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||||||||||||III USO0575109A United States Patent (19) 11) Patent Number: 5,175,109 Sakata Et Al

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||||||||||||III USO0575109A United States Patent (19) 11) Patent Number: 5,175,109 Sakata Et Al ||||||||||||III USO0575109A United States Patent (19) 11) Patent Number: 5,175,109 Sakata et al. (45) Date of Patent: " Dec. 29, 1992 54 REAGENT FOR CLASSIFYING 4,666.82 5/1987 Wiedemann et al. ................. 430/78 LEUKOCYTES BY FLOW CYTOMETRY 4,751,179 6/1988 Ledis et al. ........... ... 424/3 X 4,751,188 6/1988 Valet ............. 436/10 X 4.760.006 7/1988 Pawlowski ............................ 430/78 75) Inventors: Takashi Sakata; Tomoyuki Kuroda, both of Kakogawa, Japan 4.882,284 1 1/1989 Kirchanski et al. .. ... 436/63 73 Assignee: Toa Medical Electronics Co., Ltd., 4,933,293 6/1990 Kuroda et al. ........................ 436/63 Kobe, Japan FOREIGN PATENT DOCUMENTS *) Notice: The portion of the term of this patent O086951 8/1983 European Pat. Off. subsequent to Jun. 12, 2007 has been 1560729 2/1980 France . disclaimed. 55-18860 5/1980 Japan . (21) Appl. No.: 663,090 OTHER PUBLICATIONS Kamentsky, Blood Cells, 6, 121-140 (1980). (22 Filed: Feb. 28, 1991 Shapiro et al., J. Histochem. Cytochem., 24, 396-41 1, Related U.S. Application Data (1976). Shapiro et al., J. Histochem. Cytochem., 25, 976–989 63 Continuation of Ser. No. 91.663, Sep. 1, 1987, aban (1977). doned. Colour Index, vol. 4, published by The Society of Dyers (30) Foreign Application Priority Data and Colourists, pp. 4417-4459 (1971). Sep. 10, 1986 JP Japan ................................ 6-21376 Steinkamp, "Flow Cytometry," Rey. Sci. Instrum... pp. Nov. 27, 1986 JP Japan ................................ 6.-28.2697 1375-1400 (1974). W. Groner & D. Tycko, "Characterizing Blooc Cells 51) Int. Cl. .............................................. C09K11/06 52) U.S. Cl. ...................................... 436/17; 436/800; by Biophysical Measurements in Flow,' Blood Cells, 6, 424/3; 252/301.16 141-157 (1980). 58 Field of Search ....................... 436/8, 17, 63. 166, Primary Examiner-James C. Housel 436/172, 800; 424/3, 7.1 252/301.16. 301.9 Assistant Examiner-Jeffrey R. Snay Attorney, Agent, or Firm-Fitch, Even, Tabin & 56) References Cited Flannery U.S. PATENT DOCUMENTS 57 ABSTRACT 3.684.377 8/1972 Adams et al. 3,883.247 5/1975 Adams .................................. 356/39 A reagent for classifying leukocytes with a flow cytom 3.96.197 10/1975 Fulwyler ........ 250/36 X eter by means of optical measurements on fluoro 3.96.205 10/1975 Kleinerman ... chrome-stained blood cells is included. The reagent is 4,066,395 l/1978 Soiron et al. ........................... 8/169 useful in the practice of clinical testing and includes a 4,073,739 2/1978 Peters ............. 430/109 Neutral Red fluorochrome and either an Astrazon Or 4.146,604 3/979 Kleinerman ............................ 424/3 4.252,534 2/1981 Abel et al. .. 8/67 gane G or an Auramine O fluorochrome to enable dif 4.400.370 8/1983 Kass ........................................ 424/3 ferentiation of leukocytes. 4,528.256 7/1985 Lind....................... ... 430/83 4.58l.223 4/1986 Kass ........................................ 424/3 3 Claims, 9 Drawing Sheets U.S. Patent Dec. 29, 1992 Sheet 1 of 9 5,175,109 18 CD 14 12 16 U.S. Patent Dec. 29, 1992 Sheet 2 of 9 5,175,109 Fig. 2 a NEUTROPHILS EOSINOPHILS seases BASOPHILS cense MONOCYTES easte LYMPHOCYTES RELATIVE INTENSITY OF RIGHT-ANGLE SCATTERED LIGHT Fig.2b (prior AA7/ LYMPHO CYTES as NEUTROPHILS C RELATIVE INTENSITY OF RIGHT-ANGLE SCATTERED LIGHT 1 2 3 Fig.2c /AAP/OA AA77) SIDE SC U.S. Patent Dec. 29, 1992 Sheet 3 of 9 5,175,109 Fig. 3 Fig. 4 {2} - 32 LCD() SIDE SC. SIDE SC. Fig. 5 Fig. 6 lo?s.) | @ () G.2) SIDE SC. SIDE SC. Fig. 7 - GG) L of2O) SIDE SC. U.S. Patent Dec. 29, 1992 Sheet 4 of 9 5,175,109 Fig. 8 (s) as 'O RED FL. Fig. IO SlDE SC. U.S. Patent Dec. 29, 1992 Sheet 6 of 9 5,175,109 Fig. 13 EXCITATION AND EMISSION SPECTRA OF FLUORESCENCE OF ASTRAZON ORANGE G EXCITATION EMISSION 2OO 3OO 4OO 5OO 6 OO nm U.S. Patent Dec. 29, 1992 Sheet 7 of 9 5,175,109 Fig. 15 O RED FLUORESCENCE INTENSITY RATIO OF EOSINOPHILS TO NEUTROPHILS O GREEN FLUORESCENCE INTENSITY RATIO OF BASOPHILS TO NEUTROPHILS 4.O 3O 1.O 1 2 3 4 5 6 7 (ppm) CONCENTRATION OF NEUTRAL RED Jug/m U.S. Patent Dec. 29, 1992 Sheet 8 of 9 5,175,109 Fig. 16 W .EOSINOPHILS V \ earns s S. N LYMPHOCYTES-N pass 52O 54O 56O 58O 6OO 62o so ego (nm) U.S. Patent Dec. 29, 1992 Sheet 9 of 9 5,175,109 Fig. 17 O RED FLUORESCENCE INTENSITY RATIO OF EOSINOPHILS TO NEUTROPHILS O GREEN FLUORESCENCE INTENSITY RATIO OF BASOPHILS TO NEUTROPHILS O 5.O O O 4.O C X- O (?) H O O 2 3.O O y N Z O O (f) 9 2O L --- 8.O 8.5 9.O 9.5 O.O pH 5,175,109 1. 