Diode Array Spectrometer for the Simultaneous Determination of Hemoglobins in Whole Blood M. J. Milano” and Kwang-Yil Kim’ Department of Chemistry, State University of New York at Buffalo, Buffalo, N. Y. 14214
A solid state linear diode array spectrometer has been applied to the simultaneous determination of hemoglobins in whole blood. The use of signal averaging for low noise, derivative spectra to reduce systematic errors and least squares calculations to extract the maximum possible information from the spectra allows the analysis to be performed through fiber optic probes. The probes are inexpensive, easy to construct, and small enough to fit within a hypodermic syringe needle or catheter for in vivo monitoring. Relative amounts of oxyhemoglobin, carboxyhemoglobin, and reduced hemoglobin can be determined in 30 s with a precision of about 1YO.
Rapid scanning array spectrometers have several advantages over conventional mechanically scanned instruments which make their use in analytical systems attractive. The lack of moving parts gives increased ruggedness and excellent wavelength reproducibility. The multiplex advantage of integrating array detectors allows UV-visible spectra to be recorded in times as short as a few milliseconds or signal-averaged over longer times for improved signal-to-noise ratio. In addition, the electronic control of array detectors facilitates interfacing with computers. The operation, performance, and chemical applications of many different types of array detectors have been reviewed by Talmi (1, 2). The simplest to use and least expensive of these devices are the self-scanned linear diode arrays (LDA). Although possessing many of the features of more complicated array detectors, LDA’s have three main disadvantages: a limited number of resolution elements (about 1000 or less), low sensitivity, and a fixed sequential scanning pattern. The first two limitations are most serious in such applications as atomic emission, absorption, and fluorescence; molecular fluorescence; and Raman, where either sensitivity or resolution or both are of prime importance. The third disadvantage can be a factor in atomic emission, where a large part of the spectrum contains little or no chemical information. Although these factors do not necessarily prevent the use of LDA’s in the above fields (one of the earliest chemical applications was to atomic emission ( 3 ) ) there , are several other array detectors ( I ) which are better suited for these types of measurements. Such is not the case in molecular absorption where light levels are high and the absorption bandwidths of most species are relatively broad. Here speed, low noise, and wavelength reproducibility are of prime importance, and LDA’s can have excellent performance in these areas. The low capacitance of the surface and close proximity of the scanning circuits to the photodiodes allow scan times as little as 25.6 ps/spectrum to be achieved for a 256-element array. Yates and Kuwana have evaluated the LDA as a detector for time-resolved absorption spectroscopy ( 4 ) .LDA spectrometers have also been used in liquid chromatography (5,6),and appear to have much lower noise levels (6)than a mechanical rapid scanning LC detector 1 Present address, Chemical Engineering Division, Argonne National Laboratory, Argonne, Ill. 60439.
(7). In addition, the high inherent wavelength reproducibility, due to the fixed diode position and scanning pattern, has allowed the development of a novel technique for the deconvolution of overlapping chromatographic peaks (6).Through the generation of first derivative spectra, peaks can be eliminated or deconvoluted from chromatograms by plotting dA/dX at wavelengths corresponding to a solute’s first derivative zero crossing points. In this study the feasibility of coupling a diode array spectrometer with fiber optic probes for the in vivo determination of blood hemoglobins has been investigated. In order to take full advantage of the diode array’s strong points, a new fiber optic probe has been developed which allows absorption rather than reflectance measurements (8,9) to be made. The spectrometer has been interfaced with a minicomputer so that a variety of data processing techniques can be used to achieve maximum performance from the array detector and reduce errors in the simultaneous determination of oxyhemoglobin (HbOz), carboxyhemoglobin (HbCO), and reduced hemoglobin (HHb).
