Optics: measuring and testing – Blood analysis
Reexamination Certificate
2001-06-05
2003-10-07
Frech, Karl D. (Department: 2876)
Optics: measuring and testing
Blood analysis
C356S040000, C356S337000, C356S441000, C356S336000, C356S339000, C356S340000
Reexamination Certificate
active
06630990
ABSTRACT:
SUMMARY OF INVENTION
Methods and apparatus are disclosed for determining the volume, hemoglobin concentration, maturity and cell shape of mammalian red blood cells in a sample and simultaneously monitoring system standardization. Methods for distinguishing red blood cells from other cellular particles, prior to the red blood cell analysis are also disclosed. The method can be applied with accuracy over a wide range of visible spectrum. A whole blood sample is treated with a reagent solution containing a nonionic surfactant in an isotonic buffered solution at neutral pH, the red blood cells are passed through a beam of light in single file at a selected wavelength, obtaining an initial cytogram by means of the resultant magnitude of one light loss signal and one forward angle light scatter signal at a selected angular interval and a third side angle light scatter or two forward angle light scatter signals at selected angular intervals and a third side-angle light scatter signal, projecting the cytogram, point by point, onto a pre-calibrated 3-dimensional surface containing grid lines of volume and hemoglobin concentration, determining accurate values of cell volume and hemoglobin concentration by means of the location of each projected intercept onto the three dimensional grid surface.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method and apparatus for simultaneous monitoring of system standardization and automated analysis of mammalian red blood cell (RBC) and white blood cell (WBC) differentiation in a body fluid. The present invention particularly relates to a multi-angle light scatter and fluorescence apparatus such as multi-parameter hematology analyzer or flow cytometer that can perform both RBC and WBC differential analysis using the same optical detection system. The present invention more particularly relates to (i) a method for RBC analysis for volume, hemoglobin content, cell shape, and maturity in whole blood; (ii) an accurate method for determination of immature RBC (reticulocyte) volume and hemoglobin content; (iii) a RBC method that can continuously monitor the system standardization while a blood sample is being analyzed for RBC differentiation; and (iv) a method that can measure both mature RBC and reticulocyte volume and hemoglobin content, using one reagent and (v) an apparatus that can perform both WBC and RBC differential analysis using the same optical detection system.
2. Description of Prior Art
The conventional hematology method, microscopic examination of patient blood smears for RBC morphology for cell size, cell shape, color (for hemoglobin content) and inclusions provides a wealth of information leading towards the diagnosis and monitoring patient's clinical conditions. Quite misleading impressions can be drawn, however, from substandard blood films, besides the fact that this manual method is very subjective and time consuming. During the past three decades, a number of automated hematology analyzers have become available to handle heavy laboratory work loads and to reduce labor. Most of these instruments measure mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) of red blood cells either by electrical impedance measurement or by light scatter optical measurement in combination with a colorimetric hemoglobin measurement. Because of incompleteness or ambiguity of morphological information in cell analysis from these systems, 5 to 10 percent of samples in hematology laboratories routinely undergo smear review for cell morphology using the microscopic method. More advanced hematology analyzers in terms of RBC morphology analysis are the Technicon H*1 and the Bayer ADVIA. Both systems are designed to measure red cell volume and hemoglobin concentration simultaneously on cell-by-cell basis, according to the teachings of D. H. Tycko, described in U.S. Pat. No. 4,735,504.
U.S. Pat. No. 4.735,504 to D. H. Tycko describes Method and Apparatus for Determining the Volume and Index of Refraction of Particles. He discloses the method for measuring V and HC of is ovolumetrically-sphered RBCs by 2 selected angular interval forward light scattering signals, S1 and S2, to determine volume (V) and hemoglobin concentration (HC). Drawbacks of the method are: 1) the wavelength of the light source must be long enough (e.g., 633 nm) to avoid hemoglobin absorption from RBCs, which precludes the choice of a light source more suitable for multi-parameter blood cell analysis (e.g., a 488 nm light source); 2) the two-dimensional (2D) matrix does not provide any information on abnormal cell shape since the signals from such cells fall on a wrong location on the predetermined 2D matrix, thus generating incorrect clinical data on V & HC; 3) the 2D matrix does not provide any information regarding shifts in the system standardization, the phenomenon that can occur without any warning due to an instability in fluidics passage caused by clots in certain blood samples or instability in electronics of the system; 4) the 2D scatter method is not capable of identifying and clearly separating WBC's and nucleated red blood cells (NRBCs) from mature RBC's or stained reticulocytes. WBC's and NRBC's generate much more scatter than RBC's because of their nuclei and if they are not excluded cleanly from the RBC population before V and HC analysis, clinical results on MCV, hematocrit (Hct), MCHC and mean corpuscular hemoglobin (MCH) will be very misleading on elevated WBC or NRBC samples.
U.S. Pat. No. 5,194,909 to D. H. Tycko teaches Apparatus and Method for measuring V and HC of Red Blood Cells. The difference of this art from that of his previous teachings in U.S. Pat. No. 4,735,504 is that the 2D matrix is created using one forward light scattering signal (pre-selected) at a long wavelength (633 nm) and the second signal from a resistant pulse-sizing aperture. Drawbacks of the method are: 1) the method requires two independent sources of detection system, which creates unnecessary complications such as synchronization of the two signals from two different detection systems; 2) the wavelength of the light source must be long enough to avoid hemoglobin absorption from RBCs, which limits the choice of light source for multi-parameter blood cell analysis; 3) the 2D matrix does not provide any information on abnormally shaped RBCs, thus generating incorrect clinical information on V & HC; 4) the 2D matrix does not provide any information regarding shifts in the system standardization, the phenomenon that can occur without any warning due to instability in electronics or fluidics as explained above.
U.S. Pat. No. 5,284,771 to Fan et al. discloses Reagent Compositions and their use in sphering cells. The reagent composition includes a zwitterionic surfactant for sphering red blood cells to eliminate orientation noise and Ozxazine750 to stain reticulocytes. The light source of the optical detection system is a 633 nm HeNe laser, and the stained reticulocytes are identified by light scatter/absorption technology. Fluorescent measurement of retoculocytes was not demonstrated or claimed in this patent. The inventors of this disclosure did not make any claims on reticulocyte V & HC measurements, but they described the use of the aforenoted methods of Tycko to simultaneously measure the red cell volume and hemoglobin on a cell-by-cell basis using the TECHNICON H*1 SYSTEM. In the teachings of Fan et al., the reticulocyte staining procedure requires manual preparation, manual feeding, and over 2 min. of staining time. The inventors described that V & HC of both RBCs and reticulocytes are measured by the method of Tycko, although the reagents used for RBCs and reticulocytes are completely different in composition. The reagent used to construct Tycko's 2D matrix for RBCs for the TECHNICON H*1 spheres and fixes the RBCs as described in Tycko's disclosure, while the reagent used for the reticulocytes spheres RBCs in a buffer that does not contain any fixative. Besides, absorption by the blue
Bearden, Jr. James C.
Kim Young Ran
Landayan Marilou Z.
van't Oever Ronny
Abbott Laboratories
Anderson Regina M.
Frech Karl D.
Goller Mimi C.
Hess Daniel A.
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