Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Particle count or volume standard or control
Reexamination Certificate
2001-11-30
2004-07-06
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Particle count or volume standard or control
C436S008000, C436S016000, C436S017000, C436S063000, C436S174000, C422S073000, C435S002000, C252S408100
Reexamination Certificate
active
06759246
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of hematology, and in particular, to assays for determining the number or presence of certain components of blood such as lymphocytes. More specifically, the invention relates to control compositions used in performing such assays, and to processes for preparing and methods of using such control compositions.
BACKGROUND OF INVENTION
To Hematology control material consisting of stabilized blood cells has long been commercially available for use in automated hematology analyzers. Methods for stabilizing cells necessarily vary depending on the reagents hematology analyzers use to dilute and treat the cells for analysis, the cell properties that are used to distinguish cell classes, and the methods of detection of these physical properties by different hematology instrumentation. In particular, classification of leukocytes may be accomplished by measurement of electrical impedance, conductivity, light scatter at various angles, selective shrinkage or resistance of cells in various diluents, or cytochemical staining. To distinguish all five major classes of leukocytes (lymphocytes, monocytes, neutrophils, eosinophils and basophils), combinations of more than one type of measurement will usually be needed.
Hematology control material may be prepared using mammalian leukocytes (generally human, porcine or bovine) or may be simulated. For example, simulation of leukocytes in control material by treatment and/or fixation of erythrocytes has been used as described in U.S. Pat. Nos. 5,512,485 and 6,146,901.
In order to supply hematology controls of varying differential white cell targets, the use of lymphocytes without contaminating leukocytes of other classes is desired, but difficult to obtain. Lymphocytes free of contaminating monocytes are particularly difficult to obtain in volumes sufficient for manufacture of hematology control materials. During leukocyte isolation, classification and stabilization, it is not unusual for monocytes to become activated and adherent, forming cell clumps that can interfere with analysis in the finished control product. Additionally, lymphocyte control materials used in instruments which use strong lytic agents (especially saponin) may require the presence of additives such as phospholipids to achieve a stable lymphocyte presentation over time, even when the lymphocytes in the control material are stabilized by fixation.
Granulocytes are more readily isolated from other leukocyte classes from whole blood using density gradients. Also, in normal mammalian blood, the number of granulocytes usually exceed the number of lymphocytes by a factor of two or more, so the pool of available granulocytes from a given quantity of blood is larger than that of lymphocytes.
It would therefore be desirable to prepare a human lymphocyte analog from a starting pool of isolated mammalian granulocytes. However, light scatter, impedance volume, conductivity of and absorbance of various dyes by granulocytes are all high compared to lymphocytes. Therefore, a hematology control prepared using granulocytes with altered physical properties such that the altered granulocytes have physical properties resembling that of human lymphocytes is desired.
SUMMARY OF THE INVENTION
The present invention provides a control composition for use in an automated blood cell analyzer comprising a predetermined concentration of stabilized mammalian granulocytes that have at least one physical property of a human lymphocyte, wherein the physical properties include, without limitation, light scatter, cytochemical dye binding, electrical impedance or other physical properties of the cell that resemble that of a lymphocyte.
The lymphocyte analogs of the invention are produced by mixing a mammalian granulocyte with a solution comprising a suitable condensation product of a nitrogen-substituted alcohol with formaldehyde, and preferably a condensation product of more than one nitrogen substituted alcohol, including without limitation, 2-bromo-2-nitropropane-1,3-diol (“Bronopol”), 5-bromo-5-nitro-1,3-dioxane (“Bronidox”) or hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine (“Triadine-3”), that allows loss of granules, granule contents, or other cytoplasmic material from the cell. The granulocyte is incubated in the presence of such solution until at least one of the physical properties of light scatter, cytochemical dye binding, electrical impedance or other physical properties of the cell measured in an automated blood cell analyzer is altered to resemble the physical properties of an isolated human lymphocyte.
Preferably, the altered granulocyte is fixed with an aldehyde fixative in a concomitant and/or subsequent step to allow sufficient stabilization of the cell for its use as a lymphocyte analog in hematological quality control material.
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Fredrikson & Byron , P.A.
Research & Diagnostic Systems, Inc.
Wallenhorst Maureen M.
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