Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1998-12-09
2003-12-16
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S010000, C436S172000, C436S017000
Reexamination Certificate
active
06664110
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to flow-cytometric diagnosis of formed elements of blood; in particular the invention relates to discrimination and counting of erythroblasts from peripheral blood or other body fluid samples by flow cytometry.
2. Description of Related Art
In the field of clinical examination, sorting and counting of erythroblasts can yield vital information in the diagnosis of disease.
Erythropoiesis is a process of the bone marrow, and normally erythroblasts, prior to their release into circulation as reticulocytes in the final stage of maturation into red blood cells, are not present in the peripheral blood. Consequently, the appearance in the peripheral blood of erythroblasts in any of the distinguishable erythropoietic stages prior to becoming reticulocytes indicates the possibility of the presence of disease, such as acute myelocytic leukemia, hemolytic anemia, iron deficiency anemia and pernicious anemia. Accordingly, erythroblast sorting and counting can be extremely useful in diagnosing these sorts of diseases.
Conventionally in counting and sorting erythroblasts, generally blood smears are prepared which are then suitably stained for counting and sorting through microscopic observation.
On the other hand, various fully automated leukocyte sorter/counter devices that apply flow cytometric principles are available. “Flow Cytometers—History and Measurement Principle,”
Sysmex Journal International
, Vol. 6 No. 1 (1996) is an introduction to flow cytometry as applicable to the present invention, and is herein incorporated by reference.
Wherein erythroblasts have appeared in the peripheral blood, however, results output from flow cytometric devices and analyzed diagrammatically only suggest, by abnormal “flags,” (indicating unusual plots from the flow cytometric data) the possibility of the presence of erythroblasts, and do not enable accurate erythroblast sorting/counting. Such flags often turn out to be false positive results.
Furthermore, apart from the foregoing, Japanese Laid-Open Pat. No. 4-268453 (1992), and U.S. Pat. No. 5,559,037 disclose erythroblast sorting/counting methods.
Either of these are methods by which erythroblasts are assayed by treating samples with a suitable hemolytic agent that disables only cell membranes of erythrocytes (confers dye permeability to the cell membranes) and with a solvent that does not injure cell membranes of leukocytes (does not confer dye permeability to the cell membranes), and afterwards (or at the same time) staining with a fluorescent dye only the erythroblasts whose cell membranes have been damaged, then discriminating erythroblasts from leukocytes by measuring fluorescence intensity.
Wherein fresh blood is used immediately after the sample is taken, accurate measurements are possible with these methods. With the elapse of time after a blood sample is taken, however, not only erythroblast but also leukocyte cell membranes are easily injured, and a likely portion of the leukocytes will get stained by the fluorescent dye because their cell membranes have been damaged prior to the mixing in of hemolytic agent. There is a problem in particular wherein lymphocyte cells are injured, in that it is difficult to discriminate injured lymphocytes distinctly from erythroblasts, such that erythroblasts cannot be sorted and counted accurately.
With some samples in which lymphoblasts appear, which are close in size to erythroblasts, or with samples from chemotheraphy patients in which cell membranes of leukocytes are liable to be disabled by the hemolytic agent, accurate erythroblast counting/sorting is difficult even right after the blood is drawn.
SUMMARY OF THE INVENTION
An object of the present invention is to discriminate and count erythroblasts in peripheral blood or circulatory system-related samples accurately with high precision, even with samples wherein post-sample draw time has elapsed, or wherein easily damaged leukocytes are present.
The present invention provides reagents for discriminating and counting erythroblasts, including a hemolytic agent for dissolving erythrocytes in a body fluid sample and for conditioning leukocytes and erythroblasts in the sample to be suitable for staining, and including at least one fluorescent dye selected to stain leukocytes and erythroblasts differentially. When the selected fluorescent dye is mixed with the sample, a detectable difference in fluorescence intensity at least between leukocytes and erythroblasts arises under laser illumination in flow cytometric analysis.
The reagents provided by the present invention further include surfactant added to the hemolytic agent, selected to enable flow cytometric discrimination of erythroblasts in the body fluid sample by their maturation stages.
Furthermore, the present invention is a method of flow-cytometrically assaying body fluid samples utilizing reagents as mentioned above to discriminate and count erythroblasts in the samples.
The method includes the preparatory steps of: (a) mixing the body fluid sample with a hemolytic agent selected for dissolving erythrocytes within body fluid samples to an extent that does not interfere with the flow-cytometric assay, and for conditioning leukocytes and erythroblasts in the assay sample to be suitable for staining, and (b) staining leukocytes and erythroblasts in the sample as prepared in said step (a) by mixing the sample with at least one of the fluorescent dyes selected to stain leukocytes and erythroblasts differentially; and the method further includes the steps of: (c) flow cytometrically assaying the sample as prepared in said step (b) by measuring at least one scattered light parameter and at least one fluorescence parameter, and (d) discriminating and counting erythroblasts utilizing intensity differences in scattered light and in fluorescence as measured in step (c).
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Ikeuchi Yoshiro
Oguni Shin'ichiro
Sakata Takashi
Tsuji Tomohiro
Cross LaToya I
Shinjyu Global IP
Sysmex Corporation
Warden Jill
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