EXPERIMENTAL Instrumentation. The system layout is illustrated in the block diagram of Figure 1. The light source consists of a 90-watt tungsten halogen lamp (Norelco FCR) driven by a regulated dc power supply (Hewlett-Packard Model 6427B). Source radiation is forced on the input optical fiber of the probe. The output fiber is inserted through a Teflon plug to form the entrance slit of the polychromator. The fiber optic probes (Figure 2) are made from a single unclad fiber of methyl acrylate with a diameter of 0.25 mm and a length of 1m. The fiber is heated in boiling water, bent sharply a t its midpoint, and inserted into a 16-gauge hypodermic needle so as to form a loop a t the tip with a radius of approximately 1 mm. A small amount of epoxy glue is applied directly above the loop to hold the fiber, and the end of the loop is cut once with a razor blade to form a short path length (approximately 0.03 mm) cell into which the blood can flow. The polychromator was built in house and is of a Czerny-Turner design with a focal length of 125 mm. A 1200 l/mm grating (Edmund Scientific No. 41,037) disperses a spectrum covering 512 to 621 nm on the active area of the detector. The resolution of the system is limited to 2.2 nm by the diameter of the entrance fiber and corresponds to five diodes on the detector surface. A 256-element linear solid state array detector (Reticon RL 256ec.17) was used with scanning and signal amplifying circuits (RC 102) obtained from the array manufacturer. The only modification to these circuits consisted of replacement of the primary clock with a more stable square wave generator (Heathkit Model 1G-18) operated a t 95.0 kHz. The time required to scan one spectrum is 2.7 ms. The time between scans (integration time) can be varied from 2.7 to 40.4 ms. The output of the signal amplifying circuits is a staircase waveform, each step corresponding to the light intensity striking a single photodiode. The signal is passed through a 3-pole active filter (Frequency Devices Model 744PB-4) with a cut off frequency of 50 kHz to reduce high frequency switching noise. The clock pulses are delayed 5 ws and used to gate a sample and hold circuit within the computer interface, so that the signal is sampled in the middle of each step. The minicomputer (Data General Nova 11) has 16K of core memory, a general purpose 12-bit analog interface (ADAC Model 500-DGC8-DI-APG-A-DMA1, and three magnetic tape cassette drives for storage of programs and data. Programs for data processing were written in BASIC with assembly language subroutines for data acquisition and display. The data acquisition subroutine uses direct ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
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Flgure 1. Block dlagram of system layout
immersed in the blood sample and opened only during the actual recording of spectra.
RESULTS AND DISCUSSION
Figure 2. Flber optic probe
memory access and allows spectra to be signal averaged in real-time at rates up to 60 spectra/s. Least squares 9 point quadratidcubic algorithms (10) are used to both smooth absorbance spectra and generate first derivative (dA/dX) spectra. Samples. Blood samples were collected in “Vacutainer” tubes containing EDTA as an anticoagulant or prepared from outdated packed cells,obtained from the local Red Cross,by dilution with an equal volume of modified Alsever’s solution (2.05 g dextrose, 0.8 g sodium citrate, 0.05 g citric acid, and 0.45 g sodium chloride dissolved in 100 mL distilled water). Blood with 100% HbOp or HbCO was prepared by gentle bubbling for approximately 30 min with oxygen or carbon monoxide saturated with water vapor. Dithionite reduction was used to prepare 100% HHb. The blood sample was monitored with the array spectrometer and solid sodium dithionite added until no change in the absorption spectrum could be observed. Mixtures with known amounts of each component were prepared volumetrically. The total volume of the samples varied from 1.5 to 2.0 mL and they were analyzed at room temperature with slow stirring in a 4-mL test tube. Procedure. A t the beginning of each experiment a spectrum is recorded with the light beam blocked. This spectrum is subtracted from all subsequent spectra to correct for the dark current of the photodiodes and fixed pattern noise generated by the scanning circuits. A reference spectrum is then recorded with the probe immersed in distilled water. To minimize errors due to photodecompositionof the hemoglobins (11),the light beam is blocked when the probe is first 556
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The transmission efficiency (transmission with probe in distilled water X 100ltransmission of a straight uncut fiber) of the probes varies from about 10 to 18% because of losses associated with the sharp bend and cut of the fiber. Despite this low efficiency, the intensity of the transmitted radiation is sufficient to allow operation of the detector a t 70% of saturation with integration times of from 21.6 to 40.5 ms. The predominant source of random noise in this system is amplifier noise and is independent of signal amplitude. The overall spectral response of the system is relatively flat, varying less than 20% from 520 to 610 nm. The baseline RMS noise (standard deviation of points within a spectrum of distilled water) is, therefore, independent of wavelength and was found to be equal to f7 X lom4 A and f2 X dAldX for single absorbance and first derivative spectra respectively. The noise does follow the expected decrease up to about 100 averages for absorbance spectra and to 500 averages for the first derivative spectra, Figure 3. A value of 300 averages was chosen for the analysis as a compromise between speed (10-5 average data acquisition time) and low noise (f7X 10-6 A and fl X dA/dX). Figure 4 shows absorbance and first derivative spectra of whole blood samples containing 100% of each component. O’Haver and Green (12) have found that a result of using derivative spectra is to trade off systematic for random errors. Since systematic errors due to source drift, movement of the fibers, and scattering of light by suspended matter in the blood were expected to predominate in this system, the derivative spectra were used for the simultaneous analysis. Although none of these errors are completely wavelength independent, their effect on the derivative spectra should be relatively small. This can be seen in Figure 3. As the number of averages and time for data acquisition increases, source drift becomes more significant and causes the noise level in the absorbance spectra to level off. T h e derivative spectra, being less sensitive t o source drift, continue t o decrease in noise level u p to 10 000 averages. The relative concentration of each of the hemoglobin
0
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Figure 3. Nolse as a function of the number of signal averages (0)From absorbance spectra. (0) From flrst derivative spectra
Table I. Three-Component Simultaneous Analysis of Whole Blood Mixtures Sample No.
