives of the test solu-
tion data as a function of wavelength can be compared
with stored information on the light absorbance charac-
teristics of known interferences as a function of wave-
length and on the first and second derivatives of such
interference characteristics. The system can thus more
reliably check for and identify factors that may inter
fere with a reliable analysis of the test solution.
Upon the identification of an interfering
factor or factors and the level of its or their contri-
butions to the light-absorbance characteristics of the
test solution, the test data may be "~urified" by sub-
tracting from it a derived estimate of the contributionor contributions of an interference or interferences to
the test rcsults. In some tests, interferences may
invaliclate the ter,t results. This system may be used
to determine an in~alid test and inform the system user
The sys~em may also b~ used to in~orm the system user
o~ the ~re~e~nce~ and identity of interfering ~actors in
the t~st chemistries to permit the user to exercise his
judgment on the test results and their validity. The
stored data on the test solution light-absorbance char-
acteristics may, of course, be presented ~isually by aCRT or printer, as may the derivatives of the light-
absorbance characteristics of the test solutions.
In addition, the multiplicity of data taken
in sequential scans at known intervals of time can also
permit analysis of the kinetics of the test solution.
Such data permits an opportunity to identify interfaces
based on their different rates of change in real time.
Such analysis joined with analysis of first, second
~and possibly higher order) derivatives permits a power-
ful system in testing human serums.
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-24-
Where, as in prior automatic serum-testing
systems, only a few data points were determined, deter-
mination of the serum characteristic function and
recognition of interferences with the spectrophotometric
measurements of the test chemistry were limited in re-
liability and scope. The speed of operation and the
multiplicity of data points available with the system
of this invention permit substantially more reliable
test results and a ~reater variety of tests, among its
other advantages. The system and its apparatus is also
capable of use with new testing procedures and new serum
test chemistries as they may developed.
The preferred system described above may be
modified without departin~ from the scope of the inven~
tion as claimed.
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