2 tion by processing with a computer, this method has not REAGENT FOR CLASSIFYING LEUKOCYTES BY yet become an ideal alternative to the manual method. FLOW CYTOMETRY Furthermore, this method is not economically feasible since it requires sophisticated equipment which is large This application is a continuation, of application Ser. and costly. No. 091,663 filed Sep. 1, 1987 now abandoned. The other approach toward automatic classification The present invention relates to a reagent and a and counting of leukocytes is based on a flow system. In method for classifying leukocytes in the practice of this method, a blood sample having corpuscles sus clinical testing. More particularly, the present invention pended in a diluent is permitted to flow in such a way relates to a reagent and a method for classifying leuko- 10 that the corpuscles will individually (one by one) pass cytes with a flow cytometer by means of optical nea through a narrowed detecting area and leukocyte classi Surements on fluorochrome-stained blood cells. fication is made by analyzing the signal generated by Leukocytes in the peripheral blood of normal sub the detector. This second method of leukocyte counting jects can be classified as being of five types consisting of which makes use of a flow system is further subdivided lymphocytes, monocytes, neutrophils, eosinophils, and 15 into two categories. basophils. Different leukocyte types have different in a method of the first category, an electrolyte in functions and counting of leukocytes in the blood ac which all red cells that were present have been de cording to their type will provide valuable information stroyed with a lysing agent so that only leukocytes will for diagnostic purposes. For instance, an increase in the number of neutrephils is associated with such diseases as be suspended is permitted to flow through an orifice inflammations, myocardial infarction and leukemia, and and the change in electrical impedance that occurs at a decrease in their number is associated with viral dis the orifice when each corpuscle passes through it is eases, hypoplastic anemia, agranulocytosis, etc. On the detected, with the magnitude of the detected signal other hand, an increase in the number of eosinophils is being used as a basis for classification of leukocytes. found in such diseases as parasitosis, Hodgkin's disease 25 A method of the second category is characterized by and allergosis. An increased number of monocytes oc the use of a flow cytometer that comprises a light curs either during the convalescence period of patients source, a flow cell that permits the blood cells in a suffering from infectious diseases or in such diseases as sample to flow one by one through a constricted chan monocytic leukemia. nel, a photometric unit that detects light issuing from Classification and counting of leukocytes have been 30 each blood cell, and an analyzer for analyzing the de made most commonly by the differential counting tected signals. In this method, the corpuscles in the method which is also referred to as the visual counting sample which are stained are illuminated under light method or simply as the manual method. In this and the fluorescence emitted from the irradiated cor method, a blood sample is spread on a glass slide and the puscles is detected, optionally together with scattered blood corpuscles in the snear are fixed and stained for 3 light, with leukocyte classification being made in accor examination by microscopy. The technician identifies dance with the intensity of the detected signals. the type of individual leukocytes according to their Techniques that fall within the category of this flow morphological features (e.g., their size, the morphology cytometric method are described, for example, in Japa of their nucleus and cytoplasm, and the presence or nese Patent Publication No. 853/1984 and L. A. Ka absence of granules) or the degree of dye uptake and 40 mentsky, Blood Cells, 6, 121-140 (1980). According to performs classification and counting of them. At ordi these techniques, a blood sample is stained with 10 vol nary laboratories, 100-200 leukocytes are usually umes of an acridine orange solution, incubated for 1 counted for each sample and the percentage of the total minute, and irradiated under a light source such as an leukocyte count occupied by each type of corpuscle is argon ion laser. The green fluorescence and red fluores recorded as a measured value. 45 cence that are emitted from the individual corpuscles The differential counting method has several disad are measured and classification and counting of leuko vantages. First, microscopic observation must be pre cytes are subsequently made based on a two-dimen ceded by cumbersome procedures for preparing a speci sional plot of the florescence measurements. men that involve such steps as smearing a blood sample Other examples of techniques that are classified as on a glass slide, fixing the corpuscles and staining them.
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