Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Particle count or volume standard or control
Reexamination Certificate
2000-05-08
2003-12-23
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Particle count or volume standard or control
C436S017000, C436S063000, C436S164000, C436S165000, C422S073000, C422S082050, C422S072000, C382S133000, C382S134000
Reexamination Certificate
active
06667177
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for counting leukocytes and an apparatus for counting leukocytes. In particular, the present invention relates to a method and an apparatus suitable to count leukocytes in a platelet preparation or an erythrocyte preparation.
BACKGROUND ART
Platelet preparations and erythrocyte preparations are mainly used for alleviation of thrombocytopenia and anemia, surgical operations and so forth. Considering side effects and the like, it is not desirable from a viewpoint of quality that leukocytes are present in a platelet preparation or an erythrocyte preparation. Thus, the number of leukocytes that can be contained in a small amount in a platelet preparation or an erythrocyte preparation is measured for quality control.
Usually, the leukocyte count in a platelet preparation or an erythrocyte preparation is measured by baring nuclei of leukocytes and staining them. That is, leukocytes are accumulated by a centrifuge or the like, stained and then placed in a Nageotte chamber (hemocytometer) so that observers visually count the number using a microscope. Since platelets are rarely dissolved in this method, however, leukocytes are buried in the platelets, which results in deteriorated measurement accuracy. In addition, visual measurement is extremely inefficient. Furthermore, in this measurement method, observers often contact blood preparations with a possibility of biohazard (biological contamination). Therefore, a safe method that achieves automatization and facilitation of the measuring operation as well as improvement of measurement accuracy is presently desired.
On the other hand, in general, nuclei of leukocytes must be bared to stain the leukocytes for measurement. It has been known that a surfactant is added for this purpose. However, no method for counting leukocytes has been known, wherein a cytolytic agent that bares nuclei of leukocytes and solubilizes platelets or erythrocytes is used to solubilize platelets or erythrocytes in a platelet preparation or an erythrocyte preparation.
DISCLOSURE OF THE INVENTION
The present invention has been accomplished in the light of the above circumstances. The object of the present invention is to provide a method and an apparatus for readily measuring leukocyte count in a platelet preparation or an erythrocyte preparation.
As a result of the present inventors' efforts to achieve the aforementioned object, they have been found that measurement of the leukocyte count can be facilitated and a measurement apparatus without requiring visual measurement can be obtained by utilizing a cytolytic agent that can bare nuclei of leukocytes and solubilize platelets or erythrocytes, because such a cytolytic agent can bare nuclei of leukocytes and solubilize platelets or erythrocytes when it is added to a platelet preparation solution or an erythrocyte preparation solution. Thus, the present invention has been accomplished.
That is, the present invention provides a method for counting leukocytes in a platelet preparation by staining the leukocytes, comprising adding a cytolytic agent capable of baring nuclei of leukocytes and solubilizing platelets to a solution of the platelet preparation to bare nuclei of the leukocytes and solubilize platelets in the solution of the platelet preparation.
In the present specification, terms “platelet preparation” and “solution of the platelet preparation” are used. As for these terms, if a platelet preparation is originally in the form of a solution, “platelet preparation” is equivalent to “solution of the platelet preparation”. It is also contemplated that, even if a platelet preparation is in the form of a solid or the like, the preparation can be used as a solution after dissolution.
The present invention also provides a method for counting leukocytes in a platelet preparation by staining the leukocytes, comprising:
mixing and shaking a solution of the platelet preparation solution, a cytolytic agent capable of baring nuclei of leukocytes and solubilizing platelets and a dye, in an accumulation container comprising an opening, a sidewall portion and a bottom portion, a part or all of the sidewall portion having a horizontal sectional area gradually increasing in a direction from the bottom portion towards the opening, to solubilize platelets, bare nuclei of the leukocytes and stain the leukocytes,
setting the accumulation container on a centrifuge to accumulate the stained leukocytes on the bottom portion of the accumulation container, and
counting the stained leukocytes.
In measurement by baring nuclei of leukocytes and staining the leukocytes, what is actually measured is usually DNA aggregates of stained bared nuclei of individual leukocytes. In the present specification, the term “leukocytes” may be used to refer not only to leukocytes in the normal state, but also to the DNA aggregates of stained bared nuclei of leukocytes.
In the above method for counting leukocytes in the platelet preparation, the cytolytic agent added to the solution of the platelet preparation is preferably selected from the group consisting of anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants. The amount of the cytolytic agent added to the solution of the platelet preparation solution is preferably 0.2 to 5% (w/v).
The present invention also provides a method for counting leukocytes in a platelet preparation by staining the leukocytes, comprising:
placing a solution of the platelet preparation in an accumulation container comprising an opening, a sidewall portion, and a bottom portion having a membrane filter through which leukocytes are impassable, a part or all of the sidewall portion having a horizontal sectional area gradually increasing in a direction from the bottom portion towards the opening,
filtering the solution of the platelet preparation through the membrane filter provided at the bottom portion of the accumulation container containing the solution of the platelet preparation to accumulate the leukocytes on the bottom portion,
adding a surfactant and a dye to the leukocytes accumulated on the bottom portion to bare nuclei of the leukocytes and stain the leukocytes, and
counting the stained leukocytes.
The present invention also provides a method for counting leukocytes in an erythrocyte preparation by staining the leukocytes, comprising adding a cytolytic agent capable of baring nuclei of leukocytes and solubilizing erythrocytes to a solution of the erythrocyte preparation to bare nuclei of the leukocytes and solubilize erythrocytes in the solution of the erythrocyte preparation.
In the present specification, terms “erythrocyte preparation” and “solution of the erythrocyte preparation” are used. As for these terms, if an erythrocyte preparation is originally in the form of a solution, “erythrocyte preparation” is equivalent to “solution of the erythrocyte preparation”. It is also contemplated that, even if an erythrocyte preparation is in the form of a solid, the preparation can be used as a solution after dissolution.
The present invention also provides a method for counting leukocytes in an erythrocyte preparation by staining the leukocytes, comprising:
mixing and shaking a solution of the erythrocyte preparation, a cytolytic agent capable of baring nuclei of leukocytes and solubilizing erythrocytes and a dye, in an accumulation container comprising an opening, a sidewall portion and a bottom portion, a part or all of the sidewall portion having a horizontal sectional area gradually increasing in a direction from the bottom portion towards the opening, to solubilize erythrocytes, bare nuclei of the leukocytes and stain the leukocytes,
setting the accumulation container on a centrifuge to accumulate the stained leukocytes on the bottom portion of the accumulation container, and
counting the stained leukocytes.
In the above method for counting leukocytes in the erythrocyte preparation, the cytolytic agent is preferably selected from the group consisting of anionic surfactants, cationic surfactants, amphoteric surfactants an
Kilpatrick & Stockton LLP
Kowa Company Ltd.
Wallenhorst Maureen M.
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