Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Particle count or volume standard or control
Reexamination Certificate
1999-04-28
2001-04-10
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Particle count or volume standard or control
C436S008000, C436S017000, C436S063000, C436S164000, C436S166000, C436S174000, C436S175000, C252S408100, C435S002000
Reexamination Certificate
active
06214625
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to lytic reagent compositions and methods for differentiating leukocyte subpopulations of blood samples by means of suitable electronic instruments.
BACKGROUND OF THE INVENTION
Analysis of leukocyte populations from blood samples is an integral and essential part of diagnostic procedures regarding a multiplicity of pathologies. Measurements of basophils and eosinophils that are a subpopulation of leukocytes are important to the diagnosis of several diseases.
Traditional analysis of blood samples involves the smearing of a blood sample on a microscope slide, followed by a visual analysis of the slide. This approach is extremely time consuming as well as being subject to the interpretation of the individual analyzing the slide. These factors have led to the development of automated leukocyte analysis utilizing flow cytometry. An essential step in leukocyte analysis using automated hematology instruments is the lysis of the red blood cells. Thus far, various lytic reagents and automated methods have been developed for use with whole blood samples.
U.S. Pat. No. 4,485,175 (to Ledis et al.) describes a reagent system and method for performing differential leukocyte determinations into three subpopulations utilizing automated cell counting equipment. This reagent system contains a blood diluent and a lytic reagent. The lytic reagent comprises a mixture of quaternary ammonium surfactants. This reagent system differentiates the leukocytes into three subpopulations: lymphocytes, monocytes and granulocytes.
U.S. Pat. No. 5,155,044 (to Ledis et al.) discloses a reagent system and method for the rapid isolation and analysis of leukocytes from a whole blood sample and enables automated differentiation into five subpopulations using an automated hematology analyzer capable of impedance (DC), radio frequency (RF) and light scatter (LS) measurements. The reagent system is composed of an aqueous lytic reagent which comprises an acid, or a mixture of add and saponin, and an aqueous salt quench solution. This method is rapid and provides a five part leukocyte differential of a whole blood sample in a single step measurement.
U.S. Pat. No. 5,843,608 (to Li et al.) discloses a lytic reagent system and a method for automated differentiation of leukocytes into five subpopulations using DC, RF and light scatter measurement devices. The lytic reagent system is composed of an aqueous lytic reagent which comprises an ethoxylated long chain amine compound and an acid, and a stabilizing reagent which comprises a hypertonic, alkaline reagent composition. The disclosed lytic reagent system differentiates leukocyte into five subpopulations using a single measurement when impedance, radio frequency and light scatter measurements are used.
However, if the above two lytic reagent systems are used on a hematology analyzer equipped with only impedance and light scatter measurement devices, the basophil subpopulations will not be adequately differentiated from lymphocyte subpopulations. If only impedance and radio frequency measurements are used for the blood analysis, eosinophils will not be differentiated from neutrophils due to a complete overlap of the two populations in DC vs. Opacity (a function of DC and RF) scattergram.
Another approach for differentiation of leukocytes into four or five subpopulations is to use multiple reagents and multiple measurements. Separate reagents are used for differentiating the specific subpopulations. Typically, a first lytic reagent, like the ones revealed in U.S. Pat. No. 5,116,539 (to Hamaguchi et al.) and U.S. Pat. No. 5,389,549 (to Hamaguchi et al.) are used to differentiate the leukocytes into three subpopulations (i.e., monocytes, lymphocytes and granulocytes) using impedance and radio frequency measurements.
Hamaguchi et al. (U.S. Pat. No. 5,389,549) disdoses a lytic reagent for differentiating leukocytes into the above mentioned three subpopulations using impedance and radio frequency measurements. The lytic reagent consists essentially of a first cytolytic solution having pH of 1.5-5.0 and containing a surfactant in an amount effective to reduce erythrocyte ghosts for distinction from leukocytes without causing undesirable leukocyte damage, wherein the surfactant consists essentially of a polyoxyethylene based nonionic surfactant represented by the formula:
R
1
—R
2
—(CH
2
CH
2
O)
n
—H
wherein R
1
is an alkyl, alkenyl or alkynyl group having 12 to 22 carbon atoms, R
2
is —O—, —OC
6
H
6
— or —COO—, and n is an integer of 20-100. The reagent further consists essentially of a second cytolytic solution used in combination with the first reagent. The second cytolytic solution has a pH of 5.0-12.0 and an osmolarity of 150-2000 mOsm/kg.
In U.S. Pat. No. 5,116,539, an additional lytic reagent is used with an additional measurement to obtain the eosinophil subpopulation. This enables a four part differentiation of lymphocytes, monocytes, eosinophils and the remaining granulocytes. The lytic reagent is composed of a polyoxyethylene-based nonionic surfactant and a buffer to adjust the pH of the solution within the range of 3-11. The lytic reagent lyses not only red blood cells but also the leukocytes except eosinophils so that the eosinophils can be counted based on their remaining cellular volume measured by DC. However, to achieve eosinophil separations the method of using the lytic reagent requires incubation of the sample mixture at 40° C. for a period of 50 seconds. This elevated temperature requirement necessitated instrumentation which is significantly more complex because the reactions must be thermostatically controlled. In addition, the extended reaction time directly decreases the throughput of the automated analyzer.
In U.S. Pat. No. 5,389,549, two additional lytic reagents are needed and two additional measurements are required to obtain the eosinophil and basophil subpopulations so as to provide a five part differential. More specifically, the additional lytic reagent for differential analysis of basophils is an aqueous solution composed of a polyoxyethylene-based nonionic surfactant, potassium ophthalate, hydrochloric acid and nitric acid. This lytic reagent lyses not only red blood cells but also the leukocytes except basophils so that the basophils can be counted based on their remaining cellular volume measured by DC. Again, to achieve basophil separation the method requires incubation of the sample mixture at elevated temperature. In this disclosure, the total eosinophil and basophil subpopulations obtained from separate measurements are subtracted from the total granulocyte population to obtain the neutrophil subpopulation.
Hamaguchi et al. teach that polyoxyethylene-based nonionic surfactants are moderate in lysing blood and is suitable for leukocyte differential analysis. Hamaguchi et al. further teach that quaternary ammonium salt damage cells violently and it is entirely meaningless to use a quaternary ammonium salt as a cytolytic agent in a method of classifying leukocytes by the combination of the impedance and radio frequency methods.
U.S. Pat. No. 5,196,346 (to Lefevre et al.) discloses a lytic reagent and method of using the same for automated determination of basophils in whole blood samples by lysing all blood cells, including both erythrocytes and leukocytes, with the exception of the basophils. The lytic reagent was composed of a polyoxyethylene ether-type surfactant, a phthalic acid/HCl mixture, sodium dodecyl sulfate (SDS) and a butylated hydroxytoluene-type antioxidizing agent. The disclosed method requires a thermostatically controlled reaction temperature between 30 to 40° C. and is limited to the counting of only basophils by impedance measurement.
U.S. Pat. No. 5,821,128 (to Provost) discloses lytic reagents and method for determination of basophils and eosinophils in whole blood samples. The lytic reagent for basophil measurement consists essentially of polyoxyethylene 9-lauryl ether and acids to maintain pH of the reagent between 2 and 3.5. The method uses the lytic reagent to lys
Li Jing
Li Yi
Young Carole Jo
Alter Mitchell E.
Coulter International Corp.
Wallenhorst Maureen M.
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