Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2001-08-10
2003-07-29
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
Reexamination Certificate
active
06599713
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for determining a white blood cell count of a whole blood sample. More particularly, the present invention is concerned with a method for determining a white blood cell count of a whole blood sample, which comprises: mixing a whole blood sample with a surfactant to thereby obtain a mixture; allowing the mixture to stand for a time sufficient to lyze the white blood cells contained in the whole blood sample and release intrinsic myeloperoxidase from the white blood cells; measuring the concentration of the released myeloperoxidase in the mixture; and determining the white blood cell count in the whole blood sample, based on the, concentration of the released myeloperoxidase. By the method of the present invention for determining a white blood cell count, the determination of a white blood cell count and/or a neutrophil count of a whole blood sample can be performed easily and rapidly without the need to separate blood cells from the whole blood sample. Further, by the determination method of the present invention, in addition to a white blood cell count of a whole blood sample, the concentration of C-reactive protein contained in the same whole blood sample can be measured, so that the presence or absence of an infectious disease and the graveness of an inflammation can be diagnosed rapidly and easily and at low cost.
2. Prior Art
In recent years, in the initial steps of diagnosis of patients, as examination items for judging whether or not patients have a bacterial infectious disease, the white blood cell count of a whole blood sample and the concentration of C-reactive protein (hereinafter, frequently referred to simply as “CRP”) contained in a whole blood sample are determined. In this connection, for enabling doctors to take adequate measures, such as the administration of an antibiotic, it has been desired to develop determination techniques which can produce results rapidly and easily and at low cost.
Usually, the determination of the white blood cell count is performed using a commercially available automated blood cell counting apparatus which is based on the aperture-impedance method, wherein the apparatus is represented by COULTER COUNTER™ (manufactured and sold by COULTER ELECTRONICS, INC., the U.S.A.). However, the concentration of CRP, which is a plasma protein, cannot be determined by such an apparatus.
Recently, there has been put into the market an apparatus for determining both the white blood cell count and the CRP concentration, which apparatus is described in Yasuo YAMAO, Hiroshi OKUNARI and Henri CHAMPEIX: Readout, 16, 11-15 (1998), wherein, in the apparatus, a blood cell counting apparatus based on the aperture-impedance method is given a CRP determination function based on a latex turbidimetric immunoassay. In the case of the use of such apparatus, the procedure is as follows: the red blood cells in the whole blood sample are first lyzed, and, then, the white blood cell count is determined by the aperture-impedance method, and, subsequently, the CRP determination is performed by a method in which the blood sample containing the lyzed blood cells is reacted with latex beads having bound thereto an anti-CRP antibody for a predetermined time, and then the CRP concentration is determined by a latex turbidimetric immunoassay. That is, in the case of the use of the above-mentioned apparatus, the determination of the white blood cell count and the determination of the CRP concentration are performed based on different principles, so that completely different operations are necessary for the determination. Therefore, the conventional method using the above-mentioned apparatus has drawbacks in that very cumbersome operations are necessary and that a number of reagents are used for the determination, thus leading to an increase in cost. In addition, the maintenance of the apparatus needs much time and large cost. Due to these drawbacks, an automated blood cell counting apparatus has not yet been introduced in almost all of medical institutions which have the responsibility of providing primary care, such as practicing physicians, and general hospitals, and therefore, the needs for medical care cannot be fully satisfied.
As a means to solve the above-mentioned problems, conceivable is a method in which both the white blood cell count and the CRP concentration are determined, based on the same principle. Specifically, conceivable is a method in which, with respect to a single sample of whole blood, both the white blood cell count and the CRP concentration are determined by an immunological method. An immunological determination of the white blood cell count can be made by measuring a protein which is specifically present in white blood cells.
It is generally know that, when a bacterial infection or an inflammation occurs, the number of neutrophis in the blood is increased, wherein the neutrophis have a largest proportion among all types of white blood cells. Therefore, by determining the neutrophil count, the increase and decrease in the number of white blood cells can be detected, so that the presence or absence of a bacterial infectious disease and the graveness of an inflammation can be judged.
As an example of a method for measuring the neutrophil count of an inflamed site of tissue or measuring the white blood cell count or the granulocyte count of a whole blood sample, there can be mentioned the method described in Peter P. Bradley, Dennis A. Priebat, Robert D. Christensen and Gerald Rothstein:
J. Invest. Dermatol
., 78, 206-209 (1982). This method is as follows. First, neutrophils are separated from a whole blood sample by using the Ficoll reagent, or an inflamed skin tissue is obtained. The obtained neutrophils or the obtained inflamed skin tissue is homogenized in a 50 mM potassium phosphate buffer solution (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) and then sonicated to extract myeloperoxidase, and subsequently the enzyme activity of the extracted myeloperoxidase is determined.
Another method is disclosed in Rita Cramer, Maria Rosa Soranzo, Pietro Dri, Renzo Menegazzi, Anna Pitotti, Giuliano Zabucchi and Pierluigi Patriarca:
Journal of immunological Methods
, 70, 119-125 (1984). This method is as follows. Dextran (MW 200,000) is added to blood containing a solution of ACD (acid citrate dextrose) as an anticoagulant, to obtain a mixture. From the obtained mixture, red blood cells are removed. Subsequently, the Ficoll reagent is added to the resultant, and granulocytes are separated from the resultant mixture by centrifugation. Then, 2×10
4
cells of the granulocytes are mixed with a 0.1 M phosphate buffer solution (pH 7.0) containing 13 mM guaiacol and 0.02% cetyltrimethylammonium bromide. 1 &mgr;mol of H
2
O
2
is added thereto, and the peroxidase activity is measured.
W. M. Kuebler, C. Atels, L. Schuerer and A. E. Goetz: Int.
J. Microcirc
., 16, 89-97(1996) disclose the following method. Polymorphonuclear white blood cells are separated from whole blood by using the Ficoll reagent. The obtained white blood cells are lyzed with a 50 mM potassium phosphate buffer solution (pH 6.0) containing 0.5% HTAB, and then the enzyme activity of the myeloperoxidase in the resultant cell lysis mixture is determined. Further, the above-mentioned prior art document also discloses a method in which rat brain tissue or rabbit lung tissue is homogenized in 1 ml of a 0.02 M potassium phosphate buffer solution (pH 7.4) which is cooled with ice, and then 1 ml of 0.02 M potassium phosphate buffer solution (pH 7.4) is added thereto, and the resultant is centrifuged at 2,000 rpm for 15 minutes at 4° C. to obtain a centrifugation product containing a supernatant. Subsequently, the enzyme activity of the myeloperoxidase in the supernatant is determined. Further, the residue of the centrifugation product is incubated at 60° C. for 2 hours, and then homogenized with a 50 mM potassium phosphate buffer solution (pH 6.0) containing 0.5% HTAB, and then sonicated
Hatanaka Yoshihiro
Serizawa Ryo
Asahi Kasei Kabushiki Kaisha
Saucier Sandra E.
Young & Thompson
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