Lytic reagent and method for leukocytes differential in whole blood
AssigneeIDEXX Laboratories, Inc.
InventorsChiranjit Deka, Jianwen Feng
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Abstract
A lytic reagent composition and methods for differential analysis of leukocytes are disclosed. In embodiments, the analysis may utilize optical measurements in flow cytometry based hematology analyzers. The reagent system includes an anionic surfactant in a hypotonic solution, an inorganic buffer to maintain the pH in a range from about 6 to about 10, and optionally a leukocyte stabilizer. The reagent system is used to lyse red blood cells and stabilize the leukocytes to enable multi-part differentiation of leukocytes in a near physiologic pH environment on a flow cytometry based hematology analyzer using axial light loss, light scatter intensity, high-numerical aperture side scatter, and time-of-flight measurements.
Claims24
A lytic reagent system for use in the determination of a differential white blood cell count consisting essentially of:
- an anionic surfactant at a concentration of from about 0.005 wt% (w/v) to about 0.015 wt% (w/v);
- an alkaline metal salt to adjust osmolality of the system from about 15 mOsm to about 150 mOsm selected from the group consisting of alkaline halides, alkaline chlorides, alkaline bromides, alkaline iodides, and combinations thereof;
- a buffer to maintain a pH of the system from about 6 to about 10;
- wherein the lytic reagent system is applied to temperatures of up to about 35° C.
A hypertonic solution for discontinuing a lytic reaction in a lytic reagent system, comprising:
- an alkaline metal salt possessing an alkaline ion selected from the group consisting of sodium, potassium and lithium:
- wherein the alkaline metal salt is present at a concentration of from about 150 mM to about 250 mM, and the osmolality of the reagent system is of from about 15 mOsm to about 150 mOsm.
A method for resolving white blood cell sub-populations in a whole blood sample, comprising:
- providing a whole blood sample;
- adding a stop solution for discontinuing a lytic reaction;
- incubating the whole blood sample with a lytic reagent sufficient such that red blood cells become lysed, the lytic reagent comprising:
- an anionic surfactant,
- an alkaline metal salt to adjust osmolality of the reagent and blood sample from about 15 mOsm to about 150 mOsm, and
- a buffer to maintain a pH of the reagent and blood sample from about 6 to about 10;
- wherein the lytic reagent system is applied to temperatures of up to about 35° C.;
- measuring a response from at least two optical detectors as the white blood cells pass through a sensing region in a flow cell of an optical detection system, wherein at least one detector of the at least two detectors measures light scatter at a range of angles whose axis is generally orthogonal to a direction of propagation of a laser beam;
- and
- differentiating white blood cells in the whole blood sample.
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Dependencies are inferred from the claim text.