Method and reagent composition for performing leukocyte differential counts on fresh and aged whole blood samples, based on intrinsic peroxidase activity of leukocytes
First Claim
1. An improved method for determining a differential white blood cell count and subpopulation analysis on a fresh or aged whole blood sample containing red blood cells and white blood cells, said method based on measuring the intrinsic peroxidase activity of white blood cell subpopulations in said sample, comprising the steps of:
- a) mixing said blood sample with an aqueous reagent composition to form a uniform reaction mixture, said reagent composition comprising;
(i) a nonionic polyethoxylate surfactant at a concentration effective to lyse said red blood cells to release hemoglobin, but not to lyse said white blood cell populations in said sample;
(ii) an ionic surfactant of the anionic or zwitterionic class at a concentration effective to lyse said red cells, but not to lyse said white blood cell populations in said sample;
(iii) formaldehyde or paraformaldehyde at a concentration effective to chemically crosslink said white blood cells, but not to crosslink said lysable red blood cells in said sample;
(iv) a sugar or sugar alcohol at a concentration effective to increase the detectability of lymphocytes in said sample; and
(v) a buffer or buffer mixture to maintain a neutral or near-neutral pH of said reaction mixture between about 6.8 to about 8.0;
b) heating said reaction mixture of step a) to a temperature of from about 60°
C. to about 75°
C., whereby said red blood cells in said sample are lysed and said white blood cells are fixed; and
c) staining at least a portion of said white blood cells in said reaction mixture to yield a population of stained white blood cells and a population of unstained white blood cells in suspension;
wherein the presence of both said nonionic surfactant and said ionic surfactant in said aqueous reagent mixture provides accurate and reliable white blood cell differential counting results following step c) for fresh blood samples and for aged blood samples that have been stored at room temperature for at least about a day postdraw.
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Abstract
The present invention provides an improved reagent composition and method to perform white blood cell differential counting and subpopulation analysis using both fresh and aged blood samples with accuracy and precision. The invention is particularly applicable for the analysis of aged blood samples that have been stored at room temperature for over a day, thereby allowing accurate and useful information to be obtained from samples that are normally considered to be suboptimal. The improved reagent composition and method are particularly related to the peroxidase method of white blood cell differential determinations. One aspect of the invention includes an improved aqueous reagent composition for carrying out the peroxidase method of differential counting. Another aspect includes the use of a rinse cycle and rinse solution devoid of hemolytic surfactant to alleviate the adverse effects of rinse carryover and to streamline and economize the analytical process, particularly when the analyses are performed on automated hematology analyzers and flow cytometry systems. The composition and method of the invention provide clinically useful data for the differential analysis of whole blood samples.
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Citations
80 Claims
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1. An improved method for determining a differential white blood cell count and subpopulation analysis on a fresh or aged whole blood sample containing red blood cells and white blood cells, said method based on measuring the intrinsic peroxidase activity of white blood cell subpopulations in said sample, comprising the steps of:
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a) mixing said blood sample with an aqueous reagent composition to form a uniform reaction mixture, said reagent composition comprising;
(i) a nonionic polyethoxylate surfactant at a concentration effective to lyse said red blood cells to release hemoglobin, but not to lyse said white blood cell populations in said sample;
(ii) an ionic surfactant of the anionic or zwitterionic class at a concentration effective to lyse said red cells, but not to lyse said white blood cell populations in said sample;
(iii) formaldehyde or paraformaldehyde at a concentration effective to chemically crosslink said white blood cells, but not to crosslink said lysable red blood cells in said sample;
(iv) a sugar or sugar alcohol at a concentration effective to increase the detectability of lymphocytes in said sample; and
(v) a buffer or buffer mixture to maintain a neutral or near-neutral pH of said reaction mixture between about 6.8 to about 8.0;b) heating said reaction mixture of step a) to a temperature of from about 60°
C. to about 75°
C., whereby said red blood cells in said sample are lysed and said white blood cells are fixed; andc) staining at least a portion of said white blood cells in said reaction mixture to yield a population of stained white blood cells and a population of unstained white blood cells in suspension;