Expected, % HbCO
1 2 3 4 5 6 7 8 9 10 11 12 13
0 0 33.3 25.0 50.0 40.0 33.3 10.0 9.1 0 9.1 20 16.7
HbOz
Found, % HHb
66.7 33.6 33.3 50.0 25.0 40.0 33.3 80.0 9.1 90.0 81.8 70 75.0
33.3 66.7 33.3 25.0 25.0 20.0 33.3 10.0 81.8 10.0 9.1 10 8.3
HbCO
HbOz
HHb
0.9 -0.7 32.1 23.3 50.2 40.2 33.0 10.6 8.5 0.2 9.5 20.1 16.9
67.5 34.6 32.7 49.2 23.1 40.2 33.0 81.7 9.6 89.9 80.2 69.8 74.7
31.6 66.1 35.1 27.5 26.7 19.6 33.9 7.6 81.9 9.9 10.3 10.0 8.4
components in mixtures can be calculated from the derivative spectra either by the solution of simultaneous equations at three wavelengths, or by a least squares method using a large number of data points, n, in the spectrum. The least squares procedure assumes that the data spectrum can be approximated by a linear combination of standard spectra with equal concentrations of HbCO, HbOz, and HHb. Assuming constant path length this can be written in matrix notation as
A = aC
Figure 4. Blood hemoglobin spectra obtained through a fiber optic
probe (a) Absorbance. (b) First derivative
where A is an n by 1matrix containing the sample derivative spectrum, a is an n by 3 matrix containing the derivative spectra of the standards and C is a 3 by 1 matrix containing the concentration of each component in terms of the standards. Although in the presence of experimental errors in A and a it will not be possible to satisfy Equation 1,it is possible to obtain a matrix C which minimizes the sums of the squares ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
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Figure 5. Total hemoglobin and concentration of individual components as a function of hematocrit ( 0 )Total hemoglobin (ZCx). (0) % HbOz. (A)% HbCO. (0) YO HHb
of the deviations. It can be shown (13)that the least squares C matrix is given by
C = (ata)-’atA
(2)
where at represents the transpose of the a matrix. The primary advantage of the least squares procedure is that improved signal-to-noise ratios can be obtained compared to the solution of simultaneous equations a t three wavelengths. This results from the fact that the method effectively integrates the signal over the absorption band defined by the standard and is equivalent to the “matched window” technique described by Hirschfeld ( 1 4 ) .A second advantage is that estimates of the standard deviation of each concentration (SD C x ) can be calculated by
where p represents the number of components and e, represents the deviation of the data point at i from the least squares fit. Although these standard deviations cannot be used as a measure of the precision of the overall analysis, they do indicate the validity of the calculations and can be used to detect the presence of interfering components or whether the instrument is in need of wavelength calibration. A third advantage is that the least squares method eliminates the necessity of determining the optimum wavelengths for the calculations. I t can, therefore, be expanded easily by incorporation of the additional standard spectra in the a matrix. The two main disadvantages of the least squares method are increased computation time and the need for very precise wavelength registration between the data spectrum and the standards. Since the 3-component, 200-point least-squares fits used in this work took only 15s, the first disadvantage was not a problem. Although the lack of moving parts in the array spectrometer makes it very stable in the wavelength axis, 558
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wavelength registration is still a problem due to the difficulty in positioning the optical fiber a t the entrance of the polychromator. A horizontal displacement of only 0.05 mm results in a 0.4-nm shift in the wavelength axis of a recorded spectrum. To overcome this, a didymium derivative spectrum is recorded each time a new probe is used by holding a didymium filter in front of the input fiber. This spectrum is then compared by a least squares fit to a didymium spectrum recorded with the same probe as the hemoglobin standards, and shifted in software until the standard deviation of the fit is a minimum. The appropriate wavelength shift is then applied to all subsequent spectra recorded with that probe. Shifts corresponding to a fraction of a diode are done by interpolation. Since one resolution element covers five diodes, this interpolation does not degrade resolution. Since deviations in the cell path length for different probes can be quite large (as much as two times), the results from each simultaneous analysis are normalized (Cx/Z:;=,Cx) and reported as percent HbCO, HbOz, and HHb. The analytical results for several three-component mixtures