Optics: measuring and testing – Blood analysis
Reexamination Certificate
2000-04-13
2001-12-11
Font, Frank G. (Department: 2877)
Optics: measuring and testing
Blood analysis
C435S007240, C435S007250
Reexamination Certificate
active
06330058
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the characterization of blood types, and, more particularly, to a spectrophotometric apparatus and method of blood typing.
2. Description of Related Art
Blood typing is the most commonly used test in blood centers and transfusion medicine (Beutler et al., 1995). Manual blood typing methods are time-consuming, require special skills, and are prone to errors. Automated systems involve expensive and complex instrumentation. In addition, these methods do not lend themselves to a quantitative interpretation of the antibody-induced aggregation process. Consequently, there has been ongoing interest in developing alternative methods for blood typing. This had led us to examine the utility of a simple and easily automated approach, namely, multiwavelength ultraviolet/visible spectroscopic analysis as a potential quantitative blood typing procedure.
The basis for currently known blood typing methods is the examination of blood samples for aggregation in the presence of agglutinating antibodies (Walker et al., 1990; Gane et al., 1987). At present, the most sophisticated automated blood typing procedure uses image analysis measurements of incubated mixtures of test and reagent samples in optically clear reaction chambers (Olympus, 1993). A special camera records the light transmission pattern throughout the image to distinguish between a positive and negative agglutination test. Manual approaches employed in smaller clinical settings use tube testing that relies on the technician's subjective visual recognition of aggregates. An alternate multistep test uses bromelain-treated erythrocytes adhering to microtiter plates; typing is accomplished via analysis of coagglutination with erythrocytes of unknown sera following centrifugation and evaluation of optical image patterns (Muller et al., 1981).
One limitation of the currently employed technology is a lack of on-line capability for the characterization of blood components, as well as a lack of portable instrumentation capable of detecting, counting, and classifying specific blood components. The problem of portable instrumentation and suitable methods of analysis and diagnosis is particularly relevant to the medical industry, where the need for rapid analysis and diagnosis often involves life-threatening situations. Although the analytical instrumentation used in medical and clinical laboratories has improved considerably over the past decade, there are still no suitable techniques capable of detecting, classifying, and counting on-line critical cell populations and/or pathogens in blood and other bodily fluids. Typically the particles of interest have sizes ranging between 0.5 and 20 &mgr;m, and, in many instances, are present in fairly dilute concentrations.
As is known from spectroscopy theory, a measure of the absorption of a solution is the extinction coefficient, which also provides a measure of the turbidity and transmission properties of a sample. Spectra in the visible region of the electromagnetic spectrum reflect the presence of certain metal ions, complexes, and molecules with extensive conjugated aromatic structures. In the near-uv region small conjugated ring systems affect absorption properties. However, suspensions of very large particles are powerful scatterers of radiation, and in the case of microorganisms, the light scattering effect is sufficiently strong to mask or distort absorption effects. It is therefore known to use uv/vis spectroscopy to monitor purity, concentration, and reaction rates of such large particles.
Many attempts have been made to estimate the particle size distribution (PSD) and the chemical composition of suspended particles using optical spectral extinction (transmission) measurements. However, previously used techniques require that either the form of the PSD be known a priori or that the shape of the PSD be assumed. One of the present inventors has applied standard regularization techniques to the solution of the transmission equation and has demonstrated correct PSDs of a large variety of polymer lattices, protein aggregates, silicon dioxide particles, and microorganisms.
It is also possible to use the complementary information available from simultaneous absorption and light scattering measurements at multiple angles for the characterization of the composition and molecular weight of macromolecules (Garcia-Rubio, 1993; and “Multiangle, Multiwavelength Particle Characterization System and Method,” U.S. patent application Ser. No. 08/489,940, filed Jun. 13, 1995, now abandoned, and continuation application thereto U.S. patent application Ser. No. 08/780,828, filed Jan. 10, 1997, now U.S. Pat. No.5,808,738 the disclosures of which are incorporated herein by reference).
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a spectroscopic technique for the characterization and differentiation of blood types.
It is another object to provide on-line instrumentation capable of rapid spectrophotometric blood typing.
It is an additional object to provide such instrumentation having at least 2 nanometer resolution.
These and other objects are addressed by the apparatus and method of the present invention for a method for determining the type of a blood sample. The method takes advantage of the fact that a suspension of cells both absorb and scatter light (Anderson et al., 1967). The results of these combined effects yield the optical density or transmission. Typically large biological particles such as cells exhibit scattering throughout the uv/visible range and absorption generally below 800 nm due to their specific chromophoric components (Kerker, 1969). In the past uv/vis spectroscopy has been used extensively to examine specific components of blood. These have typically been analyzed in reference to calibrations carried out with internal or external standards. An example of this is hemoglobin measurement, which has been determined using optical density measurements of erythrocytes, where these values are calibrated against hemoglobin obtained from erythrocytes following lysis (Horecker, 1943).
Simple, rapid, inexpensive, and nondestructive direct interpretation of the size and composition information contained in a uv/vis spectrum have not been fully exploited. This has primarily been due to the difficulty in quantitatively interpreting nonlinear scattering effects combined with an equally confounding hypochromic effect arising from the dense packing of strongly absorbing species such as hemoglobin in erythrocytes (Horecker, 1943; Horecker and Brackett, 1944). For these reasons, uv/visible spectroscopy has been mainly used to obtain qualitative differences between complex mixtures or has required interpretations based on external calibrations or standards as described above. While certain scattering theories have been developed that relate the number of particles, size of particles, and number and types of absorbing species to the actual optical density spectrum, e.g., Mie scattering theory (Brandolin et al., 1991), solutions for such equations require knowledge of the refractive index of the components as well as either their absorption or scattering characteristics (Garcia-Rubio, 1992). Given the complexity of a solution such as whole blood, this can be a daunting task but one that nevertheless merits effort.
The method of the present invention comprises the steps of collecting a reference optical density spectrum over a predetermined wavelength range for a portion of the blood sample diluted in saline. Another portion of the blood sample is then mixed with an antibody corresponding to a known blood type (e.g., anti-A, anti-B, anti-D antibody). The optical density spectrum is then collected over a predetermined wavelength range for blood diluted with saline and each antibody in saline. The slopes are then calculated over a predetermined wavelength range for each spectrum. A numerical indicator of agglutination is then calculated by dividing the slope of each antibody-treated
Garcia-Rubio Luis Humberto
Leparc German
Narayanan Smita
Orton Sharyn
Potter Robert
Allen Dyer Doppelt Milbrath & Gilchrist, P.A.
Font Frank G.
Punnoose Roy M.
University of South Florida
LandOfFree
Spectrophotometric method and apparatus for blood typing does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Spectrophotometric method and apparatus for blood typing, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Spectrophotometric method and apparatus for blood typing will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2593881