wherein the presence of both said nonionic surfactant and said ionic surfactant in said aqueous reagent mixture provides accurate and reliable white blood cell differential counting results following step c) for fresh blood samples and for aged blood samples that have been stored at room temperature for at least about a day postdraw. - View Dependent Claims (2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 79)
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5. An improved method for determining a differential white blood cell count and subpopulation analysis on a fresh or aged whole blood sample containing red blood cells and white blood cells, said differential counting method based on the measurement of intrinsic peroxidase activity of white blood cell subpopulations in said sample, comprising the steps of:
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a) mixing said blood sample with an aqueous reagent composition to form a reaction mixture, said reagent composition comprising;
(i) a nonionic polyethoxylate surfactant having a hydrophilic lipophilic balance or HLB value of between about 9.3 and about 17.5 and present at a concentration effective to lyse said red cells to release hemoglobin, but not to lyse said white blood cell populations in said sample;
(ii) an ionic surfactant of the anionic or zwitterionic class at a concentration effective to lyse said red cells, but not to lyse said white blood cell populations in said sample;
(iii) formaldehyde or paraformaldehyde at a concentration effective to chemically crosslink said white blood cells, but not to crosslink said lysable red blood cells in said sample;
(iv) a sugar or sugar alcohol at a concentration effective to increase the detectability of lymphocytes in said sample; and
(v) a buffer or buffer mixture to maintain the pH of said reagent mixture at a neutral or near-neutral pH of between about 6.8 to about 8.0;b) heating said reaction mixture prepared in step a) to a temperature of from about 60°
C. to about 75°
C., whereby said red blood cells in said sample are lysed and said whim blood cells are fixed; andc) staining at least a portion of said white blood cells in said reaction mixture to yield a population of stained white blood cells and a population of unstained white blood cells in suspension; wherein the presence of both said nonionic surfactant and said ionic surfactant in said aqueous reagent mixture provides accurate and reliable whim blood cell differential counting results following step c) for fresh blood samples and for aged blood samples that have been stored at room temperature for at least about a day postdraw. - View Dependent Claims (6, 7)
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47. An improved reagent composition for lysing red blood cells and staining leukocytes for the determination of a leukocyte differential count and subpopulation analysis of a whole blood sample, said differential count based on measuring endogenous peroxidase activity in subpopulations of said leukocytes in said blood sample, which comprises in aqueous admixture:
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a) a nonionic polyethoxylate surfactant at a concentration effective to lyse said red cells to release hemoglobin, but not to lyse said white blood cell populations in said sample; b) an anionic or zwitterionic surfactant at a concentration effective to lyse said red cells to release hemoglobin, but not to lyse said white blood cell populations in said sample; c) a sugar or sugar alcohol at a concentration effective to increase the detectability of lymphocytes in said sample; d) formaldehyde or paraformaldehyde at a concentration effective to chemically crosslink said white blood cells, but not to crosslink said lysable red blood cells in said sample; and e) a buffer or buffer mixture to maintain the pH of said reagent composition at between about 6.8 to about 8.0. - View Dependent Claims (48, 49, 50, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80)
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51. An improved reagent composition for lysing red blood cells and staining leukocytes for the determination of a differential leukocyte count and subpopulation analysis of a whole blood sample based on the measurement of endogenous peroxidase activity in subpopulations of said leukocytes in said blood sample, which comprises in aqueous admixture:
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a) a nonionic polyethoxylate surfactant having a hydrophilic lipophilic balance or HLB value of between about 9.3 and about 17.5 and at a concentration effective to lyse said red cells to release hemoglobin, but not to lyse said white blood cell populations in said sample; b) an anionic or zwitterionic surfactant at a concentration effective to lyse said red cells to release hemoglobin, but not to lyse said white blood cell populations in said sample; c) a sugar or sugar alcohol at a concentration effective to increase the detectability of lymphocytes in said sample; d) formaldehyde or paraformaldehyde at a concentration effective to chemically crosslink said white blood cells, but not to crosslink said lysable red blood cells in said sample; and e) a buffer or buffer mixture to maintain the pH of said reagent composition at between about 6.8 to about 8.0. - View Dependent Claims (54)
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Specification