are given in Table I. The deviations between expected and found values are within the approximate experimental error of preparing the samples. The analysis of 21 replicate samples gave standard deviations of f0.9?h, &0.9%, and fl.l%for HbCO, HbOz, and H H b respectively. Other studies have shown that light scattering by cells can cause deviations from Beer’s law (15). With this system, however, such deviations should be small because of the narrow path length (low probability for multiple scattering) and the use of derivative spectra. Standardization of a probe with a solution of known concentration may, therefore, allow the values of BCx to be used as a measure of total hemoglobin. Experiments to verify this have shown good correlation between BCx and hemoglobin concentrations for hemolyzed solutions (within 2%), but large random errors for unhemolyzed samples. Figure 5 shows a plot of total hemoglobin measured vs. the hematocrit value (volume of cells/total volume) of a series of unhemolyzed samples prepared from totally
0
20
10
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Figure 6. Kinetic experiments (0) % HbCO, HbOz
HbCO. ( W ) % HbOz, HbOz
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HbCO. (0)% HbCO, HHb
oxygenated packed cells. Although the linearity of the plot suggests that Beer's law is obeyed, the scatter of the points indicates that measurements of total hemoglobin with this system could at best be only semiquantitative. The most probable cause of the uncertainty is variations in blood flow rates in the vicinity of the probe tip (15). However, these random errors, and even possible systematic Beer's law deviations due to light scattering should be nonspecific and have little effect on the values obtained for the relative concentration of each component. The accompanying plots in Figure 5 confirm this, and show that relative concentrations can be measured to within a few percent accuracy even at low hematocrit values. To simulate the use of the system as an in vivo hemoglobin monitor, a probe was placed in whole blood containing 100% HbOz while carbon monoxide gas was passed over the sample. HbCO and HbOz were determined a t various time intervals using a single reference spectrum recorded a t the beginning of the experiment. The results of this and a similar experiment starting with 100% HHb, Figure 6, indicate that it should be possible to use the system for kinetic studies as well as fast single analyses. In conclusion, the use of a rapid scanning diode array spectrometer with miniature fiber optic probes shows promise for the in vivo analysis of hemoglobin derivatives. Since the total time, from immersion of the probe in blood to print out of the results, is only 30 s, it is conceivable that emergency determinations could be performed in less time that it takes to draw a sample for conventional analysis. Alternately, the system has accuracy and precision comparable to presently
50
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available in vivo oximeters ( 9 ) ,with the added capability to simultaneously determine HbCO and HHb. The possibility of monitoring additional hemoglobin derivatives, such as methemoglobin and sulfhemoglobin, is presently being investigated.
LITERATURE CITED (1) (2) (3) (4) (5)
Y. Talmi, Anal. Chem., 47, 658A (1975). Y. Talmi, Anal. Chem., 47, 697A (1975). G. Horlick and E. G. Codding, Anal. Chem., 45, 1490 (1973). D. A. Yates and T. Kuwana, Anal. Chem., 48, 510 (1976). R. E. Dessy, W. G. Nunn, and C. A. Titus, J. Chromatogr. Sci., 14, 195 (1976). (6) M. J. Milano, S.Lam, and E. Grushka, J. Chromafogr., 125, 315 (1976). (7) M. S. Denton, T. P. DeAngelis, A. M. Yacynych, W.R. Heineman, and T. W.Gilbert, Anal. Chem., 48, 20 (1976). (8) J. B. Taylor, B. Lown, and M. Polanyi, J. Am. Med. Assoc., 221, 667 (1972). (9) C. C. Johnson, R. 0.Palm, D. C. Stewart, and W. E. Martin, J. Assoc. Adv. Med. Instrum., 5, 77 (1971). (10) A. Savitzky and M. J. E. Goiay, Anal. Chem., 36, 1627 (1964). (1 1) Q. H. Gibson, Biochem. J., 71, 293 (1959). (12) T. C. O'Haver and G. L. Green, Anal. Chem., 48, 312 (1976). (13) J. C. Sternberg, H. S.Stillo, and R . H. Schwendeman, Anal. Chem., 32, 84 (1960). (14) T. Hirschfeld, Appl. Spectrosc., 30, 68 (1976). (15) C. C. Johnson, J. Assoc. Adv. Med. Instrum., 4, 22 (1970).
RECEIVEDfor review October 8, 1976. Accepted December 21, 1976. This work was supported by Public Health Service Research Grant GM23001-01 and was presented in part at the 3rd Annual Meeting of the Federation of Analytical Chemistry and Spectroscopy Societies, November 16, 1976, Philadelphia, Pa.
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