Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
2001-03-09
2003-12-30
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S010000, C436S164000, C436S172000, C422S073000, C422S082050, C422S082080, C356S039000, C356S337000
Reexamination Certificate
active
06670191
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATION
This application is related to Japanese Patent Application No. 2000-184853 filed on Jun. 20, 2000, whose priority is claimed under 35 USC §119, the disclosure of which is incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for quantitatively analyzing fragmented red blood cells (FRCs) and, particularly, to a method for quantitatively analyzing fragmented red blood cells observed in peripheral blood in the case of various diseases such as cardiovascular abnormality, congenital or acquired hemolytic anemia, disseminated intravascular coagulation, hemolytic uremic syndrome and TTP (Thrombotic Thrombocytopenic Purpura).
2. Description of the Related Art
With recent studies on BMT-TMA (bone marrow transplantation-associated thrombotic microangiopathy), attention has been given to FRC % (the percentage of fragmented red blood cells) in peripheral blood, which is regarded as an important index for early diagnosis of BMT-TMA. A BMT-TMA grading system (grades of 0 to 4) was developed on the basis of the FRC % as well as the lactate dehydrogenase (LDH) (Bone Marrow Transplantation, 15, p. 247-253, 1995). The grades are defined as follows:
Grade 0 Normal LDH and FRC≦1.2%
Grade 1 Normal LDH and FRC≧1.3%;
Grade 2 Increased LDH and FRC=1.3% to 4.8%;
Grade 3 Increased LDH and ERG=4.9% to 9.6%; and
Grade 4 Increased LDH and ERG≧9.7%
The FRC % can be determined through visual observation of a peripheral blood smear film. In general, the FRCs are not quantitatively analyzed, but analyzed for detection thereof. Although there are several reports which state that evidence of FRCs can be detected on the basis of a red blood cell size distribution standardized for automatic red blood cell counters (American Journal of Clinical Pathology, 90, p. 268-273, 1988), the quantitative analysis of the ERCs still relies on the visual observation of a smear film, and the results of the observation may vary significantly with different observers due to absence of criteria to be referenced.
SUMMARY OF THE INVENTION
In view of the foregoing, the present invention is directed to a method for quantitatively analyzing fragmented red blood cells, which can determine the FRC % with a high level of accuracy by establishing a specific area on a scattergram of a flow cytometer.
According to the present invention, there is provided a method for quantitatively analyzing fragmented red blood cells, comprising the steps of: adding a nucleic acid staining fluorochrome dye to a blood sample for preparation of a specimen containing particles; allowing the specimen to flow through a flow cytometer, and measuring a scattered light intensity and a fluorescent light intensity for each particle in the specimen; preparing a two-dimensional particle distribution diagram on the basis of the measured scattered light intensity and fluorescent light intensity; establishing a red blood cell distribution area on the two-dimensional distribution diagram, and counting particles falling within the red blood cell distribution area for determination of the total number A of red blood cells; establishing a fragmented red blood cell distribution area in a region in which the scattered light intensity and the fluorescent light intensity are low within the red blood cell distribution area, and counting particles falling within the fragmented red blood cell distribution area for determination of the number B of fragmented red blood cells; and calculating a value B/A, and calculating a content by percentage F of the fragmented red blood cells from a conversion function F=f(B/A).
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patent: 6118522 (2000-09-01), Kanai et al.
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patent: B22674704 (1997-07-01), None
ZR Zeigler, et al. “Bone marrow transplant-associated thrombotic microangiopath: a case series” Bone Marrow Transplatation, 15, pp. 247-253 (1995).
J. David Bessman, M.D. “Red Blood Cell Fragmentation” American Journal of Clinical Pathology, 90, p. 268-273 (1988).
Imoto Shion
Jiang Meiyi
Kumagai Shunichi
Matsumoto Hideaki
Saigo Katsuyasu
Sysmex Corporation
Wallenhorst Maureen M.
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